Role of α1- and α2-GABAA receptors in mediating the respiratory changes associated with benzodiazepine sedation

PubMed Central

Masneuf, S; Buetler, J; Koester, C; Crestani, F

2012-01-01

BACKGROUND AND PURPOSE The molecular substrates underlying the respiratory changes associated with benzodiazepine sedation are unknown. We examined the effects of different doses of diazepam and alprazolam on resting breathing in wild-type (WT) mice and clarified the contribution of α1- and α2-GABAA receptors, which mediate the sedative and muscle relaxant action of diazepam, respectively, to these drug effects using point-mutated mice possessing either α1H101R- or α2H101R-GABAA receptors insensitive to benzodiazepine. EXPERIMENTAL APPROACH Room air breathing was monitored using whole-body plethysmography. Different groups of WT mice were injected i.p. with diazepam (1–100 mg·kg−1), alprazolam (0.3, 1 or 3 mg·kg−1) or vehicle. α1H101R and α2H101R mice received 1 or 10 mg·kg−1 diazepam or 0.3 or 3 mg·kg−1 alprazolam. Respiratory frequency, tidal volume, time of expiration and time of inspiration before and 20 min after drug injection were analysed. KEY RESULTS Diazepam (10 mg·kg−1) decreased the time of expiration, thereby increasing the resting respiratory frequency, in WT and α2H101R mice, but not in α1H101R mice. The time of inspiration was shortened in WT and α1H101R mice, but not in α2H101R mice. Alprazolam (1–3 mg·kg−1) stimulated the respiratory frequency by shortening expiration and inspiration duration in WT mice. This tachypnoeic effect was partially conserved in α1H101R mice while absent in α2H101R mice. CONCLUSIONS AND IMPLICATIONS These results identify a specific role for α1-GABAA receptors and α2-GABAA receptors in mediating the shortening by benzodiazepines of the expiratory and inspiratory phase of resting breathing respectively. PMID:22044283

Benzodiazepine-site pharmacology on GABAA receptors in histaminergic neurons.

PubMed

May, A C; Fleischer, W; Kletke, O; Haas, H L; Sergeeva, O A

2013-09-01

The histaminergic tuberomamillary nucleus (TMN) of the posterior hypothalamus controls the cognitive aspects of vigilance which is reduced by common sedatives and anxiolytics. The receptors targeted by these drugs in histaminergic neurons are unknown. TMN neurons express nine different subunits of the GABAA receptor (GABAA R) with three α- (α1, α2 and α5) and two γ- (γ1, γ 2) subunits, which confer different pharmacologies of the benzodiazepine-binding site. We investigated the actions of zolpidem, midazolam, diazepam, chlordiazepoxide, flumazenil (Ro15-1788) and methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) in TMN neurons using mouse genetics, electrophysiological and molecular biological methods. We find the sensitivity of GABAA R to zolpidem, midazolam and DMCM significantly reduced in TMN neurons from γ2F77I mice, but modulatory activities of diazepam, chlordiazepoxide and flumazenil not affected. Potencies and efficacies of these compounds are in line with the dominance of α2- and α1-subunit containing receptors associated with γ2- or γ1-subunits. Functional expression of the γ1-subunit is supported by siRNA-based knock-down experiments in γ2F77I mice. GABAA R of TMN neurons respond to a variety of common sedatives with a high affinity binding site (γ2F77I) involved. The γ1-subunit likely contributes to the action of common sedatives in TMN neurons. This study is relevant for understanding the role of neuronal histamine and benzodiazepines in disorders of sleep and metabolism. © 2013 The British Pharmacological Society.

Patients with premenstrual syndrome have reduced sensitivity to midazolam compared to control subjects.

PubMed

Sundström, I; Nyberg, S; Bäckström, T

1997-12-01

Premenstrual syndrome (PMS) depends on gonadal hormones produced by the corpus luteum. Given the facilitory actions on GABAergic inhibitory neurotransmission exerted by certain progesterone metabolites, further studies on the GABAA receptor system in premenstrual syndrome are warranted. This study evaluated the benzodiazepine sensitivity in PMS patients and control subjects, using saccadic eye velocity (SEV) and visual analogue ratings of sedation as dependent measures. PMS patients displayed a significantly reduced SEV responsiveness to benzodiazepines compared to control subjects in the follicular phase, whereas there was no difference between groups in the luteal phase. In the luteal phase, the sedation response to benzodiazepines was significantly reduced in PMS patients compared to control subjects. There was also an influence of PMS symptom severity on these measures, as high-severity PMS patients displayed blunted SEV and sedation responses to benzodiazepines compared to low-severity patients. These results indicate that PMS patients have a reduced functional sensitivity at the GABAA/benzodiazepine receptor complex throughout the menstrual cycle.

GABA(A) receptors mediate orexin-A induced stimulation of food intake.

PubMed

Kokare, Dadasaheb M; Patole, Angad M; Carta, Anna; Chopde, Chandrabhan T; Subhedar, Nishikant K

2006-01-01

Although the role of orexins in sleep/wake cycle and feeding behavior is well established, underlying mechanisms have not been fully understood. An attempt has been made to investigate the role of GABA(A) receptors and their benzodiazepine site on the orexin-A induced response to feeding. Different groups of rats were food deprived overnight and next day injected intracerebroventricularly (icv) with vehicle (artificial CSF; 5 microl/rat) or orexin-A (20-50 nM/rat) and the animals were given free access to food. Cumulative food intake was measured during light phase of light/dark cycle at 1-, 2-, 4- and 6-h post-injection time points. Orexin-A (30-50 nM/rat, icv) stimulated food intake at all the time points (P < 0.05). Prior administration of GABA(A) receptor agonists muscimol (25 ng/rat, icv) and diazepam (0.5 mg/kg, ip) at subeffective doses significantly potentiated the hyperphagic effect of orexin-A (30 nM/rat, icv). However, the effect was negated by the GABA(A) receptor antagonist bicuculline (1 mg/kg, ip). Interestingly, benzodiazepine receptor antagonist flumazenil (5 ng/rat, icv), augmented the orexin-A (30 nM/rat, icv) induced hyperphagia; the effect may be attributed to the intrinsic activity of the agent. The results suggest that the hyperphagic effect of orexin-A, at least in part, is mediated by enhanced GABA(A) receptor activity.

Calcium/calmodulin-dependent kinase II phosphorylation of the GABAA receptor alpha1 subunit modulates benzodiazepine binding.

PubMed

Churn, Severn B; Rana, Aniruddha; Lee, Kangmin; Parsons, J Travis; De Blas, Angel; Delorenzo, Robert J

2002-09-01

gamma-Aminobutyric acid (GABA) is the primary neurotransmitter that is responsible for the fast inhibitory synaptic transmission in the central nervous system. A major post-translational mechanism that can rapidly regulate GABAAR function is receptor phosphorylation. This study was designed to test the effect of endogenous calcium and calmodulin-dependent kinase II (CaM kinase II) activation on both allosteric modulator binding and GABAA receptor subunit phosphorylation. Endogenous CaM kinase II activity was stimulated, and GABAA receptors were subsequently analyzed for bothallosteric modulator binding properties and immunoprecipitated and analyzed for subunit phosphorylation levels. A significant increase in allosteric-modulator binding of the GABAAR was observed under conditions maximal for CaM kinase II activation. In addition, CaM kinase II activation resulted in a direct increase in phosphorylation of the GABAA receptor alpha1 subunit. The data suggest that the CaM kinase II-dependent phosphorylation of the GABAA receptor alpha1 subunit modulated allosteric modulator binding to the GABAA receptor.

Benzodiazepine-induced anxiolysis and reduction of conditioned fear are mediated by distinct GABAA receptor subtypes in mice

PubMed Central

Smith, Kiersten S.; Engin, Elif; Meloni, Edward G.; Rudolph, Uwe

2012-01-01

GABAA receptor modulating drugs such as benzodiazepines (BZs) have been used to treat anxiety disorders for over five decades. In order to determine whether the same or different GABAA receptor subtypes are necessary for the anxiolytic-like action of BZs in unconditioned anxiety and conditioned fear models, we investigated the role of different GABAA receptor subtypes by challenging wild type, α1(H101R), α2(H101R) and α3(H126R) mice bred on the C57BL/6J background with diazepam or chlordiazepoxide in the elevated plus maze and the fear-potentiated startle paradigms. Both drugs significantly increased open arm exploration in the elevated plus maze in wild type, α1(H101R) and α3(H126R), but this effect was abolished in α2(H101R) mice; these were expected results based on previous published results. In contrast, while administration of diazepam and chlordiazepoxide significantly attenuated fear-potentiated startle (FPS) in wild type mice and α3(H126R) mice, the fear-reducing effects of these drugs were absent in both α1(H101R) and α2(H101R) point mutants, indicating that both α1- and α2-containing GABAA receptors are necessary for BZs to exert their effects on conditioned fear responses.. Our findings illustrate both an overlap and a divergence between the GABAA receptor subtype requirements for the impact of BZs, specifically that both α1- and α2-containing GABAA receptors are necessary for BZs to reduce conditioned fear whereas only α2-containing GABAA receptors are needed for BZ-induced anxiolysis in unconditioned tests of anxiety. This raises the possibility that GABAergic pharmacological interventions for specific anxiety disorders can be differentially tailored. PMID:22465203

Structural basis for ligand recognition at the benzodiazepine binding site of GABAA alpha 3 receptor, and pharmacophore-based virtual screening approach.

PubMed

Vijayan, R S K; Ghoshal, Nanda

2008-10-01

Given the heterogeneity of GABA(A) receptor, the pharmacological significance of identifying subtype selective modulators is increasingly being recognized. Thus, drugs selective for GABA(A) alpha(3) receptors are expected to display fewer side effects than the drugs presently in clinical use. Hence we carried out 3D QSAR (three-dimensional quantitative structure-activity relationship) studies on a series of novel GABA(A) alpha(3) subtype selective modulators to gain more insight into subtype affinity. To identify the 3D functional attributes required for subtype selectivity, a chemical feature-based pharmacophore, primarily based on selective ligands representing diverse structural classes was generated. The obtained pseudo receptor model of the benzodiazepine binding site revealed a binding mode akin to "Message-Address" concept. Scaffold hopping was carried out across multi-conformational May Bridge database for the identification of novel chemotypes. Further a focused data reduction approach was employed to choose a subset of enriched compounds based on "Drug likeness" and "Similarity-based" methods. These results taken together could provide impetus for rational design and optimization of more selective and high affinity leads with a potential to have decreased adverse effects.

Relationship between a GABAA alpha 6 Pro385Ser substitution and benzodiazepine sensitivity.

PubMed

Iwata, N; Cowley, D S; Radel, M; Roy-Byrne, P P; Goldman, D

1999-09-01

In humans, interindividual variation in sensitivity to benzodiazepine drugs may correlate with behavioral variation, including vulnerability to disease states such as alcoholism. In the rat, variation in alcohol and benzodiazepine sensitivity has been correlated with an inherited variant of the GABAA alpha 6 receptor. The authors detected a Pro385Ser [1236C > T] amino acid substitution in the human GABAA alpha 6 that may influence alcohol sensitivity. In this pilot study, they evaluated the contribution of this polymorphism to benzodiazepine sensitivity. Sensitivity to diazepam was assessed in 51 children of alcoholics by using two eye movement measures: peak saccadic velocity and average smooth pursuit gain. Association analysis was performed with saccadic velocity and smooth pursuit gain as dependent variables and comparing Pro385/Ser385 heterozygotes and Pro385/Pro385 homozygotes. The Pro385Ser genotype was associated with less diazepam-induced impairment of saccadic velocity but not with smooth pursuit gain. The Pro385Ser genotype may play a role in benzodiazepine sensitivity and conditions, such as alcoholism, that may be correlated with this trait.

Women with PTSD have a changed sensitivity to GABA-A receptor active substances.

PubMed

Möller, Anna Tiihonen; Bäckström, Torbjörn; Nyberg, Sigrid; Söndergaard, Hans Peter; Helström, Lotti

2016-06-01

The use of benzodiazepines in treating anxiety symptoms in patients with posttraumatic stress disorder (PTSD) has been debated. Studies on other anxiety disorders have indicated changed sensitivity to GABA-A receptor active substances. In the present study, we investigated the GABA receptor sensitivity in PTSD patients. Injections of allopreganolone, diazepam, and flumazenil were carried out, each on separate occasions, in 10 drug naïve patients with PTSD compared to 10 healthy controls. Effects were measured in saccadic eye velocity (SEV) and in subjective ratings of sedation. The PTSD patients were less sensitive to allopregnanolone compared with healthy controls. This was seen as a significant difference in SEV between the groups (p = 0.047). Further, the patients were less sensitive to diazepam, with a significant less increase in sedation compared to controls (p = 0.027). After flumazenil injection, both patients and controls had a significant agonistic effect on SEV, leading to decreased SEV after injection. The patients also responded with an increase in sedation after flumazenil injection, while this was not seen in the controls. Patients with PTSD have a changed sensitivity to GABA-A receptor active substances. As a consequence of this, benzodiazepines and other GABA-A receptor active compounds such as sleeping pills will be less useful for this group of patients.

The Role of GABAA Receptors in the Development of Alcoholism

PubMed Central

Enoch, Mary-Anne

2008-01-01

Alcoholism is a common, heritable, chronic relapsing disorder. GABAA receptors undergo allosteric modulation by ethanol, anesthetics, benzodiazepines and neurosteroids and have been implicated in the acute as well as the chronic effects of ethanol including tolerance, dependence and withdrawal. Medications targeting GABAA receptors ameliorate the symptoms of acute withdrawal. Ethanol induces plasticity in GABAA receptors: tolerance is associated with generally decreased GABAA receptor activation and differentially altered subunit expression. The dopamine (DA) mesolimbic reward pathway originating in the ventral tegmental area (VTA), and interacting stress circuitry play an important role in the development of addiction. VTA GABAergic interneurons are the primary inhibitory regulators of DA neurons and a subset of VTA GABAA receptors may be implicated in the switch from heavy drinking to dependence. GABAA receptors modulate anxiety and response to stress; important elements of sustained drinking and relapse. The GABAA receptor subunit genes clustered on chromosome 4 are highly expressed in the reward pathway. Several recent studies have provided strong evidence that one of these genes, GABRA2, is implicated in alcoholism in humans. The influence of the interaction between ethanol and GABAA receptors in the reward pathway on the development of alcoholism together with genetic and epigenetic vulnerabilities will be explored in this review. PMID:18440057

The role of GABA(A) receptors in the development of alcoholism.

PubMed

Enoch, Mary-Anne

2008-07-01

Alcoholism is a common, heritable, chronic relapsing disorder. GABA(A) receptors undergo allosteric modulation by ethanol, anesthetics, benzodiazepines and neurosteroids and have been implicated in the acute as well as the chronic effects of ethanol including tolerance, dependence and withdrawal. Medications targeting GABA(A) receptors ameliorate the symptoms of acute withdrawal. Ethanol induces plasticity in GABA(A) receptors: tolerance is associated with generally decreased GABA(A) receptor activation and differentially altered subunit expression. The dopamine (DA) mesolimbic reward pathway originating in the ventral tegmental area (VTA), and interacting stress circuitry play an important role in the development of addiction. VTA GABAergic interneurons are the primary inhibitory regulators of DA neurons and a subset of VTA GABA(A) receptors may be implicated in the switch from heavy drinking to dependence. GABA(A) receptors modulate anxiety and response to stress; important elements of sustained drinking and relapse. The GABA(A) receptor subunit genes clustered on chromosome 4 are highly expressed in the reward pathway. Several recent studies have provided strong evidence that one of these genes, GABRA2, is implicated in alcoholism in humans. The influence of the interaction between ethanol and GABA(A) receptors in the reward pathway on the development of alcoholism together with genetic and epigenetic vulnerabilities will be explored in this review.

Memory Effects of Benzodiazepines: Memory Stages and Types Versus Binding-Site Subtypes

PubMed Central

Savić, Miroslav M.; Obradović, Dragan I.; Ugrešić, Nenad D.; Bokonjić, Dubravko R.

2005-01-01

Benzodiazepines are well established as inhibitory modulators of memory processing. This effect is especially prominent when applied before the acquisition phase of a memory task. This minireview concentrates on the putative subtype selectivity of the acquisition-impairing action of benzodiazepines. Namely, recent genetic studies and standard behavioral tests employing subtype-selective ligands pointed to the predominant involvement of two subtypes of benzodiazepine binding sites in memory modulation. Explicit memory learning seems to be affected through the GABAA receptors containing the α1 and α1 subunits, whereas the effects on procedural memory can be mainly mediated by the α1 subunit. The pervading involvement of the α1 subunit in memory modulation is not at all unexpected because this subunit is the major subtype, present in 60% of all GABAA receptors. On the other hand, the role of α5 subunits, mainly expressed in the hippocampus, in modulating distinct forms of memory gives promise of selective pharmacological coping with certain memory deficit states. PMID:16444900

K+ channel TASK-1 knockout mice show enhanced sensitivities to ataxic and hypnotic effects of GABA(A) receptor ligands.

PubMed

Linden, Anni-Maija; Aller, M Isabel; Leppä, Elli; Rosenberg, Per H; Wisden, William; Korpi, Esa R

2008-10-01

TASK two-pore-domain leak K(+) channels occur throughout the brain. However, TASK-1 and TASK-3 knockout (KO) mice have few neurological impairments and only mildly reduced sensitivities to inhalational anesthetics, contrasting with the anticipated functions and importance of these channels. TASK-1/-3 channel expression can compensate for the absence of GABA(A) receptors in GABA(A) alpha6 KO mice. To investigate the converse, we analyzed the behavior of TASK-1 and -3 KO mice after administering drugs with preferential efficacies at GABA(A) receptor subtypes: benzodiazepines (diazepam and flurazepam, active at alpha1betagamma2, alpha2betagamma2, alpha3betagamma2, and alpha5betagamma2 subtypes), zolpidem (alpha1betagamma2 subtype), propofol (beta2-3-containing receptors), gaboxadol (alpha4betadelta and alpha6betadelta subtypes), pregnanolone, and pentobarbital (many subtypes). TASK-1 KO mice showed increased motor impairment in rotarod and beam-walking tests after diazepam and flurazepam administration but not after zolpidem. They also showed prolonged loss of righting reflex induced by propofol and pentobarbital. Autoradiography indicated no change in GABA(A) receptor ligand binding levels. These altered behavioral responses to GABAergic drugs suggest functional up-regulation of alpha2beta2/3gamma2 and alpha3beta2/3gamma2 receptor subtypes in TASK-1 KO mice. In addition, female, but not male, TASK-1 KO mice were more sensitive to gaboxadol, suggesting an increased influence of alpha4betadelta or alpha6betadelta subtypes. The benzodiazepine sensitivity of TASK-3 KO mice was marginally increased. Our results underline that TASK-1 channels perform such key functions in the brain that compensation is needed for their absence. Furthermore, because inhalation anesthetics act partially through GABA(A) receptors, the up-regulation of GABA(A) receptor function in TASK-1 KO mice might mask TASK-1 channel's significance as a target for inhalation anesthetics.

The mouse beam walking assay offers improved sensitivity over the mouse rotarod in determining motor coordination deficits induced by benzodiazepines.

PubMed

Stanley, Joanna L; Lincoln, Rachael J; Brown, Terry A; McDonald, Louise M; Dawson, Gerard R; Reynolds, David S

2005-05-01

The mouse rotarod test of motor coordination/sedation is commonly used to predict clinical sedation caused by novel drugs. However, past experience suggests that it lacks the desired degree of sensitivity to be predictive of effects in humans. For example, the benzodiazepine, bretazenil, showed little impairment of mouse rotarod performance, but marked sedation in humans. The aim of the present study was to assess whether the mouse beam walking assay demonstrates: (i) an increased sensitivity over the rotarod and (ii) an increased ability to predict clinically sedative doses of benzodiazepines. The study compared the effects of the full benzodiazepine agonists, diazepam and lorazepam, and the partial agonist, bretazenil, on the mouse rotarod and beam walking assays. Diazepam and lorazepam significantly impaired rotarod performance, although relatively high GABA-A receptor occupancy was required (72% and 93%, respectively), whereas beam walking performance was significantly affected at approximately 30% receptor occupancy. Bretazenil produced significant deficits at 90% and 53% receptor occupancy on the rotarod and beam walking assays, respectively. The results suggest that the mouse beam walking assay is a more sensitive tool for determining benzodiazepine-induced motor coordination deficits than the rotarod. Furthermore, the GABA-A receptor occupancy values at which significant deficits were determined in the beam walking assay are comparable with those observed in clinical positron emission tomography studies using sedative doses of benzodiazepines. These data suggest that the beam walking assay may be able to more accurately predict the clinically sedative doses of novel benzodiazepine-like drugs.

Reversal of pathological pain through specific spinal GABAA receptor subtypes.

PubMed

Knabl, Julia; Witschi, Robert; Hösl, Katharina; Reinold, Heiko; Zeilhofer, Ulrike B; Ahmadi, Seifollah; Brockhaus, Johannes; Sergejeva, Marina; Hess, Andreas; Brune, Kay; Fritschy, Jean-Marc; Rudolph, Uwe; Möhler, Hanns; Zeilhofer, Hanns Ulrich

2008-01-17

Inflammatory diseases and neuropathic insults are frequently accompanied by severe and debilitating pain, which can become chronic and often unresponsive to conventional analgesic treatment. A loss of synaptic inhibition in the spinal dorsal horn is considered to contribute significantly to this pain pathology. Facilitation of spinal gamma-aminobutyric acid (GABA)ergic neurotransmission through modulation of GABA(A) receptors should be able to compensate for this loss. With the use of GABA(A)-receptor point-mutated knock-in mice in which specific GABA(A) receptor subtypes have been selectively rendered insensitive to benzodiazepine-site ligands, we show here that pronounced analgesia can be achieved by specifically targeting spinal GABA(A) receptors containing the alpha2 and/or alpha3 subunits. We show that their selective activation by the non-sedative ('alpha1-sparing') benzodiazepine-site ligand L-838,417 (ref. 13) is highly effective against inflammatory and neuropathic pain yet devoid of unwanted sedation, motor impairment and tolerance development. L-838,417 not only diminished the nociceptive input to the brain but also reduced the activity of brain areas related to the associative-emotional components of pain, as shown by functional magnetic resonance imaging in rats. These results provide a rational basis for the development of subtype-selective GABAergic drugs for the treatment of chronic pain, which is often refractory to classical analgesics.

Dual Modulators of GABA-A and Alpha7 Nicotinic Receptors for Treating Autism

DTIC Science & Technology

2014-08-01

Synthesis of 2-261, AVL-3288 & GRN-529. This task was accomplished in December, 2013. The task was accomplished one month later than predicted in the...approved SOW because of the need to synthesize some of the starting materials that were commercially unavailable for the synthesis of the compounds...Interestingly recent studies with the benzodiazepine agonist clonazepam , a non-selective GABAA receptor PAM, resulted in a bell-shaped dose response

The differential effects of alprazolam and oxazepam on methamphetamine self-administration in rats.

PubMed

Spence, Allyson L; Guerin, Glenn F; Goeders, Nicholas E

2016-09-01

Methamphetamine is the second most commonly used illicit drug in the world, and despite recent attempts by the Drug Enforcement Administration to combat this epidemic, methamphetamine use is still on the rise. As methamphetamine use increases so does polydrug use, particularly that involving methamphetamine and benzodiazepines. The present study was designed to examine the effects of two benzodiazepines on methamphetamine self-administration. Five doses of methamphetamine (0.0075, 0.015, 0.03, 0.09, and 0.12mg/kg/infusion) were tested, producing an inverted U-shaped dose-response curve. Rats were then pretreated with oxazepam, alprazolam, or vehicle prior to methamphetamine self-administration. To determine if the effects of these drugs were due to the GABAA receptor and/or translocator protein (TSPO), we also pretreated rats with an antagonist for the benzodiazepine-binding site on the GABAA receptor (i.e., flumazenil) and a TSPO antagonist (i.e., PK11195) prior to alprazolam or oxazepam administration. Oxazepam significantly reduced methamphetamine self-administration as demonstrated by a downward shift of the dose-response curve. In contrast, alprazolam significantly enhanced methamphetamine self-administration as evidenced by a leftward shift of the dose-response curve. Flumazenil completely blocked the effects of alprazolam on methamphetamine self-administration. When administered individually, both flumazenil and PK11195 partially reversed the effects of oxazepam on methamphetamine self-administration. However, when these two antagonists were combined, the effects of oxazepam were completely reversed. The GABAA receptor is responsible for the alprazolam-induced enhancement of methamphetamine self-administration, while the activation of both the GABAA receptor and TSPO are responsible for the oxazepam-induced reduction of methamphetamine self-administration. Published by Elsevier Ireland Ltd.

Extrasynaptic αβ subunit GABAA receptors on rat hippocampal pyramidal neurons

PubMed Central

Mortensen, Martin; Smart, Trevor G

2006-01-01

Extrasynaptic GABAA receptors that are tonically activated by ambient GABA are important for controlling neuronal excitability. In hippocampal pyramidal neurons, the subunit composition of these extrasynaptic receptors may include α5βγ and/or α4βδ subunits. Our present studies reveal that a component of the tonic current in the hippocampus is highly sensitive to inhibition by Zn2+. This component is probably not mediated by either α5βγ or α4βδ receptors, but might be explained by the presence of αβ isoforms. Using patch-clamp recording from pyramidal neurons, a small tonic current measured in the absence of exogenous GABA exhibited both high and low sensitivity to Zn2+ inhibition (IC50 values, 1.89 and 223 μm, respectively). Using low nanomolar and micromolar GABA concentrations to replicate tonic currents, we identified two components that are mediated by benzodiazepine-sensitive and -insensitive receptors. The latter indicated that extrasynaptic GABAA receptors exist that are devoid of γ2 subunits. To distinguish whether the benzodiazepine-insensitive receptors were αβ or αβδ isoforms, we used single-channel recording. Expressing recombinant α1β3γ2, α5β3γ2, α4β3δ and α1β3 receptors in human embryonic kidney (HEK) or mouse fibroblast (Ltk) cells, revealed similar openings with high main conductances (∼25–28 pS) for γ2 or δ subunit-containing receptors whereas αβ receptors were characterized by a lower main conductance state (∼11 pS). Recording from pyramidal cell somata revealed a similar range of channel conductances, indicative of a mixture of GABAA receptors in the extrasynaptic membrane. The lowest conductance state (∼11 pS) was the most sensitive to Zn2+ inhibition in accord with the presence of αβ receptors. This receptor type is estimated to account for up to 10% of all extrasynaptic GABAA receptors on hippocampal pyramidal neurons. PMID:17023503

Valium without dependence? Individual GABAA receptor subtype contribution toward benzodiazepine addiction, tolerance, and therapeutic effects.

PubMed

Cheng, Tianze; Wallace, Dominique Marie; Ponteri, Benjamin; Tuli, Mahir

2018-01-01

Benzodiazepines are one of the most prescribed medications as first-line treatment of anxiety, insomnia, and epilepsy around the world. Over the past two decades, advances in the neuropharmacological understanding of gamma aminobutyric acid (GABA) A receptors revealed distinct contributions from each subtype and produced effects. Recent findings have highlighted the importance of α 1 containing GABA A receptors in the mechanisms of addiction and tolerance in benzodiazepine treatments. This has shown promise in the development of tranquilizers with minimal side effects such as cognitive impairment, dependence, and tolerance. A valium-like drug without its side effects, as repeatedly demonstrated in animals, is achievable.

Anticonflict effect of alpidem as compared with the benzodiazepine alprazolam in rats.

PubMed

Hascoët, M; Bourin, M

1997-02-01

A comparative study between two drugs acting on the GABAA receptor, alprazolam and alpidem was undertaken, using simple tests such as measurement of spontaneous locomotor activity, four plates test and rotarod in mice. Additional conflict test was further performed using a new conflict paradigm where the opportunity existed for rats to choose during punished periods between immediate, punished reinforcement and delayed non-punished reinforcement. The benzodiazepine alprazolam, demonstrated, as expected, strong anxiolytic effects in mice and increased punished response in rats at non sedative doses (0.5, 1 mg/kg). High doses of alprazolam decreased spontaneous locomotor activity and induced myorelaxant effects in mice. Alpidem, an imidazopyridine derivative, induced motor impairment in mice and only very weak anxiolytic effects in the four plates test in mice (4 mg/kg) and in punished procedure in rats (32 mg/kg). As alprazolam is a full agonist for the GABAA receptor complex and alpidem is a partial agonist acting with specificity on omega 1 GABAA receptor subtypes, the results are discussed. Activity on omega 1 receptor subtypes is perhaps not sufficient in order to obtain a true anti-conflict effect and compounds such as alpidem only relieve some of the symptoms of anxiety disorders.

Using drug combinations to assess potential contributions of non-GABAA receptors in the discriminative stimulus effects of the neuroactive steroid pregnanolone in rats.

PubMed

Eppolito, Amy K; Kodeih, Hanna R; Gerak, Lisa R

2014-10-01

Neuroactive steroids are increasingly implicated in the development of depression and anxiety and have been suggested as possible treatments for these disorders. While neuroactive steroids, such as pregnanolone, act primarily at γ-aminobutyric acidA (GABAA) receptors, other mechanisms might contribute to their behavioral effects and could increase their clinical effectiveness, as compared with drugs acting exclusively at GABAA receptors (e.g., benzodiazepines). The current study examined the role of non-GABAA receptors, including N-methyl-d-aspartate (NMDA) and serotonin3 (5-HT3) receptors, in the discriminative stimulus effects of pregnanolone. Separate groups of rats discriminated either 3.2mg/kg pregnanolone from vehicle or 0.32mg/kg of the benzodiazepine midazolam from vehicle while responding under a fixed-ratio 10 schedule for food pellets. When administered alone in both groups, pregnanolone and midazolam produced ≥80% drug-lever responding, the NMDA receptor antagonists dizocilpine and phencyclidine produced ≥60 and ≥30% drug-lever responding, respectively, and the 5-HT3 receptor agonist 1-(m-chlorophenyl)-biguanide (CPBG) and morphine produced <20% drug-lever responding up to doses that markedly decreased response rates. When studied together, neither dizocilpine, phencyclidine, CPBG nor morphine significantly altered the midazolam dose-effect curve in either group. Given that CPBG is without effect, it is unlikely that 5-HT3 receptors contribute substantially to the discriminative stimulus effects of pregnanolone. Similarities across groups in effects of dizocilpine and phencyclidine suggest that NMDA receptors do not differentially contribute to the effects of pregnanolone. Thus, NMDA and 5-HT3 receptors are not involved in the discriminative stimulus effects of pregnanolone. Copyright © 2014 Elsevier Inc. All rights reserved.

GABAA-benzodiazepine receptors in the dorsomedial (Dm) telencephalon modulate restraint-induced antinociception in the fish Leporinus macrocephalus.

PubMed

Wolkers, Carla Patricia Bejo; Barbosa Junior, Augusto; Menescal-de-Oliveira, Leda; Hoffmann, Anette

2015-08-01

The possibility that fish experience pain has been denied based on the absence of the neural substrates to support this "experience". In this context, the identification of brain regions involved in nociception modulation could provide important insights regarding the processing of nociceptive information in fish. Our study evaluated the participation of the GABAA-benzodiazepine receptor in the dorsomedial (Dm) telencephalon in restraint-induced antinociception in the fish Leporinus macrocephalus through the microinjection of the anxiolytic drug midazolam. The microinjection of midazolam in the Dm did not alter the nocifensive response; however, this drug did block the inhibition of the nocifensive response to formaldehyde promoted by restraint stress. The fish that received midazolam (40nmol) microinjection prior to restraint (3 or 5min), followed by subcutaneous injection with formaldehyde presented a higher distance traveled than the fish that received saline microinjection. This effect might reflect the specific action of midazolam on benzodiazepine receptors in the Dm telencephalon, as pre-treatment with flumazenil, a benzodiazepine receptor antagonist, inhibited the effects of this drug. In the present study, we present the first evidence demonstrating a role for the dorsomedial telencephalic region in the modulation of stress-induced antinociception in fish, revealing new perspectives in the understanding of nociceptive information processing in this group. Copyright © 2015 Elsevier Inc. All rights reserved.

Dissociating anxiolytic and sedative effects of GABAAergic drugs using temperature and locomotor responses to acute stress

PubMed Central

Klanker, Marianne; Groenink, Lucianne; Korte, S. Mechiel; Cook, James M.; Van Linn, Michael L.; Hopkins, Seth C.; Olivier, Berend

2009-01-01

Rationale The stress-induced hyperthermia (SIH) model is an anxiety model that uses the transient rise in body temperature in response to acute stress. Benzodiazepines produce anxiolytic as well as sedative side effects through nonselective binding to GABAA receptor subunits. The GABAA receptor α1 subunit is associated with sedation, whereas the GABAA receptor α2 and α3 subunits are involved in anxiolytic effects. Objectives We therefore examined the effects of (non) subunit-selective GABAA receptor agonists on temperature and locomotor responses to novel cage stress. Results Using telemetric monitoring of temperature and locomotor activity, we found that nonsubunit-selective GABAA receptor agonist diazepam as well as the α3 subunit-selective receptor agonist TP003 dose-dependently attenuated SIH and locomotor responses. Administration of GABAA receptor α1-selective agonist zolpidem resulted in profound hypothermia and locomotor sedation. The GABAA receptor α1-selective antagonist βCCt antagonized the hypothermia, but did not reverse the SIH response attenuation caused by diazepam and zolpidem. These results suggest an important regulating role for the α1 subunit in thermoregulation and sedation. Ligands of extrasynaptic GABAA receptors such as alcohol and nonbenzodiazepine THIP attenuated the SIH response only at high doses. Conclusions The present study confirms a putative role for the GABAA receptor α1 subunit in hypothermia and sedation and supports a role for α2/3 subunit GABAA receptor agonists in anxiety processes. In conclusion, we show that home cage temperature and locomotor responses to novel home cage stress provide an excellent tool to assess both anxiolytic and sedative effects of various (subunit-selective) GABAAergic compounds. PMID:19169673

Menthol enhances phasic and tonic GABAA receptor-mediated currents in midbrain periaqueductal grey neurons

PubMed Central

Lau, Benjamin K; Karim, Shafinaz; Goodchild, Ann K; Vaughan, Christopher W; Drew, Geoffrey M

2014-01-01

Background and Purpose Menthol, a naturally occurring compound in the essential oil of mint leaves, is used for its medicinal, sensory and fragrant properties. Menthol acts via transient receptor potential (TRPM8 and TRPA1) channels and as a positive allosteric modulator of recombinant GABAA receptors. Here, we examined the actions of menthol on GABAA receptor-mediated currents in intact midbrain slices. Experimental Approach Whole-cell voltage-clamp recordings were made from periaqueductal grey (PAG) neurons in midbrain slices from rats to determine the effects of menthol on GABAA receptor-mediated phasic IPSCs and tonic currents. Key Results Menthol (150–750 μM) produced a concentration-dependent prolongation of spontaneous GABAA receptor-mediated IPSCs, but not non-NMDA receptor-mediated EPSCs throughout the PAG. Menthol actions were unaffected by TRPM8 and TRPA1 antagonists, tetrodotoxin and the benzodiazepine antagonist, flumazenil. Menthol also enhanced a tonic current, which was sensitive to the GABAA receptor antagonists, picrotoxin (100 μM), bicuculline (30 μM) and Zn2+ (100 μM), but unaffected by gabazine (10 μM) and a GABAC receptor antagonist, 1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid hydrate (TPMPA; 50 μM). In addition, menthol potentiated currents induced by the extrasynaptic GABAA receptor agonist THIP/gaboxadol (10 μM). Conclusions and Implications These results suggest that menthol positively modulates both synaptic and extrasynaptic populations of GABAA receptors in native PAG neurons. The development of agents that potentiate GABAA-mediated tonic currents and phasic IPSCs in a manner similar to menthol could provide a basis for novel GABAA-related pharmacotherapies. PMID:24460753

Duration of treatment and activation of α1-containing GABAA receptors variably affect the level of anxiety and seizure susceptibility after diazepam withdrawal in rats

PubMed Central

Kovačević, Jovana; Timić, Tamara; Tiruveedhula, Veera V.; Batinić, Bojan; Namjoshi, Ojas A.; Milić, Marija; Joksimović, Srđan; Cook, James M.; Savić, Miroslav M.

2014-01-01

Long-term use of benzodiazepine-type drugs may lead to physical dependence, manifested by withdrawal syndrome after abrupt cessation of treatment. The aim of the present study was to investigate the influence of duration of treatment, as well as the role of α1-containing GABAA receptors, in development of physical dependence to diazepam, assessed through the level of anxiety and susceptibility to pentylenetetrazole (PTZ)-induced seizures, 24 h after withdrawal from protracted treatment in rats. Withdrawal of 2 mg/kg diazepam after 28, but not after 14 or 21 days of administration led to an anxiety-like behavior in the elevated plus maze. Antagonism of the diazepam effects at α1-containing GABAA receptors, achieved by daily administration of the neutral modulator βCCt (5 mg/kg), did not affect the anxiety level during withdrawal. An increased susceptibility to PTZ-induced seizures was observed during diazepam withdrawal after 21 and 28 days of treatment. Daily co-administration of βCCt further decreased the PTZ-seizure threshold after 21 days of treatment, whilst it prevented the diazepam withdrawal-elicited decrease of the PTZ threshold after 28 days of treatment. In conclusion, the current study suggests that the role of α1-containing GABAA receptors in mediating the development of physical dependence may vary based on the effect being studied and duration of protracted treatment. Moreover, the present data supports previous findings that the lack of activity at α1-containing GABAA receptors is not sufficient to eliminate physical dependence liability of ligands of the benzodiazepine type. PMID:24695241

Differential effects of short- and long-term zolpidem treatment on recombinant α1β2γ2s subtype of GABAA receptors in vitro

PubMed Central

Vlainić, Josipa; Jembrek, Maja Jazvinšćak; Vlainić, Toni; Štrac, Dubravka Švob; Peričić, Danka

2012-01-01

Aim: Zolpidem is a non-benzodiazepine agonist at benzodiazepine binding site in GABAA receptors, which is increasingly prescribed. Recent studies suggest that prolonged zolpidem treatment induces tolerance. The aim of this study was to explore the adaptive changes in GABAA receptors following short and long-term exposure to zolpidem in vitro. Methods: Human embryonic kidney (HEK) 293 cells stably expressing recombinant α1β2γ2s GABAA receptors were exposed to zolpidem (1 and 10 μmol/L) for short-term (2 h daily for 1, 2, or 3 consecutive days) or long-term (continuously for 48 h). Radioligand binding studies were used to determine the parameters of [3H]flunitrazepam binding sites. Results: A single (2 h) or repeated (2 h daily for 2 or 3 d) short-term exposure to zolpidem affected neither the maximum number of [3H]flunitrazepam binding sites nor the affinity. In both control and short-term zolpidem treated groups, addition of GABA (1 nmol/L–1 mmol/L) enhanced [3H]flunitrazepam binding in a concentration-dependent manner. The maximum enhancement of [3H]flunitrazepam binding in short-term zolpidem treated group was not significantly different from that in the control group. In contrast, long-term exposure to zolpidem resulted in significantly increase in the maximum number of [3H]flunitrazepam binding sites without changing the affinity. Furthermore, long-term exposure to zolpidem significantly decreased the ability of GABA to stimulate [3H]flunitrazepam binding. Conclusion: The results suggest that continuous, but not intermittent and short-term, zolpidem-exposure is able to induce adaptive changes in GABAA receptors that could be related to the development of tolerance and dependence. PMID:22922343

Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit.

PubMed

Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nabekura, J; Noguchi, K; Akaike, N; Witt, M R; Nielsen, M

1997-06-25

Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.

GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

NASA Technical Reports Server (NTRS)

Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

1995-01-01

The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of schizophrenics, the previously reported upregulation of muscimol binding sites and downregulation of benzodiazepine binding sites in the prefrontal and adjacent cingulate cortex of schizophrenics are possibly due to posttranscriptional modifications of mRNAs and their translated polypeptides.

Targeted deletion of the GABRA2 gene encoding alpha2-subunits of GABA(A) receptors facilitates performance of a conditioned emotional response, and abolishes anxiolytic effects of benzodiazepines and barbiturates.

PubMed

Dixon, C I; Rosahl, T W; Stephens, D N

2008-07-01

Mice with point-mutated alpha2 GABA(A) receptor subunits (rendering them diazepam insensitive) are resistant to the anxiolytic-like effects of benzodiazepines (BZs) in the conditioned emotional response (CER) test, but show normal anxiolytic effects of a barbiturate. We investigated the consequence of deleting the alpha2-subunit on acquisition of the CER with increasing intensity of footshock, and on the anxiolytic efficacy of a benzodiazepine, diazepam, and a barbiturate, pentobarbital. alpha2 knockout (KO) and wildtype (WT) mice were trained in a conditioned emotional response (CER) task, in which lever pressing for food on a variable interval (VI) schedule was suppressed during the presentation of a compound light/tone conditioned stimulus (CS+) that predicted footshock. The ability of diazepam and of pentobarbital to reduce suppression during the CS+ was interpreted as an anxiolytic response. There were no differences between the genotypes in shock sensitivity, as assessed by their flinch responses to increasing levels of shock. However, alpha2 KO mice showed a greater suppression of lever pressing than WT littermates in the presence of a compound cue signalling footshock. Diazepam (0, 0.5, 1 and 2 mg/kg) induced a dose-dependent anxiolytic-like effect in WT mice but no such effect was seen in KO mice. Similarly, although pentobarbital (20 mg/kg) reduced the ability of the CS+ to reduce lever pressing rates in WT mice, this effect was not seen in the KO. These findings suggest that alpha2-containing GABA(A) receptors mediate the anxiolytic effects of barbiturates, as well as benzodiazepines, and that they may be involved in neuronal circuits underlying conditioned anxiety.

Sex and estrous cycle-dependent changes in neurosteroid and benzodiazepine effects on food consumption and plus-maze learning behaviors in rats.

PubMed

Reddy, D S; Kulkarni, S K

1999-01-01

Experiments were designed to investigate the influence of estrous cycle and gender of the rat on the effects of a gamma-aminobutyric acid type A (GABA(A)) receptor active neurosteroid, 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone), the benzodiazepine, triazolam, and a GABA(A) receptor antagonistic neurosteroid, delta5-androsten-3beta-ol-17-one sulfate (dehydroepiandrosterone sulfate), on food intake and elevated plus-maze learning behaviors. Allopregnanolone (0.25 mg/kg, s.c.) and triazolam (0.25 mg/kg, i.p.) produced a hyperphagic effect, while dehydroepiandrosterone sulfate (5 mg/kg, s.c.) elicited an anorectic effect. However, allopregnanolone was more potent in diestrous females, whereas triazolam exhibited significantly higher hyperphagic potency in estrus females. The extent of anorexia following dehydroepiandrosterone sulfate was alike in male and female rats. The triazolam- and allopregnanolone-induced hyperphagic effect was blocked by bicuculline (1 mg/kg, i.p.), a selective GABA(A) receptor antagonist. In contrast to triazolam, the hyperphagic effect of allopregnanolone was insensitive to flumazenil (5 mg/kg, i.p.), a benzodiazepine antagonist. Vehicle-treated diestrous rats displayed moderately higher latencies in the elevated plus-maze learning task than estrus or proestrus females. Although allopregnanolone and triazolam elicited equipotent learning deficits in plus-maze learning in male and female rats, the magnitude of impairment-induced by triazolam was significantly higher in diestrous females than proestrus females. Dehydroepiandrosterone sulfate enhanced memory performance only in male rats. Although the use of the elevated plus-maze as a learning paradigm with benzodiazepines and neurosteroids may be sensitive to changes in anxiety, the differential data suggest that neurosteroid-induced effects are at least partly specific to learning behavior. These results confirm the role of estrous cycle and sex of rats in modifying the potency of neurosteroids and benzodiazepines on food consumption and learning and memory processes.

Benzodiazepine temazepam suppresses the transient auditory 40-Hz response amplitude in humans.

PubMed

Jääskeläinen, I P; Hirvonen, J; Saher, M; Pekkonen, E; Sillanaukee, P; Näätänen, R; Tiitinen, H

1999-06-18

To discern the role of the GABA(A) receptors in the generation and attentive modulation of the transient auditory 40-Hz response, the effects of the benzodiazepine temazepam (10 mg) were studied in 10 healthy social drinkers, using a double-blind placebo-controlled design. Three hundred Hertz standard and 330 Hz rare deviant tones were presented to the left, and 1000 Hz standards and 1100 Hz deviants to the right ear of the subjects. Subjects attended to a designated ear and were to detect deviants therein while ignoring tones to the other. Temazepam significantly suppressed the amplitude of the 40-Hz response, the effect being equal for attended and non-attended tone responses. This suggests involvement of GABA(A) receptors in transient auditory 40-Hz response generation, however, not in the attentive modulation of the 40-Hz response.

Investigation of the Anxiolytic and Antidepressant Effects of Curcumin, a Compound From Turmeric (Curcuma longa), in the Adult Male Sprague-Dawley Rat.

PubMed

Ceremuga, Tomás Eduardo; Helmrick, Katie; Kufahl, Zachary; Kelley, Jesse; Keller, Brian; Philippe, Fabiola; Golder, James; Padrón, Gina

As the use of herbal medications continues to increase in America, the potential interaction between herbal and prescription medications necessitates the discovery of their mechanisms of action. The purpose of this study was to investigate the anxiolytic and antidepressant effects of curcumin, a compound from turmeric (Curcuma longa), and its effects on the benzodiazepine site of the γ-aminobutyric acid receptor A (GABAA) receptor. Utilizing a prospective, between-subjects group design, 55 male Sprague-Dawley rats were randomly assigned to 1 of the 5 intraperitoneally injected treatment groups: vehicle, curcumin, curcumin + flumazenil, midazolam, and midazolam + curcumin. Behavioral testing was performed using the elevated plus maze, open field test, and forced swim test. A 2-tailed multivariate analysis of variance and least significant difference post hoc tests were used for data analysis. In our models, curcumin did not demonstrate anxiolytic effects or changes in behavioral despair. An interaction of curcumin at the benzodiazepine site of the GABAA receptor was also not observed. Additional studies are recommended that examine the anxiolytic and antidepressant effects of curcumin through alternate dosing regimens, modulation of other subunits on the GABAA receptor, and interactions with other central nervous system neurotransmitter systems.

Zolpidem modulation of phasic and tonic GABA currents in the rat dorsal motor nucleus of the vagus

PubMed Central

Gao, Hong; Smith, Bret N.

2010-01-01

Zolpidem is a widely prescribed sleep aid with relative selectivity for GABAA receptors containing α1–3 subunits. We examined the effects of zolpidem on the inhibitory currents mediated by GABAA receptors using whole-cell patch-clamp recordings from DMV neurons in transverse brainstem slices from rat. Zolpidem prolonged the decay time of mIPSCs and of muscimol-evoked whole-cell GABAergic currents, and it occasionally enhanced the amplitude of mIPSCs. The effects were blocked by flumazenil, a benzodiazepine antagonist. Zolpidem also hyperpolarized the resting membrane potential, with a concomitant decrease in input resistance and action potential firing activity in a subset of cells. Zolpidem did not clearly alter the GABAA receptor-mediated tonic current (Itonic) under baseline conditions, but after elevating extracellular GABA concentration with nipecotic acid, a non-selective GABA transporter blocker, zolpidem consistently and significantly increased the tonic GABA current. This increase was suppressed by flumazenil and gabazine. These results suggest that α1–3 subunits are expressed in synaptic GABAA receptors on DMV neurons. The baseline tonic GABA current is likely not mediated by these same low affinity, zolpidem-sensitive GABAA receptors. However, when the extracellular GABA concentration is increased, zolpidem-sensitive extrasynaptic GABAA receptors containing α1–3 subunits contribute to the Itonic. PMID:20226798

A Review of the Updated Pharmacophore for the Alpha 5 GABA(A) Benzodiazepine Receptor Model

PubMed Central

Clayton, Terry; Poe, Michael M.; Rallapalli, Sundari; Biawat, Poonam; Savić, Miroslav M.; Rowlett, James K.; Gallos, George; Emala, Charles W.; Kaczorowski, Catherine C.; Stafford, Douglas C.; Arnold, Leggy A.; Cook, James M.

2015-01-01

An updated model of the GABA(A) benzodiazepine receptor pharmacophore of the α5-BzR/GABA(A) subtype has been constructed prompted by the synthesis of subtype selective ligands in light of the recent developments in both ligand synthesis, behavioral studies, and molecular modeling studies of the binding site itself. A number of BzR/GABA(A) α5 subtype selective compounds were synthesized, notably α5-subtype selective inverse agonist PWZ-029 (1) which is active in enhancing cognition in both rodents and primates. In addition, a chiral positive allosteric modulator (PAM), SH-053-2′F-R-CH3 (2), has been shown to reverse the deleterious effects in the MAM-model of schizophrenia as well as alleviate constriction in airway smooth muscle. Presented here is an updated model of the pharmacophore for α5β2γ2 Bz/GABA(A) receptors, including a rendering of PWZ-029 docked within the α5-binding pocket showing specific interactions of the molecule with the receptor. Differences in the included volume as compared to α1β2γ2, α2β2γ2, and α3β2γ2 will be illustrated for clarity. These new models enhance the ability to understand structural characteristics of ligands which act as agonists, antagonists, or inverse agonists at the Bz BS of GABA(A) receptors. PMID:26682068

GABAA receptor γ2 subunit knockdown mice have enhanced anxiety-like behavior but unaltered hypnotic response to benzodiazepines

PubMed Central

Chandra, Dev; Korpi, Esa R; Miralles, Celia P; De Blas, Angel L; Homanics, Gregg E

2005-01-01

Background Gamma-aminobutyric acid type A receptors (GABAA-Rs) are the major inhibitory receptors in the mammalian brain and are modulated by a number of sedative/hypnotic drugs including benzodiazepines and anesthetics. The significance of specific GABAA-Rs subunits with respect to behavior and in vivo drug responses is incompletely understood. The γ2 subunit is highly expressed throughout the brain. Global γ2 knockout mice are insensitive to the hypnotic effects of diazepam and die perinatally. Heterozygous γ2 global knockout mice are viable and have increased anxiety-like behaviors. To further investigate the role of the γ2 subunit in behavior and whole animal drug action, we used gene targeting to create a novel mouse line with attenuated γ2 expression, i.e., γ2 knockdown mice. Results Knockdown mice were created by inserting a neomycin resistance cassette into intron 8 of the γ2 gene. Knockdown mice, on average, showed a 65% reduction of γ2 subunit mRNA compared to controls; however γ2 gene expression was highly variable in these mice, ranging from 10–95% of normal. Immunohistochemical studies demonstrated that γ2 protein levels were also variably reduced. Pharmacological studies using autoradiography on frozen brain sections demonstrated that binding of the benzodiazepine site ligand Ro15-4513 was decreased in mutant mice compared to controls. Behaviorally, knockdown mice displayed enhanced anxiety-like behaviors on the elevated plus maze and forced novelty exploration tests. Surprisingly, mutant mice had an unaltered response to hypnotic doses of the benzodiazepine site ligands diazepam, midazolam and zolpidem as well as ethanol and pentobarbital. Lastly, we demonstrated that the γ2 knockdown mouse line can be used to create γ2 global knockout mice by crossing to a general deleter cre-expressing mouse line. Conclusion We conclude that: 1) insertion of a neomycin resistance gene into intron 8 of the γ2 gene variably reduced the amount of γ2, and that 2) attenuated expression of γ2 increased anxiety-like behaviors but did not lead to differences in the hypnotic response to benzodiazepine site ligands. This suggests that reduced synaptic inhibition can lead to a phenotype of increased anxiety-like behavior. In contrast, normal drug effects can be maintained despite a dramatic reduction in GABAA-R targets. PMID:15850489

α2-containing GABAA receptors expressed in hippocampal region CA3 control fast network oscillations

PubMed Central

Heistek, Tim S; Ruiperez-Alonso, Marta; Timmerman, A Jaap; Brussaard, Arjen B; Mansvelder, Huibert D

2013-01-01

GABAA receptors are critically involved in hippocampal oscillations. GABAA receptor α1 and α2 subunits are differentially expressed throughout the hippocampal circuitry and thereby may have distinct contributions to oscillations. It is unknown which GABAA receptor α subunit controls hippocampal oscillations and where these receptors are expressed. To address these questions we used transgenic mice expressing GABAA receptor α1 and/or α2 subunits with point mutations (H101R) that render these receptors insensitive to allosteric modulation at the benzodiazepine binding site, and tested how increased or decreased function of α subunits affects hippocampal oscillations. Positive allosteric modulation by zolpidem prolonged decay kinetics of hippocampal GABAergic synaptic transmission and reduced the frequency of cholinergically induced oscillations. Allosteric modulation of GABAergic receptors in CA3 altered oscillation frequency in CA1, while modulation of GABA receptors in CA1 did not affect oscillations. In mice having a point mutation (H101R) at the GABAA receptor α2 subunit, zolpidem effects on cholinergically induced oscillations were strongly reduced compared to wild-type animals, while zolpidem modulation was still present in mice with the H101R mutation at the α1 subunit. Furthermore, genetic knockout of α2 subunits strongly reduced oscillations, whereas knockout of α1 subunits had no effect. Allosteric modulation of GABAergic receptors was strongly reduced in unitary connections between fast spiking interneurons and pyramidal neurons in CA3 of α2H101R mice, but not of α1H101R mice, suggesting that fast spiking interneuron to pyramidal neuron synapses in CA3 contain α2 subunits. These findings suggest that α2-containing GABAA receptors expressed in the CA3 region provide the inhibition that controls hippocampal rhythm during cholinergically induced oscillations. PMID:23109109

Magnolol, a major bioactive constituent of the bark of Magnolia officinalis, induces sleep via the benzodiazepine site of GABA(A) receptor in mice.

PubMed

Chen, Chang-Rui; Zhou, Xu-Zhao; Luo, Yan-Jia; Huang, Zhi-Li; Urade, Yoshihiro; Qu, Wei-Min

2012-11-01

Magnolol (6,6',7,12-tetramethoxy-2,2'-dimethyl-1-beta-berbaman, C(18)H(18)O(2)), an active ingredient of the bark of Magnolia officinalis, has been reported to exert potent anti-epileptic effects via the GABA(A) receptor. The receptor also mediates sleep in humans and animals. The aim of this study was to determine whether magnolol could modulate sleep behaviors by recording EEG and electromyogram in mice. The results showed that magnolol administered i.p. at a dose of 5 or 25 mg/kg could significantly shorten the sleep latency, increase the amount of non-rapid eye movement (non-REM, NREM) and rapid eye movement (REM) sleep for 3 h after administration with an increase in the number of NREM and REM sleep episodes. Magnolol at doses of 5 and 25 mg/kg increased the number of bouts of wakefulness but decreased their duration. On the other hand, magnolol increased the number of state transitions from wakefulness to NREM sleep and subsequently from NREM sleep to wakefulness. Immunohistochemical study showed that magnolol increased c-Fos expression in the neurons of ventrolateral preoptic area, a sleep center in the anterior hypothalamus, and decreased c-Fos expression in the arousal tuberomammillary nucleus, which was located in the caudolateral hypothalamus. The sleep-promoting effects and changes in c-Fos induced by magnolol were reversed by flumazenil, an antagonist at the benzodiazepine site of the GABA(A) receptor. These results indicate that magnolol increased NREM and REM sleep via the GABA(A) receptor. Copyright © 2012 Elsevier Ltd. All rights reserved.

Effects of chronic ethanol consumption on rat GABA(A) and strychnine-sensitive glycine receptors expressed by lateral/basolateral amygdala neurons.

PubMed

McCool, Brian A; Frye, Gerald D; Pulido, Marisa D; Botting, Shaleen K

2003-02-14

It is well known that the anxiolytic potential of ethanol is maintained during chronic exposure. We have confirmed this using a light-dark box paradigm following chronic ethanol ingestion via a liquid diet. However, cessation from chronic ethanol exposure is known to cause severe withdrawal anxiety. These opposing effects on anxiety likely result from neuro-adaptations of neurotransmitter systems within the brain regions regulating anxiety. Recent work highlights the importance of amygdala ligand-gated chloride channels in the expression of anxiety. We have therefore examined the effects of chronic ethanol exposure on GABA(A) and strychnine-sensitive glycine receptors expressed by acutely isolated adult rat lateral/basolateral amygdala neurons. Chronic ethanol exposure increased the functional expression of GABA(A) receptors in acutely isolated basolateral amygdala neurons without altering strychnine-sensitive glycine receptors. Neither the acute ethanol nor benzodiazepine sensitivity of either receptor system was affected. We explored the likelihood that subunit composition might influence each receptor's response to chronic ethanol. Importantly, when expressed in a mammalian heterologous system, GABA(A) receptors composed of unique alpha subunits were differentially sensitive to acute ethanol. Likewise, the presence of the beta subunit appeared to influence the acute ethanol sensitivity of glycine receptors containing the alpha(2) subunit. Our results suggest that the facilitation of GABA(A) receptors during chronic ethanol exposure may help explain the maintenance of ethanol's anti-anxiety effects during chronic ethanol exposure. Furthermore, the subunit composition of GABA(A) and strychnine-sensitive glycine receptors may ultimately influence the response of each system to chronic ethanol exposure.

Molecular size of the gamma-aminobutyric acidA receptor purified from mammalian cerebral cortex.

PubMed

Mamalaki, C; Barnard, E A; Stephenson, F A

1989-01-01

The hydrodynamic behaviour of both the soluble and purified gamma-aminobutyric acidA (GABAA) receptor of bovine or rat cerebral cortex has been investigated in solution in Triton X-100 or in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS). In all the hydrodynamic separations made, it was found that the binding activities for GABA, benzodiazepine, and (where detectable) t-butylbicyclophosphorothionate comigrated. Conditions were established for gel exclusion chromatography and for sucrose density gradient velocity sedimentation that maintain the GABAA receptor in a nonaggregated form. Using these conditions, the molecular weight of the bovine GABAA receptor in the above-mentioned detergents was calculated using the H2O/2H2O method. A value of Mr 230,000-240,000 was calculated for the bovine pure GABAA receptor purified in sodium deoxycholate/Triton X-100 media. A value of Mr 284,000-290,000 was calculated for the nonaggregated bovine or rat cortex receptor in CHAPS, but the Stokes radius is smaller in the latter than in the former medium and the detergent binding in CHAPS is underestimated. Thus the deduced Mr, 240,000, is the best estimate by this method.

Allosteric modulation by benzodiazepines of GABA-gated chloride channels of an identified insect motor neurone.

PubMed

Buckingham, Steven D; Higashino, Yoshiaki; Sattelle, David B

2009-11-01

The actions of benzodiazepines were studied on the responses to GABA of the fast coxal depressor (D(f)) motor neurone of the cockroach, Periplaneta americana. Ro5-4864, diazepam and clonazepam were investigated. Responses to GABA receptors were enhanced by both Ro5-4864 and diazepam, whereas clonazepam, a potent-positive allosteric modulator of human GABA(A) receptors, was ineffective on the native insect GABA receptors of the D(f) motor neurone. Thus, clear pharmacological differences exist between insect and mammalian native GABA-gated chloride channels with respect to the actions of benzodiazepines. The results enhance our understanding of invertebrate GABA-gated chloride channels which have recently proved important in (a) comparative studies aimed at identifying human allosteric drug-binding sites and (b) understanding the actions of compounds used to control ectoparasites and insect crop pests.

Effects of Chronic Ethanol Consumption on Rat GABAA and Strychnine-sensitive Glycine Receptors Expressed by Lateral/Basolateral Amygdala Neurons

PubMed Central

McCool, Brian A.; Frye, Gerald D.; Pulido, Marisa D.; Botting, Shaleen K.

2010-01-01

It is well known that the anxiolytic potential of ethanol is maintained during chronic exposure. We have confirmed this using a light-dark box paradigm following chronic ethanol ingestion via a liquid diet. However, cessation from chronic ethanol exposure is known to cause severe withdrawal anxiety. These opposing effects on anxiety likely result from neuro-adaptations of neurotransmitter systems within the brain regions regulating anxiety. Recent work highlights the importance of amygdala ligand-gated chloride channels in the expression of anxiety. We have therefore examined the effects of chronic ethanol exposure on GABAA and strychnine-sensitive glycine receptors expressed by acutely isolated adult rat lateral/basolateral amygdala neurons. Chronic ethanol exposure increased the functional expression of GABAA receptors in acutely isolated basolateral amygdala neurons without altering strychnine-sensitive glycine receptors. Neither the acute ethanol nor benzodiazepine sensitivity of either receptor system was affected. We explored the likelihood that subunit composition might influence each receptor’s response to chronic ethanol. Importantly, when expressed in a mammalian heterologous system, GABAA receptors composed of unique α subunits were differentially sensitive to acute ethanol. Likewise, the presence of the β subunit appeared to influence the acute ethanol sensitivity of glycine receptors containing the α2 subunit. Our results suggest that the facilitation of GABAA receptors during chronic ethanol exposure may help explain the maintenance of ethanol’s anti-anxiety effects during chronic ethanol exposure. Furthermore, the subunit composition of GABAA and strychnine-sensitive glycine receptors may ultimately influence the response of each system to chronic ethanol exposure. PMID:12560122

Effect of GABA agonists and GABA-A receptor modulators on cocaine- and food-maintained responding and cocaine discrimination in rats.

PubMed

Barrett, Andrew C; Negus, S Stevens; Mello, Nancy K; Caine, S Barak

2005-11-01

Recent studies indicate that GABAergic ligands modulate abuse-related effects of cocaine. The goal of this study was to evaluate the effects of a mechanistically diverse group of GABAergic ligands on the discriminative stimulus and reinforcing effects of cocaine in rats. One group of rats was trained to discriminate 5.6 mg/kg cocaine from saline in a two-lever, food-reinforced, drug discrimination procedure. In two other groups, responding was maintained by cocaine (0-3.2 mg/kg/injection) or liquid food (0-100%) under a fixed ratio 5 schedule. Six GABA agonists were tested: the GABA-A receptor agonist muscimol, the GABA-B receptor agonist baclofen, the GABA transaminase inhibitor gamma-vinyl-GABA (GVG), and three GABA-A receptor modulators (the barbiturate pentobarbital, the high-efficacy benzodiazepine midazolam, and the low-efficacy benzodiazepine enazenil). When tested alone, none of the compounds substituted fully for the discriminative stimulus effects of cocaine. As acute pretreatments, select doses of midazolam and pentobarbital produced 2.2- to 3.6-fold rightward shifts in the cocaine dose-effect function. In contrast, muscimol, baclofen, GVG, and enazenil failed to alter the discriminative stimulus effects of cocaine. In assays of cocaine- and food-maintained responding, midazolam and pentobarbital decreased cocaine self-administration at doses 9.6- and 3.3-fold lower, respectively, than those that decreased food-maintained responding. In contrast, muscimol, baclofen, and GVG decreased cocaine self-administration at doses that also decreased food-maintained responding. Enazenil failed to alter cocaine self-administration. Together with previous studies, these data suggest that among mechanistically diverse GABA agonists, high-efficacy GABA-A modulators may be the most effective for modifying the abuse-related effects of cocaine.

Honokiol promotes non-rapid eye movement sleep via the benzodiazepine site of the GABA(A) receptor in mice.

PubMed

Qu, Wei-Min; Yue, Xiao-Fang; Sun, Yu; Fan, Kun; Chen, Chang-Rui; Hou, Yi-Ping; Urade, Yoshihiro; Huang, Zhi-Li

2012-10-01

Decoctions of the Chinese herb houpu contain honokiol and are used to treat a variety of mental disorders, including depression. Depression commonly presents alongside sleep disorders and sleep disturbances, which appear to be a major risk factor for depression. Here, we have evaluated the somnogenic effect of honokiol and the mechanisms involved. Honokiol was administered i.p. at 20:00 h in mice. Flumazenil, an antagonist at the benzodiazepine site of the GABA(A) receptor, was administered i.p. 15 min before honokiol. The effects of honokiol were measured by EEG and electromyogram (EMG), c-Fos expression and in vitro electrophysiology. Honokiol (10 and 20 mg·kg⁻¹) significantly shortened the sleep latency to non-rapid eye movement (non-REM, NREM) sleep and increased the amount of NREM sleep. Honokiol increased the number of state transitions from wakefulness to NREM sleep and, subsequently, from NREM sleep to wakefulness. However, honokiol had no effect on either the amount of REM sleep or EEG power density of both NREM and REM sleep. Honokiol increased c-Fos expression in ventrolateral preoptic area (VLPO) neurons, as examined by immunostaining, and excited sleep-promoting neurons in the VLPO by whole-cell patch clamping in the brain slice. Pretreatment with flumazenil abolished the somnogenic effects and activation of the VLPO neurons by honokiol. Honokiol promoted NREM sleep by modulating the benzodiazepine site of the GABA(A) receptor, suggesting potential applications in the treatment of insomnia, especially for patients who experience difficulty in falling and staying asleep. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

[Analysis of the binding capacity of the benzodiazepine site of gabaa receptor in mice C57BL/6 and BALB/C pretreated with anxiolytics].

PubMed

Iarkova, M A

2011-01-01

The level of specific 3H-flunitrazepam binding in synaptosomal membranes of C57BL/6 and BALB/c mice brain underwent to the stress of different types has been studied. Mild stress (Elevated Plus Maze) was shown to induce the decrease of benzodiazepine binding in BALB/c mice only, while the strong one (Exposure to a predator) was revealed to cause this decrease in both strains. Behavioral effects of different non-benzodiazepine drugs possessing anxiolytic properties (Afobazol, Ladasten and Noopept) was accompanied with the normalization of the level of benzodiazepine reception, reduced by the stress of both modalities.

Monoterpenoids (thymol, carvacrol and S-(+)-linalool) with anesthetic activity in silver catfish (Rhamdia quelen): evaluation of acetylcholinesterase and GABAergic activity

PubMed Central

Bianchini, A.E.; Garlet, Q.I.; da Cunha, J.A.; Bandeira, G.; Brusque, I.C.M.; Salbego, J.; Heinzmann, B.M.; Baldisserotto, B.

2017-01-01

This study evaluated the anesthetic potential of thymol and carvacrol, and their influence on acetylcholinesterase (AChE) activity in the muscle and brain of silver catfish (Rhamdia quelen). The AChE activity of S-(+)-linalool was also evaluated. We subsequently assessed the effects of thymol and S-(+)-linalool on the GABAergic system. Fish were exposed to thymol and carvacrol (25, 50, 75, and 100 mg/L) to evaluate time for anesthesia and recovery. Both compounds induced sedation at 25 mg/L and anesthesia with 50–100 mg/L. However, fish exposed to carvacrol presented strong muscle contractions and mortality. AChE activity was increased in the brain of fish at 50 mg/L carvacrol and 100 mg/L thymol, and decreased in the muscle at 100 mg/L carvacrol. S-(+)-linalool did not alter AChE activity. Anesthesia with thymol was reversed by exposure to picrotoxin (GABAA antagonist), similar to the positive control propofol, but was not reversed by flumazenil (antagonist of benzodiazepine binding site), as observed for the positive control diazepam. Picrotoxin did not reverse the effect of S-(+)-linalool. Thymol exposure at 50 mg/L is more suitable than carvacrol for anesthesia in silver catfish, because this concentration did not cause any mortality or interference with AChE activity. Thymol interacted with GABAA receptors, but not with the GABAA/benzodiazepine site. In contrast, S-(+)-linalool did not act in GABAA receptors in silver catfish. PMID:29069225

Monoterpenoids (thymol, carvacrol and S-(+)-linalool) with anesthetic activity in silver catfish (Rhamdia quelen): evaluation of acetylcholinesterase and GABAergic activity.

PubMed

Bianchini, A E; Garlet, Q I; da Cunha, J A; Bandeira, G; Brusque, I C M; Salbego, J; Heinzmann, B M; Baldisserotto, B

2017-10-19

This study evaluated the anesthetic potential of thymol and carvacrol, and their influence on acetylcholinesterase (AChE) activity in the muscle and brain of silver catfish (Rhamdia quelen). The AChE activity of S-(+)-linalool was also evaluated. We subsequently assessed the effects of thymol and S-(+)-linalool on the GABAergic system. Fish were exposed to thymol and carvacrol (25, 50, 75, and 100 mg/L) to evaluate time for anesthesia and recovery. Both compounds induced sedation at 25 mg/L and anesthesia with 50-100 mg/L. However, fish exposed to carvacrol presented strong muscle contractions and mortality. AChE activity was increased in the brain of fish at 50 mg/L carvacrol and 100 mg/L thymol, and decreased in the muscle at 100 mg/L carvacrol. S-(+)-linalool did not alter AChE activity. Anesthesia with thymol was reversed by exposure to picrotoxin (GABAA antagonist), similar to the positive control propofol, but was not reversed by flumazenil (antagonist of benzodiazepine binding site), as observed for the positive control diazepam. Picrotoxin did not reverse the effect of S-(+)-linalool. Thymol exposure at 50 mg/L is more suitable than carvacrol for anesthesia in silver catfish, because this concentration did not cause any mortality or interference with AChE activity. Thymol interacted with GABAA receptors, but not with the GABAA/benzodiazepine site. In contrast, S-(+)-linalool did not act in GABAA receptors in silver catfish.

NOVEL POSITIVE ALLOSTERIC MODULATORS OF GABAA RECEPTORS: DO SUBTLE DIFFERENCES IN ACTIVITY AT α1 PLUS α5 VERSUS α2 PLUS α3 SUBUNITS ACCOUNT FOR DISSIMILARITIES IN BEHAVIORAL EFFECTS IN RATS?

PubMed Central

Savić, Miroslav M.; Majumder, Samarpan; Huang, Shengming; Edwankar, Rahul V.; Furtmüller, Roman; Joksimović, Srđan; Clayton, Terry; Ramerstorfer, Joachim; Milinković, Marija M.; Roth, Bryan L.; Sieghart, Werner; Cook, James M.

2010-01-01

Over the last years, genetic studies have greatly improved our knowledge on the receptor subtypes mediating various pharmacological effects of positive allosteric modulators at GABAA receptors. This stimulated the development of new benzodiazepine (BZ)-like ligands, especially those inactive/low-active at GABAA receptors containing the α1 subunit, with the aim of generating more selective drugs. Hereby, the affinity and efficacy of four recently-synthesized BZ site ligands: SH-053-2’N, SH-053-S-CH3-2’F, SH-053-R-CH3-2’F and JY-XHe-053 were assessed. They were also studied in behavioral tests of spontaneous locomotor activity, elevated plus maze, and water maze in rats, which are considered predictive of, respectively, the sedative, anxiolytic, and amnesic influence of BZs. The novel ligands had moderately low to low affinity and mild to partial agonistic efficacy at GABAA receptors containing the α1 subunit, with variable, but more pronounced efficacy at other BZ-sensitive binding sites. While presumably α1 receptor-mediated sedative effects of GABAA modulation were not fully eliminated with any of the ligands tested, only SH-053-2’N and SH-053-S-CH3-2’F, both dosed at 30 mg/kg, exerted anxiolytic effects. The lack of clear anxiolytic-like activity of JY-XHe-053, despite its efficacy at α2- and α3-GABAA receptors, may have been partly connected with its preferential affinity at α5-GABAA receptors coupled with weak agonist activity at α1-containing subtypes. The memory impairment in water-maze experiments, generally reported with BZ site agonists, was completely circumvented with all four ligands. The results suggest that a substantial amount of activity at α1 GABAA receptors is needed for effecting spatial learning and memory impairments, while much weaker activity at α1- and α5-GABAA receptors is sufficient for eliciting sedation. PMID:20074611

Role of neurosteroids in the anticonvulsant activity of midazolam.

PubMed

Dhir, Ashish; Rogawski, Michael A

2012-04-01

Midazolam is a short-acting benzodiazepine that is widely used as an i.v. sedative and anticonvulsant. Besides interacting with the benzodiazepine site associated with GABA(A) receptors, some benzodiazepines act as agonists of translocator protein (18 kDa) (TSPO) to enhance the synthesis of steroids, including neurosteroids with positive modulatory actions on GABA(A) receptors. We sought to determine if neurosteroidogenesis induced by midazolam contributes to its anticonvulsant action. Mice were pretreated with neurosteroid synthesis inhibitors and potentiators followed by midazolam or clonazepam, a weak TSPO ligand. Anticonvulsant activity was assessed with the i.v. pentylenetetrazol (PTZ) threshold test. Midazolam (500-5000 µg·kg(-1) , i.p.) caused a dose-dependent increase in seizure threshold. Pretreatment with the neurosteroid synthesis inhibitors finasteride, a 5α-reductase inhibitor, and a functional TSPO antagonist PK 11195, reduced the anticonvulsant action of midazolam. The anticonvulsant action of midazolam was enhanced by the neurosteroidogenic drug metyrapone, an 11β-hydroxylase inhibitor. In contrast, the anticonvulsant action of clonazepam (100 µg·kg(-1) ) was reduced by finasteride but not by PK 11195, indicating a possible contribution of neurosteroids unrelated to TSPO. Enhanced endogenous neurosteroid synthesis, possibly mediated by an interaction with TSPO, contributed to the anticonvulsant action of midazolam. Enhanced neurosteroidogenesis may also be a factor in the actions of other benzodiazepines, even those that only weakly interact with TSPO. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Role of neurosteroids in the anticonvulsant activity of midazolam

PubMed Central

Dhir, Ashish; Rogawski, Michael A

2012-01-01

BACKGROUND AND PURPOSE Midazolam is a short-acting benzodiazepine that is widely used as an i.v. sedative and anticonvulsant. Besides interacting with the benzodiazepine site associated with GABAA receptors, some benzodiazepines act as agonists of translocator protein (18 kDa) (TSPO) to enhance the synthesis of steroids, including neurosteroids with positive modulatory actions on GABAA receptors. We sought to determine if neurosteroidogenesis induced by midazolam contributes to its anticonvulsant action. EXPERIMENTAL APPROACH Mice were pretreated with neurosteroid synthesis inhibitors and potentiators followed by midazolam or clonazepam, a weak TSPO ligand. Anticonvulsant activity was assessed with the i.v. pentylenetetrazol (PTZ) threshold test. KEY RESULTS Midazolam (500–5000 µg·kg−1, i.p.) caused a dose-dependent increase in seizure threshold. Pretreatment with the neurosteroid synthesis inhibitors finasteride, a 5α-reductase inhibitor, and a functional TSPO antagonist PK 11195, reduced the anticonvulsant action of midazolam. The anticonvulsant action of midazolam was enhanced by the neurosteroidogenic drug metyrapone, an 11β-hydroxylase inhibitor. In contrast, the anticonvulsant action of clonazepam (100 µg·kg−1) was reduced by finasteride but not by PK 11195, indicating a possible contribution of neurosteroids unrelated to TSPO. CONCLUSION AND IMPLICATIONS Enhanced endogenous neurosteroid synthesis, possibly mediated by an interaction with TSPO, contributed to the anticonvulsant action of midazolam. Enhanced neurosteroidogenesis may also be a factor in the actions of other benzodiazepines, even those that only weakly interact with TSPO. PMID:22014182

Antagonism of the Ethanol-Like Discriminative Stimulus Effects of Ethanol, Pentobarbital, and Midazolam in Cynomolgus Monkeys Reveals Involvement of Specific GABAA Receptor SubtypesS⃞

PubMed Central

Rogers, Laura S. M.; Grant, Kathleen A.

2009-01-01

The γ-aminobutyric acid (GABA)A receptors mediating the discriminative stimulus effects of ethanol were studied by comparing the potency of ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazol(1,5-a)benzodiazepine-3-carboxylate (Ro15-4513) and ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol(1,5-a)-benzodiazepine-3-carboxylate (flumazenil, Ro15-1788) to antagonize ethanol, pentobarbital (PB), and midazolam substitution for ethanol. Ro15-4513 has high affinity for receptors containing α4/6 and α5 subunits and lower affinity for α1, α2, and α3 subunits. Flumazenil is nonselective for GABAA receptors containing α1, α2, α3, and α5 subunits and has low affinity for α4/6-containing receptors. Male (n = 9) and female (n = 8) cynomolgus monkeys (Macaca fascicularis) were trained to discriminate ethanol (1.0 or 2.0 g/kg i.g., 30-min pretreatment) from water. Ethanol, PB, and midazolam dose-dependently substituted for ethanol (80% ethanol-appropriate responding). Ro15-4513 (0.003–0.56 mg/kg i.m., 5-min pretreatment) shifted the ethanol, PB, and midazolam dose-response functions rightward in a vast majority of monkeys tested (15/15, 16/17, and 11/12, respectively). In contrast, flumazenil (0.30–10.0 mg/kg i.m., 5-min pretreatment) shifted the ethanol, PB, and midazolam dose-response functions rightward in 9 of 16, 12 of 16, and 7 of 9 monkeys tested, respectively. In the monkeys showing antagonism with both Ro15-4513 and flumazenil, ethanol and PB substitution were antagonized more potently by Ro15-4513 than by flumazenil, whereas midazolam substitution was antagonized with similar potency. There were no sex or training dose differences, with the exception that flumazenil failed to antagonize ethanol substitution in males trained to discriminate 2.0 g/kg ethanol. GABAA receptors with high affinity for Ro15-4513 (i.e., containing α4/6 and α5 subunits) may be particularly important mediators of the multiple discriminative stimulus effects of ethanol through GABAA receptor systems. PMID:19641166

GABAA-benzodiazepine-chloride receptor-targeted therapy for tinnitus control: preliminary report.

PubMed

Shulman, Abraham; Strashun, Arnold M; Goldstein, Barbara A

2002-01-01

Our goal was to attempt to establish neuropharmacological tinnitus control (i.e., relief) with medication directed to restoration of a deficiency in the gamma-aminobutyric acid-benzodiazepine-chloride receptor in tinnitus patients with a diagnosis of a predominantly central type tinnitus. Thirty tinnitus patients completed a medical audiological tinnitus patient protocol and brain magnetic resonance imaging and single-photon emission computed tomography of brain. Treatment with GABAergic and benzodiazepine medication continued for 4-6 weeks. A maintenance dose was continued when tinnitus control was positive. Intake and outcome questionnaires were completed. Of 30 patients, 21 completed the trial (70%). Tinnitus control lasting from 4-6 weeks to 3 years was reported by 19 of the 21 (90%). The trial was not completed by 9 of the 30 (30%). No patient experienced an increase in tinnitus intensity or annoyance. Sequential brain single-photon emission computed tomography in 10 patients revealed objective evidence of increased brain perfusion. Patients with a predominantly central type tinnitus experience significant tinnitus control with medication directed to the gamma-aminobutyric acid-benzodiazepine-chloride receptor.

Flavonoid nutraceuticals and ionotropic receptors for the inhibitory neurotransmitter GABA.

PubMed

Johnston, Graham A R

2015-10-01

Flavonoids that are found in nutraceuticals have many and varied effects on the activation of ionotropic receptors for GABA, the major inhibitory neurotransmitter in our brains. They can act as positive or negative modulators enhancing or reducing the effect of GABA. They can act as allosteric agonists. They can act to modulate the action of other modulators. There is considerable evidence that these flavonoids are able to enter the brain to influence brain function. They may have a range of effects including relief of anxiety, improvement in cognition, acting as neuroprotectants and as sedatives. All of these effects are sought after in nutraceuticals. A number of studies have likened flavonoids to the widely prescribed benzodiazepines as 'a new family of benzodiazepine receptor ligands'. They are much more than that with many flavonoid actions on ionotropic GABA receptors being insensitive to the classic benzodiazepine antagonist flumazenil and thus independent of the classic benzodiazepine actions. It is time to consider flavonoids in their own right as important modulators of these vital receptors in brain function. Flavonoids are rarely consumed as a single flavonoid except as dietary supplements. The effects of mixtures of flavonoids and other modulators on GABAA receptors need to be more thoroughly investigated. Copyright © 2015 Elsevier Ltd. All rights reserved.

A microPET comparison of the effects of etifoxine and diazepam on [(11)C]flumazenil uptake in rat brains.

PubMed

Bouillot, Caroline; Bonnefoi, Frédéric; Liger, François; Zimmer, Luc

2016-01-26

Using positron emission tomography (PET), the present study assessed the binding of [(11)C]flumazenil to GABA-A receptors in anesthetized rats following a single intravenous injection of an active dose of either etifoxine (25mg/kg) or diazepam (1mg/kg), which are both anxiolytic drugs. [(11)C]flumazenil binding was measured in five discrete brain structures, namely the caudate putamen, hippocampus, cerebellum, occipital cortex and parietal cortex. As expected, diazepam injection produced a significant decrease in [(11)C]flumazenil binding, which was interpreted as benzodiazepine GABA-A receptor occupancy, whereas etifoxine increased the binding of [(11)C]flumazenil. This first use of in vivo imaging after etifoxine administration revealed the activated binding pattern of [(11)C]flumazenil and highlighted the pharmacological differences between etifoxine and benzodiazepines. Using the same [(11)C]flumazenil radiotracer, PET neuroimaging could be applied to larger animals and, ultimately, to human subjects, thus providing new perspectives for better defining the molecular pharmacology of etifoxine. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Functional expression of the GABAA receptor α2 and α3 subunits at synapses between intercalated medial paracapsular neurons of mouse amygdala

PubMed Central

Geracitano, Raffaella; Fischer, David; Kasugai, Yu; Ferraguti, Francesco; Capogna, Marco

2012-01-01

In the amygdala, GABAergic neurons in the intercalated medial paracapsular cluster (Imp) have been suggested to play a key role in fear learning and extinction. These neurons project to the central (CE) amygdaloid nucleus and to other areas within and outside the amygdala. In addition, they give rise to local collaterals that innervate other neurons in the Imp. Several drugs, including benzodiazepines (BZ), are allosteric modulators of GABAA receptors. BZ has both anxiolytic and sedative actions, which are mediated through GABAA receptors containing α2/α3 and α1 subunits, respectively. To establish whether α1 or α2/α3 subunits are expressed at Imp cell synapses, we used paired recordings of anatomically identified Imp neurons and high resolution immunocytochemistry in the mouse. We observed that a selective α3 subunit agonist, TP003 (100 nM), significantly increased the decay time constant of the unitary IPSCs. A similar effect was also induced by zolpidem (10 μM) or by diazepam (1 μM). In contrast, lower doses of zolpidem (0.1–1 μM) did not significantly alter the kinetics of the unitary IPSCs. Accordingly, immunocytochemical experiments established that the α2 and α3, but not the α1 subunits of the GABAA receptors, were present at Imp cell synapses of the mouse amygdala. These results define, for the first time, some of the functional GABAA receptor subunits expressed at synapses of Imp cells. The data also provide an additional rationale to prompt the search of GABAA receptor α3 selective ligands as improved anxiolytic drugs. PMID:22666188

GABA(A) receptors in the pontine reticular formation of C57BL/6J mouse modulate neurochemical, electrographic, and behavioral phenotypes of wakefulness.

PubMed

Flint, RaShonda R; Chang, Theresa; Lydic, Ralph; Baghdoyan, Helen A

2010-09-15

Drugs that potentiate transmission at GABA(A) receptors are widely used to enhance sleep and to cause general anesthesia. The mechanisms underlying these effects are unknown. This study tested the hypothesis that GABA(A) receptors in the pontine reticular nucleus, oral part (PnO) of mouse modulate five phenotypes of arousal: sleep and wakefulness, cortical electroencephalogram (EEG) activity, acetylcholine (ACh) release in the PnO, breathing, and recovery time from general anesthesia. Microinjections into the PnO of saline (vehicle control), the GABA(A) receptor agonist muscimol, muscimol with the GABA(A) receptor antagonist bicuculline, and bicuculline alone were performed in male C57BL/6J mice (n = 33) implanted with EEG recording electrodes. Muscimol caused a significant increase in wakefulness and decrease in rapid eye movement (REM) and non-REM (NREM) sleep. These effects were reversed by coadministration of bicuculline. Bicuculline administered alone caused a significant decrease in wakefulness and increase in NREM sleep and REM sleep. Muscimol significantly increased EEG power in the delta range (0.5-4 Hz) during wakefulness and in the theta range (4-9 Hz) during REM sleep. Dialysis delivery of bicuculline to the PnO of male mice (n = 18) anesthetized with isoflurane significantly increased ACh release in the PnO, decreased breathing rate, and increased anesthesia recovery time. All drug effects were concentration dependent. The effects on phenotypes of arousal support the conclusion that GABA(A) receptors in the PnO promote wakefulness and suggest that increasing GABAergic transmission in the PnO may be one mechanism underlying the phenomenon of paradoxical behavioral activation by some benzodiazepines.

Temporal characteristics of stress-induced decrease in benzodiazepine reception in C57BL/6 and BALB/c mice.

PubMed

Yarkova, M A; Seredenin, S B

2014-10-01

We studied the duration of the drop of specific (3)H-flunitrazepam binding by synaptosomal membranes from the brain of C57Bl/6 and BALB/c mice after open-field and "contact with predator" tests. It was found that reduced benzodiazepine reception in BALB/c mice after open-field test persisted for 1.5 h, but no changes of this parameter were found in C57Bl/6 mice. After contact with predator, the binding capacity of the benzodiazepine site of GABAA receptor was reduced for 8 h in BALB/c mice and for 24 h in C57Bl/6 mice.

Behavioral Effects of the Benzodiazepine-Positive Allosteric Modulator SH-053-2’F-S-CH3 in an Immune-Mediated Neurodevelopmental Disruption Model

PubMed Central

Richetto, Juliet; Labouesse, Marie A.; Poe, Michael M.; Cook, James M.; Grace, Anthony A.; Riva, Marco A.

2015-01-01

Background: Impaired γ-aminobutyric acid (GABA) signaling may contribute to the emergence of cognitive deficits and subcortical dopaminergic hyperactivity in patients with schizophrenia and related psychotic disorders. Against this background, it has been proposed that pharmacological interventions targeting GABAergic dysfunctions may prove useful in correcting such cognitive impairments and dopaminergic imbalances. Methods: Here, we explored possible beneficial effects of the benzodiazepine-positive allosteric modulator SH-053-2’F-S-CH3, with partial selectivity at the α2, α3, and α5 subunits of the GABAA receptor in an immune-mediated neurodevelopmental disruption model. The model is based on prenatal administration of the viral mimetic polyriboinosinic-polyribocytidilic acid [poly(I:C)] in mice, which is known to capture various GABAergic, dopamine-related, and cognitive abnormalities implicated in schizophrenia and related disorders. Results: Real-time polymerase chain reaction analyses confirmed the expected alterations in GABAA receptor α subunit gene expression in the medial prefrontal cortices and ventral hippocampi of adult poly(I:C) offspring relative to control offspring. Systemic administration of SH-053-2’F-S-CH3 failed to normalize the poly(I:C)-induced deficits in working memory and social interaction, but instead impaired performance in these cognitive and behavioral domains both in control and poly(I:C) offspring. In contrast, SH-053-2’F-S-CH3 was highly effective in mitigating the poly(I:C)-induced amphetamine hypersensitivity phenotype without causing side effects in control offspring. Conclusions: Our preclinical data suggest that benzodiazepine-like positive allosteric modulators with activity at the α2, α3, and α5 subunits of the GABAA receptor may be particularly useful in correcting pathological overactivity of the dopaminergic system, but they may be ineffective in targeting multiple pathological domains that involve the co-existence of psychotic, social, and cognitive dysfunctions. PMID:25636893

GABA-A Receptor Modulation and Anticonvulsant, Anxiolytic, and Antidepressant Activities of Constituents from Artemisia indica Linn

PubMed Central

Khan, Imran; Karim, Nasiara; Ahmad, Waqar; Abdelhalim, Abeer; Chebib, Mary

2016-01-01

Artemisia indica, also known as “Mugwort,” has been widely used in traditional medicines. However, few studies have investigated the effects of nonvolatile components of Artemisia indica on central nervous system's function. Fractionation of Artemisia indica led to the isolation of carnosol, ursolic acid, and oleanolic acid which were evaluated for their effects on GABA-A receptors in electrophysiological studies in Xenopus oocytes and were subsequently investigated in mouse models of acute toxicity, convulsions (pentylenetetrazole induced seizures), depression (tail suspension and forced swim tests), and anxiety (elevated plus maze and light/dark box paradigms). Carnosol, ursolic acid, and oleanolic acid were found to be positive modulators of α1β2γ2L GABA-A receptors and the modulation was antagonized by flumazenil. Carnosol, ursolic acid, and oleanolic acid were found to be devoid of any signs of acute toxicity (50–200 mg/kg) but elicited anticonvulsant, antidepressant, and anxiolytic activities. Thus carnosol, ursolic acid, and oleanolic acid demonstrated CNS activity in mouse models of anticonvulsant, antidepressant, and anxiolysis. The anxiolytic activity of all three compounds was ameliorated by flumazenil suggesting a mode of action via the benzodiazepine binding site of GABA-A receptors. PMID:27143980

Midazolam as an anticonvulsant antidote for organophosphate intoxication− A pharmacotherapeutic appraisal

PubMed Central

Reddy, Sandesh D.; Reddy, Doodipala Samba

2015-01-01

SUMMARY Objective This review summarizes the therapeutic potential of midazolam as an anticonvulsant antidote for organophosphate (OP) intoxication. Methods Benzodiazepines are widely used for acute seizures and status epilepticus (SE), a neurological emergency of persistent seizures that can lead to severe neuronal damage or death. Midazolam is a benzodiazepine hypnotic with a rapid onset and short duration of action. Results Midazolam is considered the new drug of choice for persistent acute seizures and SE, including those caused by neurotoxic OPs and nerve agents. Midazolam is a positive allosteric modulator of synaptic GABA-A receptors in the brain. It potentiates GABAergic inhibition and thereby controls hyperexcitability and seizures. Midazolam is administered intravenously or intramuscularly to control acute seizures and SE. Due to its favorable pharmacokinetic features, midazolam is being considered as a replacement anticonvulsant for diazepam in the antidote kit for nerve agents. Clinical studies such as the recent RAMPART trial have confirmed the anticonvulsant efficacy of midazolam in SE in prehospital settings. Significance In experimental models, midazolam is effective when given at the onset of seizures caused by nerve agents. However, benzodiazepines are less effective at terminating seizures when given 30 min or later after OP exposure or seizure onset likely because of internalization or down-regulation of synaptic, but not extrasynaptic, GABA-A receptors, which can lead to diminished potency and seizure recurrence. PMID:26032507

Treating acute seizures with benzodiazepines: does seizure duration matter?

PubMed

Naylor, David E

2014-10-01

Several clinical trials have shown improved seizure control and outcome by early initiation of treatment with benzodiazepines, before arrival in the emergency department and before intravenous access can be established. Here, evidence is provided and reviewed for rapid treatment of acute seizures in order to avoid the development of benzodiazepine pharmacoresistance and the emergence of self-sustaining status epilepticus. Alterations in the physiology, pharmacology, and postsynaptic level of GABA-A receptors can develop within minutes to an hour and hinder the ability of synaptic inhibition to stop seizures while also impairing the efficacy of GABAergic agents, such as benzodiazepines, to boost impaired inhibition. In addition, heightened excitatory transmission further exacerbates the inhibitory/excitatory balance and makes seizure control even more resistant to treatment. The acute increase in the surface expression of NMDA receptors during prolonged seizures also may cause excitotoxic injury, cell death, and other pathological expressions and re-arrangements of receptor subunits that all contribute to long-term sequelae such as cognitive impairment and chronic epilepsy. In conclusion, a short window of opportunity exists when seizures are maximally controlled by first-line benzodiazepine treatment. After that, multiple pathological mechanisms quickly become engaged that make seizures increasingly more difficult to control with high risk for long-term harm.

Desensitization of GABAergic receptors as a mechanism of zolpidem-induced somnambulism.

PubMed

Juszczak, Grzegorz R

2011-08-01

Sleepwalking is a frequently reported side effect of zolpidem which is a short-acting hypnotic drug potentiating activity of GABA(A) receptors. Paradoxically, the most commonly used medications for somnambulism are benzodiazepines, especially clonazepam, which also potentiate activity of GABA(A) receptors. It is proposed that zolpidem-induced sleepwalking can be explained by the desensitization of GABAergic receptors located on serotonergic neurons. According to the proposed model, the delay between desensitization of GABA receptors and a compensatory decrease in serotonin release constitutes the time window for parasomnias. The occurrence of sleepwalking depends on individual differences in receptor desensitization, autoregulation of serotonin release and drug pharmacokinetics. The proposed mechanism of interaction between GABAergic and serotonergic systems can be also relevant for zolpidem abuse and zolpidem-induced hallucinations. It is therefore suggested that special care should be taken when zolpidem is used in patients taking at the same time selective serotonin reuptake inhibitors. Copyright © 2011 Elsevier Ltd. All rights reserved.

Benzodiazepine and kainate receptor binding sites in the RCS rat retina.

PubMed

Stasi, Kalliopi; Naskar, Rita; Thanos, Solon; Kouvelas, Elias D; Mitsacos, Ada

2003-02-01

The effect of age and photoreceptor degeneration on the kainate subtype of glutamate receptors and on the benzodiazepine-sensitive gamma-aminobutyric acid-A receptors (GABA(A)) in normal and RCS (Royal College of Surgeons) rats were investigated. [(3)H]Kainate and [(3)H]flunitrazepam were used as radioligands for kainate and GABA(A)/benzodiazepine()receptors, respectively, using the quantitative receptor autoradiography technique. In both normal and RCS rat retina we observed that [(3)Eta]flunitrazepam and [(3)Eta]kainate binding levels were several times higher in inner plexiform layer (IPL) than in outer plexiform layer (OPL) at all four ages studied (P17, P35, P60 and P180). Age-related changes in receptor binding were observed in normal rat retina: [(3)Eta]flunitrazepam binding showed a significant decrease of 25% between P17 and P60 in IPL,and [(3)Eta]kainate binding showed significant decreases between P17 and P35 in both synaptic layers (71% in IPL and 63% in OPL). Degeneration-related changes in benzodiazepine and kainate receptor binding were observed in RCS rat retina. In IPL, [(3)Eta]flunitrazepam and [(3)Eta]kainate binding levels were higher than in normal retina at P35 (by 24% and 86%, respectively). In OPL, [(3)Eta]flunitrazepam binding was higher in RCS than in normal retina on P35 (74%) and also on P60 (62%). The results indicate that postnatal changes occur in kainate and benzodiazepine receptor binding sites in OPL and IPL of the rat retina up to 6 months of age. The data also suggest that the receptor binding changes observed in the RCS retina could be a consequence of the primary photoreceptor degeneration.

Midazolam inhibits hippocampal long-term potentiation and learning through dual central and peripheral benzodiazepine receptor activation and neurosteroidogenesis.

PubMed

Tokuda, Kazuhiro; O'Dell, Kazuko A; Izumi, Yukitoshi; Zorumski, Charles F

2010-12-15

Benzodiazepines (BDZs) enhance GABA(A) receptor inhibition by direct actions on central BDZ receptors (CBRs). Although some BDZs also bind mitochondrial receptors [translocator protein (18 kDa) (TSPO)] and promote the synthesis of GABA-enhancing neurosteroids, the role of neurosteroids in the clinical effects of BDZs is unknown. In rat hippocampal slices, we compared midazolam, an anesthetic BDZ, with clonazepam, an anticonvulsant/anxiolytic BDZ that activates CBRs selectively. Midazolam, but not clonazepam, increased neurosteroid levels in CA1 pyramidal neurons without changing TSPO immunostaining. Midazolam, but not clonazepam, also augmented a form of spike inhibition after stimulation adjacent to the pyramidal cell layer and inhibited induction of long-term potentiation. These effects were prevented by finasteride, an inhibitor of neurosteroid synthesis, or 17PA [17-phenyl-(3α,5α)-androst-16-en-3-ol], a blocker of neurosteroid effects on GABA(A) receptors. Moreover, the synaptic effects were mimicked by a combination of clonazepam with FGIN (2-[2-(4-fluorophenyl)-1H-indol-3-yl]-N,N-dihexylacetamide), a selective TSPO agonist, or a combination of clonazepam with exogenous allopregnanolone. Consistent with these in vitro results, finasteride abolished the effects of midazolam on contextual fear learning when administrated 1 d before midazolam injection. Thus, dual activation of CBRs and TSPO appears to result in unique actions of clinically important BDZs. Furthermore, endogenous neurosteroids are shown to be important regulators of pyramidal neuron function and synaptic plasticity.

Midazolam inhibits hippocampal long-term potentiation and learning through dual central and peripheral benzodiazepine receptor activation and neurosteroidogenesis

PubMed Central

Tokuda, Kazuhiro; O’Dell, Kazuko A.; Izumi, Yukitoshi; Zorumski, Charles F.

2010-01-01

Benzodiazepines (BDZs) enhance γ-aminobutyric acid-A (GABAA) receptor inhibition by direct actions on central BDZ receptors (CBRs). Although some BDZs also bind mitochondrial receptors (translocator protein 18kDa, TSPO) and promote the synthesis of GABA-enhancing neurosteroids, the role of neurosteroids in the clinical effects of BDZs is unknown. In rat hippocampal slices, we compared midazolam, an anesthetic BDZ with clonazepam, an anticonvulsant/anxiolytic BDZ that activates CBRs selectively. Midazolam, but not clonazepam, increased neurosteroid levels in CA1 pyramidal neurons without changing TSPO immunostaining. Midazolam, but not clonazepam, also augmented a form of spike inhibition following stimulation adjacent to the pyramidal cell layer and inhibited induction of long-term potentiation. These effects were prevented by finasteride, an inhibitor of neurosteroid synthesis, or 17PA (17-phenyl-(3α, 5α)-androst-16-en-3-ol), a blocker of neurosteroid effects on GABAA receptors. Moreover, the synaptic effects were mimicked by a combination of clonazepam with FGIN, a selective TSPO agonist, or a combination of clonazepam with exogenous allopregnanolone. Consistent with these in vitro results, finasteride abolished the effects of midazolam on contextual fear learning when administrated one day prior to midazolam injection. Thus, dual activation of CBRs and TSPO appears to result in unique actions of clinically-important BDZs. Furthermore, endogenous neurosteroids are shown to be important regulators of pyramidal neuron function and synaptic plasticity. PMID:21159950

Bud extracts from Tilia tomentosa Moench inhibit hippocampal neuronal firing through GABAA and benzodiazepine receptors activation.

PubMed

Allio, Arianna; Calorio, Chiara; Franchino, Claudio; Gavello, Daniela; Carbone, Emilio; Marcantoni, Andrea

2015-08-22

Tilia tomentosa Moench bud extracts (TTBEs) is used in traditional medicine for centuries as sedative compound. Different plants belonging to the Tilia genus have shown their efficacy in the treatment of anxiety but still little is known about the mechanism of action of their bud extracts. To evaluate the action of TTBEs as anxiolytic and sedative compound on in vitro hippocampal neurons. The anxiolytic effect of TTBEs was assayed by testing the effects of these compounds on GABAA receptor-activated chloride current of hippocampal neurons by means of the patch-clamp technique and microelectrode-arrays (MEAs). TTBEs acutely administered on mouse hippocampal neurons, activated a chloride current comparable to that measured in the presence of GABA (100 µM). Bicuculline (100 µM) and picrotoxin (100 µM) blocked about 90% of this current, while the remaining 10% was blocked by adding the benzodiazepine (BDZ) antagonist flumazenil (30 µM). Flumazenil alone blocked nearly 60% of the TTBEs activated current, suggesting that TTBEs binds to both GABAA and BDZ receptor sites. Application of high-doses of TTBEs on spontaneous active hippocampal neurons grown for 3 weeks on MEAs blocked the synchronous activity of these neurons. The effects were mimicked by GABA and prevented by picrotoxin (100µM) and flumazenil (30 µM). At minimal doses, TTBEs reduced the frequency of synchronized bursts and increased the cross-correlation index of synchronized neuronal firing. Our data suggest that TTBEs mimics GABA and BDZ agonists by targeting hippocampal GABAergic synapses and inhibiting network excitability by increasing the strength of inhibitory synaptic outputs. Our results contribute toward the validation of TTBEs as effective sedative and anxiolytic compound. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Ester to amide substitution improves selectivity, efficacy and kinetic behavior of a benzodiazepine positive modulator of GABAA receptors containing the α5 subunit

PubMed Central

Stamenić, Tamara Timić; Poe, Michael M.; Rehman, Sabah; Santrač, Anja; Divović, Branka; Scholze, Petra; Ernst, Margot; Cook, James M.; Savić, Miroslav M.

2016-01-01

We have synthesized and characterized MP-III-022 ((R)-8-ethynyl-6-(2-fluorophenyl)-N,4-dimethyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxamide) in vitro and in vivo as a binding- and efficacy-selective positive allosteric modulator of GABAA receptors containing the α5 subunit (α5GABAARs). By approximation of the electrophysiological responses which the estimated free rat brain concentrations can induce, we demonstrated that convenient systemic administration of MP-III-022 in the dose range 1-10 mg/kg may result in a selective potentiation, over a wide range from mild to moderate to strong, of α5βγ2 GABAA receptors. For eliciting a comparable range of potentiation, the widely studied parent ligand SH-053-2′F-R-CH3 containing an ester moiety needs to be administered over a much wider dose range (10-200 mg/kg), but at the price of activating non-α5 GABAARs as well as the desired α5GABAARs at the highest dose. At the dose of 10 mg/kg, which elicits a strong positive modulation of α5GABAARs, MP-III-022 caused mild, but significant muscle relaxation, while at doses 1-10 mg/kg was devoid of ataxia, sedation or an influence on the anxiety level, characteristic for non-selective benzodiazepines. As an amide compound with improved stability and kinetic properties, MP-III-022 may represent an optimized tool to study the influence of α5GABAARs on the neuronal pathways related to CNS disorders such as schizophrenia, Alzheimer's disease, Down syndrome or autism. PMID:27639297

Clobazam and its active metabolite N-desmethylclobazam display significantly greater affinities for α₂- versus α₁-GABA(A)-receptor complexes.

PubMed

Jensen, Henrik Sindal; Nichol, Kathryn; Lee, Deborah; Ebert, Bjarke

2014-01-01

Clobazam (CLB), a 1,5-benzodiazepine (BZD), was FDA-approved in October 2011 for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome (LGS) in patients 2 years and older. BZDs exert various CNS effects through allosteric modulation of GABAA receptors. The structurally distinct, 1,4-BZD clonazepam (CLN) is also approved to treat LGS. The precise mechanisms of action and clinical efficacy of both are unknown. Data show that the GABAA α₁-subunit-selective compound zolpidem [ZOL] exhibits hypnotic/sedative effects. Conversely, data from knock-in mice carrying BZD binding site mutations suggest that the α₂ subunit mediates anticonvulsant effects, without sedative actions. Hence, the specific pattern of interactions across the GABAA receptor complexes of BZDs might be reflected in their clinical efficacies and adverse effect profiles. In this study, GABAA-receptor binding affinities of CLB, N-desmethylclobazam (N-CLB, the major metabolite of CLB), CLN, and ZOL were characterized with native receptors from rat-brain homogenates and on cloned receptors from HEK293 cells transfected with combinations of α (α₁, α₂, α₃, or α₅), β₂, and γ₂ subtypes. Our results demonstrate that CLB and N-CLB have significantly greater binding affinities for α₂- vs. α₁-receptor complexes, a difference not observed for CLN, for which no distinction between α₂ and α₁ receptors was observed. Our experiments with ZOL confirmed the high preference for α₁ receptors. These results provide potential clues to a new understanding of the pharmacologic modes of action of CLB and N-CLB.

Clobazam and Its Active Metabolite N-desmethylclobazam Display Significantly Greater Affinities for α2- versus α1-GABAA–Receptor Complexes

PubMed Central

Jensen, Henrik Sindal; Nichol, Kathryn; Lee, Deborah; Ebert, Bjarke

2014-01-01

Clobazam (CLB), a 1,5-benzodiazepine (BZD), was FDA-approved in October 2011 for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome (LGS) in patients 2 years and older. BZDs exert various CNS effects through allosteric modulation of GABAA receptors. The structurally distinct, 1,4-BZD clonazepam (CLN) is also approved to treat LGS. The precise mechanisms of action and clinical efficacy of both are unknown. Data show that the GABAA α1-subunit–selective compound zolpidem [ZOL] exhibits hypnotic/sedative effects. Conversely, data from knock-in mice carrying BZD binding site mutations suggest that the α2 subunit mediates anticonvulsant effects, without sedative actions. Hence, the specific pattern of interactions across the GABAA receptor complexes of BZDs might be reflected in their clinical efficacies and adverse effect profiles. In this study, GABAA-receptor binding affinities of CLB, N-desmethylclobazam (N-CLB, the major metabolite of CLB), CLN, and ZOL were characterized with native receptors from rat-brain homogenates and on cloned receptors from HEK293 cells transfected with combinations of α (α1, α2, α3, or α5), β2, and γ2 subtypes. Our results demonstrate that CLB and N-CLB have significantly greater binding affinities for α2- vs. α1-receptor complexes, a difference not observed for CLN, for which no distinction between α2 and α1 receptors was observed. Our experiments with ZOL confirmed the high preference for α1 receptors. These results provide potential clues to a new understanding of the pharmacologic modes of action of CLB and N-CLB. PMID:24533090

PWZ-029, A COMPOUND WITH MODERATE INVERSE AGONIST FUNCTIONAL SELECTIVITY AT GABAA RECEPTORS CONTAINING α5 SUBUNITS, IMPROVES PASSIVE, BUT NOT ACTIVE, AVOIDANCE LEARNING IN RATS

PubMed Central

Savić, Miroslav M.; Clayton, Terry; Furtmüller, Roman; Gavrilović, Ivana; Samardžić, Janko; Savić, Snežana; Huck, Sigismund; Sieghart, Werner; Cook, James M.

2008-01-01

Benzodiazepine (BZ) site ligands affect vigilance, anxiety, memory processes, muscle tone and epileptogenic propensity through modulation of neurotransmission at GABAA receptors containing α1, α2, α3 or α5 subunits, and may have numerous experimental and clinical applications. The ability of nonselective BZ site inverse agonists to enhance cognition, documented in animal models and human studies, is clinically not feasible due to potentially unacceptable psychomotor effects. Most investigations to date have proposed the α1 and/or α5 subunit-containing GABAA receptors as comprising the memory-modulating population of these receptors. The novel ligand PWZ-029, which we synthesised and characterized electrophysiologically, possesses in vitro binding selectivity and moderate inverse agonist functional selectivity at α5-containing GABAA receptors. This ligand has also been examined in rats in the passive and active avoidance, spontaneous locomotor activity, elevated plus maze and grip strength tests, primarily predictive of the effects on the memory acquisition, basal locomotor activity, anxiety level and muscle tone, respectively. The improvement of task learning was detected at the dose of 5 mg/kg in the passive, but not active avoidance test. The inverse agonist PWZ-029 had no effect on anxiety or muscle tone, whereas at higher doses (10 and 20 mg/kg) it decreased locomotor activity. This effect was antagonized by flumazenil and also by the lower (but not the higher) dose of an agonist (SH-053-R-CH3-2’F) selective for GABAA receptors containing the α5 subunit. The hypolocomotor effect of PWZ-029 was not antagonized by the antagonist β-CCt exhibiting a preferential affinity for α1-subunit containing receptors. These data suggest that moderate negative modulation at GABAA receptors containing the α5 subunit is a sufficient condition for eliciting enhanced encoding/consolidation of declarative memory, while the influence of higher doses of modulators at these receptors on motor activity shows an intricate pattern whose relevance and mechanism await to be defined. PMID:18394590

Magnolol, a major bioactive constituent of the bark of Magnolia officinalis, exerts antiepileptic effects via the GABA/benzodiazepine receptor complex in mice

PubMed Central

Chen, CR; Tan, R; Qu, WM; Wu, Z; Wang, Y; Urade, Y; Huang, ZL

2011-01-01

BACKGROUND AND PURPOSE The aim of this study was to evaluate the anti-convulsant effects of magnolol (6, 6′, 7, 12-tetramethoxy-2, 2′-dimethyl-1-β-berbaman, C18H18O2) and the mechanisms involved. EXPERIMENTAL APPROACH Mice were treated with magnolol (20, 40 and 80 mg·kg−1) 30 min before injection with pentylenetetrazol (PTZ, 60 mg·kg−1, i.p.). The anti-seizure effects of magnolol were analysed using seizure models of behaviour, EEG and in vitro electrophysiology and c-Fos expression in the hippocampus and cortex. KEY RESULTS Magnolol at doses of 40 and 80 mg·kg−1 significantly delayed the onset of myoclonic jerks and generalized clonic seizures, and decreased the seizure stage and mortality compared with those of the vehicle-treated animals. EEG recordings showed that magnolol (40 and 80 mg·kg−1) prolonged the latency of seizure onset and decreased the number of seizure spikes. The anti-epileptic effect of magnolol was reversed by the GABAA/benzodiazepine receptor antagonist flumazenil. Pretreatment with flumazenil decreased the effects of magnolol on prolongation of seizure latency and decline of seizure stage. In a Mg2+-free model of epileptiform activity, using multi-electrode array recordings in mouse hippocampal slices, magnolol decreased spontaneous epileptiform discharges. Magnolol also significantly decreased seizure-induced Fos immunoreactivity in the piriform cortex, dentate gyrus and hippocampal area CA1. These effects were attenuated by pretreatment with flumazenil. CONCLUSIONS AND IMPLICATIONS These findings indicate that the inhibitory effects of magnolol on epileptiform activity were mediated by the GABAA/benzodiazepine receptor complex. PMID:21518336

Differential antagonism of tetramethylenedisulfotetramine-induced seizures by agents acting at NMDA and GABAA receptors

PubMed Central

Shakarjian, Michael P.; Velíšková, Jana; Stanton, Patric K.; Velíšek, Libor

2012-01-01

Tetramethylenedisulfotetramine (TMDT) is a highly lethal neuroactive rodenticide responsible for many accidental and intentional poisonings in mainland China. Ease of synthesis, water solubility, potency, and difficulty to treat make TMDT a potential weapon for terrorist activity. We characterized TMDT-induced convulsions and mortality in male C57BL/6 mice. TMDT (ip) produced a continuum of twitches, clonic, and tonic-clonic seizures decreasing in onset latency and increasing in severity with increasing dose; 0.4 mg/kg was 100% lethal. The NMDA antagonist, ketamine (35 mg/kg) injected ip immediately after the first TMDT-induced seizure, did not change number of tonic-clonic seizures or lethality, but increased the number of clonic seizures. Doubling the ketamine dose decreased tonic-clonic seizures and eliminated lethality through a 60 min observation period. Treating mice with another NMDA antagonist, MK-801, 0.5 or 1 mg/kg ip, showed similar effects as low and high doses of ketamine, respectively, and prevented lethality, converting status epilepticus EEG activity to isolated interictal discharges. Treatment with these agents 15 min prior to TMDT administration did not increase their effectiveness. Post-treatment with the GABAA receptor allosteric enhancer diazepam (5 mg/kg) greatly reduced seizure manifestations and prevented lethality 60 min post-TMDT, but ictal events were evident in EEG recordings and, hours post-treatment, mice experienced status epilepticus and died. Thus, TMDT is a highly potent and lethal convulsant for which single-dose benzodiazepine treatment is inadequate in managing electrographic seizures or lethality. Repeated benzodiazepine dosing or combined application of benzodiazepines and NMDA receptor antagonists are more likely to be effective in treating TMDT poisoning. PMID:23022509

Insights into structure–activity relationship of GABAA receptor modulating coumarins and furanocoumarins

PubMed Central

Singhuber, Judith; Baburin, Igor; Ecker, Gerhard F.; Kopp, Brigitte; Hering, Steffen

2011-01-01

The coumarins imperatorin and osthole are known to exert anticonvulsant activity. We have therefore analyzed the modulation of GABA-induced chloride currents (IGABA) by a selection of 18 coumarin derivatives on recombinant α1β2γ2S GABAA receptors expressed in Xenopus laevis oocytes by means of the two-microelectrode voltage clamp technique. Osthole (EC50=14±1 μM) and oxypeucedanin (EC50=25±8 μM) displayed the highest efficiency with IGABA potentiation of 116±4% and 547±56%, respectively. IGABA enhancement by osthole and oxypeucedanin was not inhibited by flumazenil (1 μM) indicating an interaction with a binding site distinct from the benzodiazepine binding site. In general, prenyl residues are essential for the positive modulatory activity, while longer side chains or bulkier residues (e.g. geranyl residues) diminish IGABA modulation. Generation of a binary classification tree revealed the importance of polarisability, which is sufficient to distinguish actives from inactives. A 4-point pharmacophore model based on oxypeucedanin – comprising three hydrophobic and one aromatic feature – identified 6 out of 7 actives as hits. In summary, (oxy-)prenylated coumarin derivatives from natural origin represent new GABAA receptor modulators. PMID:21749864

Effects of GABAergic modulators on food and cocaine self-administration in baboons.

PubMed

Weerts, Elise M; Froestl, Wolfgang; Griffiths, Roland R

2005-12-12

Drugs that indirectly alter dopaminergic systems may alter the reinforcing effects of cocaine. The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) has extensive neural connections in mesolimbic regions that appear to modulate dopamine. The current study evaluated the effects of GABA(B) receptor agonists baclofen and CGP44532, the benzodiazepine agonist alprazolam, and the GABA reuptake inhibitor tiagabine on lever responding maintained by low dose cocaine injections (0.032 mg/kg) or by food pellet (1 g) delivery in baboons. The benzodiazepine antagonist flumazenil was tested as a negative control. Cocaine or food was available under a fixed ratio (FR 10) schedule of reinforcement during daily 2-h sessions. During baseline conditions, cocaine and pellets maintained similar numbers of reinforcers per session. Baclofen, CGP44532 and tiagabine dose-dependently reduced the number of cocaine injections, where as the benzodiazepine antagonist flumazenil did not. Baclofen, CGP44532 and tiagabine also produced dose-related decreases in food-maintained behavior. In contrast, the benzodiazepine agonist alprazolam, which positively modulates GABA(A) receptors via the benzodiazepine site, produced decreases in cocaine self-injection, but not food-maintained behavior. Thus, the effects of alprazolam were specific for cocaine-maintained behavior, where as the effects of baclofen and CGP44532 were not.

Enhancement of GABAergic transmission by zolpidem, an imidazopyridine with preferential affinity for type I benzodiazepine receptors.

PubMed

Biggio, G; Concas, A; Corda, M G; Serra, M

1989-02-28

The effect of zolpidem, an imidazopyridine derivative with high affinity at the type I benzodiazepine recognition site, on the function of the GABAA/ionophore receptor complex was studied in vitro. Zolpidem, mimicking the action of diazepam, increased [3H]GABA binding, enhanced muscimol-stimulated 36Cl- uptake and reduced [35S]TBPS binding in rat cortical membrane preparations. Zolpidem was less effective than diazepam on the above parameters. Zolpidem induced a lower increase of [3H]GABA binding (23 vs. 35%) and muscimol-stimulated 36Cl- uptake (22 vs. 40%) and a smaller decrease of [35S]TBPS binding (47 vs. 77%) than diazepam. The finding that zolpidem enhanced the function of GABAergic synapses with an efficacy qualitatively and quantitatively different from that of diazepam suggests that this compound is a partial agonist at the benzodiazepine recognition site. Thus, our results are consistent with the view that the biochemical and pharmacological profile of a benzodiazepine recognition site ligand reflects its efficacy to enhance GABAergic transmission. Whether the preferential affinity of zolpidem at the type I site is involved in its atypical biochemical and pharmacological profile remains to be clarified.

Effects of Insecticidal Ketones Present in Mint Plants on GABAA Receptor from Mammalian Neurons

PubMed Central

Sánchez-Borzone, Mariela Eugenia; Marin, Leticia Delgado; García, Daniel Asmed

2017-01-01

Background: The genus Mentha, an important member of the Lamiaceae family, is represented by many species commonly known as mint. The insecticidal activity of Mentha oil and its main components has been tested and established against various insects/pests. Among these, the ketone monoterpenes that are most common in different Mentha species demonstrated insect toxicity, with pulegone being the most active, followed by carvone and menthone. Considering that the GABAA receptor (GABAA-R) is one of the main insecticide targets on neurons, and that pulegone would modulate the insect GABA system, it may be expected that the insecticidal properties of Mentha ketones are mediated by their interaction with this receptor. Objective: In order to discern the pharmacological actions of these products when used as insecticides on mammalian organisms, we evaluated the pharmacologic activity of ketones, commonly present in Mentha plants, on native GABAA-R from rats. Materials and Methods: Determination of ketones effects on allosterically enhanced benzodiazepine binding, using primary cultures of cortical neurons, which express functional receptors and MTT assay to evaluate their cell toxicity. Results: Our results seem to indicate that ketone components of Mentha, with proven repellent or insecticide activity, were able to behave as GABAA-R negative allosteric modulators in murine cells and consequently could exhibit convulsant activity in mammalians. Only pulegone at the highest assayed concentration (2 mM) showed a significant reduction in cell viability after exposure for 24 hr. Conclusion: The present results strongly suggest that the ketone components of Mentha are able to exhibit convulsant activity in mammalian organisms, but functional assays and in vivo experiments would be necessary to corroborate this proposed action. SUMMARY The pharmacological activity of insecticide ketones, commonly present in Mentha plants, was evaluated on native GABAA receptor from mammalian neurons.All studied compounds: pulegone, menthone and dihydrocarvone, were able to behave as negative allosteric modulators and could exhibit convulsant activity in mammalian organisms.Citotoxicity assays demonstrated that only pulegone affected the cell viability. Abbreviations used: GABA: gamma aminobutyric acid, GABAA-R: GABAA receptor, MTT: 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazam, DMEM: Dulbecco's modified minimum essential mèdium, [3H]TBOB: [3H] t-Butylbicycloorthobenzoate PMID:28216893

Evaluation of anti-hyperalgesic and analgesic effects of two benzodiazepines in human experimental pain: a randomized placebo-controlled study.

PubMed

Vuilleumier, Pascal H; Besson, Marie; Desmeules, Jules; Arendt-Nielsen, Lars; Curatolo, Michele

2013-01-01

Compounds that act on GABA-receptors produce anti-hyperalgesia in animal models, but little is known on their effects in humans. The aim of this study was to explore the potential usefulness of GABA-agonism for the control of pain in humans. Two agonists at the benzodiazepine-binding site of GABAA-receptors (clobazam and clonazepam) were studied using multiple experimental pain tests. Positive results would support further investigation of GABA agonism for the control of clinical pain. In a randomized double-blind crossover design, 16 healthy male volunteers received clobazam 20 mg, clonazepam 1 mg and tolterodine 1 mg (active placebo). The area of static hyperalgesia after intradermal capsaicin injection was the primary endpoint. Secondary endpoints were: area of dynamic hyperalgesia, response to von Frey hair stimulation, pressure pain thresholds, conditioned pain modulation, cutaneous and intramuscular electrical pain thresholds (1, 5 and 20 repeated stimulation), and pain during cuff algometry. For the primary endpoint, an increase in the area of static hyperalgesia was observed after administration of placebo (p<0.001), but not after clobazam and clonazepam. Results suggestive for an anti-hyperalgesic effect of the benzodiazepines were obtained with all three intramuscular pain models and with cuff algometry. No effect could be detected with the other pain models employed. Collectively, the results are suggestive for a possible anti-hyperalgesic effect of drugs acting at the GABAA-receptors in humans, particularly in models of secondary hyperalgesia and deep pain. The findings are not conclusive, but support further clinical research on pain modulation by GABAergic drugs. Because of the partial results, future research should focus on compounds acting selectively on subunits of the GABA complex, which may allow the achievement of higher receptor occupancy than unselective drugs. Our data also provide information on the most suitable experimental models for future investigation of GABAergic compounds. ClinicalTrials.gov NCT01011036.

Evaluation of Anti-Hyperalgesic and Analgesic Effects of Two Benzodiazepines in Human Experimental Pain: A Randomized Placebo-Controlled Study

PubMed Central

Vuilleumier, Pascal H.; Besson, Marie; Desmeules, Jules; Arendt-Nielsen, Lars; Curatolo, Michele

2013-01-01

Background and Aims Compounds that act on GABA-receptors produce anti-hyperalgesia in animal models, but little is known on their effects in humans. The aim of this study was to explore the potential usefulness of GABA-agonism for the control of pain in humans. Two agonists at the benzodiazepine-binding site of GABAA-receptors (clobazam and clonazepam) were studied using multiple experimental pain tests. Positive results would support further investigation of GABA agonism for the control of clinical pain. Methods In a randomized double-blind crossover design, 16 healthy male volunteers received clobazam 20 mg, clonazepam 1 mg and tolterodine 1 mg (active placebo). The area of static hyperalgesia after intradermal capsaicin injection was the primary endpoint. Secondary endpoints were: area of dynamic hyperalgesia, response to von Frey hair stimulation, pressure pain thresholds, conditioned pain modulation, cutaneous and intramuscular electrical pain thresholds (1, 5 and 20 repeated stimulation), and pain during cuff algometry. Results For the primary endpoint, an increase in the area of static hyperalgesia was observed after administration of placebo (p<0.001), but not after clobazam and clonazepam. Results suggestive for an anti-hyperalgesic effect of the benzodiazepines were obtained with all three intramuscular pain models and with cuff algometry. No effect could be detected with the other pain models employed. Conclusions Collectively, the results are suggestive for a possible anti-hyperalgesic effect of drugs acting at the GABAA-receptors in humans, particularly in models of secondary hyperalgesia and deep pain. The findings are not conclusive, but support further clinical research on pain modulation by GABAergic drugs. Because of the partial results, future research should focus on compounds acting selectively on subunits of the GABA complex, which may allow the achievement of higher receptor occupancy than unselective drugs. Our data also provide information on the most suitable experimental models for future investigation of GABAergic compounds. Trial Registration ClinicalTrials.gov NCT01011036 PMID:23554851

Role of Anticonvulsant and Antiepileptogenic Neurosteroids in the Pathophysiology and Treatment of Epilepsy

PubMed Central

Reddy, Doodipala Samba

2011-01-01

This review highlights the role of major endogenous neurosteroids in seizure disorders and the promise of neurosteroid replacement therapy in epilepsy. Neurosteroids are endogenous modulators of seizure susceptibility. Neurosteroids such as allopregnanolone (3α-hydroxy-5α-pregnane-20-one) and allotetrahydrodeoxycorticosterone (3α,21-dihydroxy-5α-pregnan-20-one) are positive modulators of GABA-A receptors. Aside from peripheral tissues, neurosteroids are synthesized within the brain, mostly in principal neurons. Neurosteroids potentiate synaptic GABA-A receptor function and also activate δ-subunit-containing extrasynaptic GABA-A receptors that mediate tonic currents and thus may play an important role in neuronal network excitability and seizure susceptibility. Our studies over the past decade have shown that neurosteroids are broad-spectrum anticonvulsants and confer seizure protection in various animal models. They protect against seizures induced by GABA-A receptor antagonists, 6-Hz model, pilocarpine-induced limbic seizures, and seizures in kindled animals. Unlike benzodiazepines, tolerance does not occur to their actions during chronic administration. Our recent studies provide compelling evidence that neurosteroids may have antiepileptogenic properties. There is emerging evidence that endogenous neurosteroids may play a key role in the pathophysiology of catamenial epilepsy, stress–sensitive seizure conditions, temporal lobe epilepsy, and alcohol-withdrawal seizures. It is suggested that neurosteroid replacement with natural or synthetic neurosteroids may be useful in the treatment of epilepsy. Synthetic analogs of neurosteroids that are devoid of hormonal side effects show promise in the treatment of diverse seizure disorders. Agents that stimulate endogenous production of neurosteroids may also be useful for treatment of epilepsy. PMID:22654805

Selective labelling of diazepam-insensitive GABAA receptors in vivo using [3H]Ro 15-4513.

PubMed

Pym, Luanda J; Cook, Susan M; Rosahl, Thomas; McKernan, Ruth M; Atack, John R

2005-11-01

Classical benzodiazepines (BZs), such as diazepam, bind to GABAA receptors containing alpha1, alpha2, alpha3 or alpha5 subunits that are therefore described as diazepam-sensitive (DS) receptors. However, the corresponding binding site of GABAA receptors containing either an alpha4 or alpha6 subunit do not bind the classical BZs and are therefore diazepam-insensitive (DIS) receptors; a difference attributable to a single amino acid (histidine in alpha1, alpha2, alpha3 and alpha5 subunits and arginine in alpha4 and alpha6). Unlike classical BZs, the imidazobenzodiazepines Ro 15-4513 and bretazenil bind to both DS and DIS populations of GABAA receptors. In the present study, an in vivo assay was developed using lorazepam to fully occupy DS receptors such that [3H]Ro 15-4513 was then only able to bind to DIS receptors. When dosed i.v., [3H]Ro 15-4513 rapidly entered and was cleared from the brain, with approximately 70% of brain radioactivity being membrane-bound. Essentially all membrane binding to DS+DIS receptors could be displaced by unlabelled Ro 15-4513 or bretazenil, with respective ID50 values of 0.35 and 1.2 mg kg(-1). A dose of 30 mg kg(-1) lorazepam was used to block all DS receptors in a [3H]Ro 15-1788 in vivo binding assay. When predosed in a [3H]Ro 15-4513 binding assay, lorazepam blocked [3H]Ro 15-4513 binding to DS receptors, with the remaining binding to DIS receptors accounting for 5 and 23% of the total (DS plus DIS) receptors in the forebrain and cerebellum, respectively. The in vivo binding of [3H]Ro 15-4513 to DIS receptors in the presence of lorazepam was confirmed using alpha1H101R knock-in mice, in which alpha1-containing GABAA receptors are rendered diazepam insensitive by mutation of the histidine that confers diazepam sensitivity to arginine. In these mice, and in the presence of lorazepam, there was an increase of in vivo [3H]Ro 15-4513 binding in the forebrain and cerebellum from 4 and 15% to 36 and 59% of the total (i.e. DS plus DIS) [3H]Ro 15-4513 binding observed in the absence of lorazepam.

The flavone hispidulin, a benzodiazepine receptor ligand with positive allosteric properties, traverses the blood–brain barrier and exhibits anticonvulsive effects

PubMed Central

Kavvadias, Dominique; Sand, Philipp; Youdim, Kuresh A; Qaiser, M Zeeshan; Rice-Evans, Catherine; Baur, Roland; Sigel, Erwin; Rausch, Wolf-Dieter; Riederer, Peter; Schreier, Peter

2004-01-01

The functional characterization of hispidulin (4′,5,7-trihydroxy-6-methoxyflavone), a potent benzodiazepine (BZD) receptor ligand, was initiated to determine its potential as a modulator of central nervous system activity. After chemical synthesis, hispidulin was investigated at recombinant GABAA/BZD receptors expressed by Xenopus laevis oocytes. Concentrations of 50 nM and higher stimulated the GABA-induced chloride currents at tested receptor subtypes (α1−3,5,6β2γ2S) indicating positive allosteric properties. Maximal stimulation at α1β2γ2S was observed with 10 μM hispidulin. In contrast to diazepam, hispidulin modulated the α6β2γ2S-GABAA receptor subtype. When fed to seizure-prone Mongolian gerbils (Meriones unguiculatus) in a model of epilepsy, hispidulin (10 mg kg−1 body weight (BW) per day) and diazepam (2 mg kg−1 BW per day) markedly reduced the number of animals suffering from seizures after 7 days of treatment (30 and 25% of animals in the respective treatment groups, vs 80% in the vehicle group). Permeability across the blood–brain barrier for the chemically synthesized, 14C-labelled hispidulin was confirmed by a rat in situ perfusion model. With an uptake rate (Kin) of 1.14 ml min−1 g−1, measurements approached the values obtained with highly penetrating compounds such as diazepam. Experiments with Caco-2 cells predict that orally administered hispidulin enters circulation in its intact form. At a concentration of 30 μM, the flavone crossed the monolayer without degradation as verified by the absence of glucuronidated metabolites. PMID:15231642

Neurosteroids in hepatic encephalopathy: Novel insights and new therapeutic opportunities.

PubMed

Butterworth, Roger F

2016-06-01

Hepatic encephalopathy (HE) is a serious neuropsychiatric disorder resulting from liver failure. Symptoms of HE include mild cognitive impairment, stupor and coma. Morphological changes to neuroglia (both astrocytes and microglia) occur in HE consisting of cytotoxic brain edema (astrocyte swelling) in acute liver failure and Alzheimer type-2 astrocytosis in cirrhosis. Visual-evoked responses in animals with liver failure and HE manifest striking similarities to those in animals treated with agonists of the GABA-A receptor complex. Neurosteroids are synthesized in brain following activation of translocator protein (TSPO), a mitochondrial neuroglial cholesterol-transporter protein. TSPO sites are activated in both animal models of HE as well as in autopsied brain tissue from HE patients. Activation of TSPO sites results in increased cholesterol transport into the mitochondrion followed by stimulation of a metabolic pathway culminating in the synthesis of allopregnanolone (ALLO) and tetrahydrodeoxycorticosterone (THDOC), neurosteroids with potent positive allosteric modulatory action on the GABA-A receptor complex. Concentrations of ALLO and THDOC in brain tissue from mice with HE resulting from toxic liver injury are sufficient to induce sedation in animals of the same species and significant increases in concentrations of ALLO have been reported in autopsied brain tissue from cirrhotic patients with HE leading to the proposal that "increased GABAergic tone" in HE results from that increased brain concentrations of this neurosteroid. Agents with the potential to decrease neurosteroid synthesis and/or prevent their modulatory actions on the GABA-A receptor complex may provide novel approaches to the management and treatment of HE. Such agents include indomethacin, benzodiazepine receptor inverse agonists and a novel series of compounds known as GABA-A receptor-modulating steroid antagonists (GAMSA). Copyright © 2015 Elsevier Ltd. All rights reserved.

Ganaxolone improves behavioral deficits in a mouse model of post-traumatic stress disorder

PubMed Central

Pinna, Graziano; Rasmusson, Ann M.

2014-01-01

Allopregnanolone and its equipotent stereoisomer, pregnanolone (together termed ALLO), are neuroactive steroids that positively and allosterically modulate the action of gamma-amino-butyric acid (GABA) at GABAA receptors. Levels of ALLO are reduced in the cerebrospinal fluid of female premenopausal patients with post-traumatic stress disorder (PTSD), a severe, neuropsychiatric condition that affects millions, yet is without a consistently effective therapy. This suggests that restoring downregulated brain ALLO levels in PTSD may be beneficial. ALLO biosynthesis is also decreased in association with the emergence of PTSD-like behaviors in socially isolated (SI) mice. Similar to PTSD patients, SI mice also exhibit changes in the frontocortical and hippocampal expression of GABAA receptor subunits, resulting in resistance to benzodiazepine-mediated sedation and anxiolysis. ALLO acts at a larger spectrum of GABAA receptor subunits than benzodiazepines, and increasing corticolimbic ALLO levels in SI mice by injecting ALLO or stimulating ALLO biosynthesis with a selective brain steroidogenic stimulant, such as S-norfluoxetine, at doses far below those that block serotonin reuptake, reduces PTSD-like behavior in these mice. This suggests that synthetic analogs of ALLO, such as ganaxolone, may also improve anxiety, aggression, and other PTSD-like behaviors in the SI mouse model. Consistent with this hypothesis, ganaxolone (3.75–30 mg/kg, s.c.) injected 60 min before testing of SI mice, induced a dose-dependent reduction in aggression toward a same-sex intruder and anxiety-like behavior in an elevated plus maze. The EC50 dose of ganaxolone used in these tests also normalized exaggerated contextual fear conditioning and, remarkably, enhanced fear extinction retention in SI mice. At these doses, ganaxolone failed to change locomotion in an open field test. Therefore, unlike benzodiazepines, ganaxolone at non-sedating concentrations appears to improve dysfunctional emotional behavior associated with deficits in ALLO in mice and may provide an alternative treatment for PTSD patients with deficits in the synthesis of ALLO. Selective serotonin reuptake inhibitors (SSRIs) are the only medications currently approved by the FDA for treatment of PTSD, although they are ineffective in a substantial proportion of PTSD patients. Hence, an ALLO analog such as ganaxolone may offer a therapeutic GABAergic alternative to SSRIs for the treatment of PTSD or other disorders in which ALLO biosynthesis may be impaired. PMID:25309317

The role of GABA-A and mitochondrial diazepam-binding inhibitor receptors on the effects of neurosteroids on food intake in mice.

PubMed

Reddy, D S; Kulkarni, S K

1998-06-01

The present studies were undertaken to investigate the neuroactive steroidal modulation of feeding behavior and possible involvement of gamma-aminobutyric acid type-A (GABA-A) and mitochondrial diazepam binding inhibitor (DBI) receptors (MDR) in food-deprived male mice. Allopregnanolone (0.5-2 mg/kg), a neurosteroid, progesterone (1-10 mg/kg), a neurosteroid precursor, and 4'-chlordiazepam (0.25-1 mg/kg), a specific high affinity MDR agonist, produced a dose-dependent hyperphagic effects. In contrast, neurosteroids pregnenolone sulfate (PS) (1-10 mg/kg) and dehydroepiandrosterone sulfate (DHEAS) (1-10 mg/kg) produced a hypophagic effect, in a dose-dependent manner. The allopregnanolone-, progesterone- and 4'-chlordiazepam-induced hyperphagic effect was blocked by picrotoxin (1 mg/kg), a GABA-A chloride channel antagonist, but not by flumazenil (2 mg/kg), a benzodiazepine (BZD) antagonist. The 4'-chlordiazepam-induced hyperphagic effect was prevented by pretreatment with PK11195 (2 mg/kg), a selective partial MDR antagonist. The hypophagic effect of DHEAS (10 mg/kg) was reversed by dizocilpine (10 microg/kg), an NMDA receptor antagonist, but resistant to muscimol (0.1 mg/kg), a selective GABA-A receptor agonist. In contrast, the PS (10 mg/kg)-induced hypophagic response was resistant to dizocilpine, but sensitive to muscimol (0.1 mg/kg). Both the sulfated neurosteroids PS and DHEAS also reversed the hyperphagic effect of allopregnanolone. In addition, the BZD agonist triazolam (0.05-0.25 mg/kg) also produced a flumazenil- and picrotoxin-sensitive hyperphagic effects, thereby suggesting the changes in feeding behavior by neurosteroids represent GABA-A receptor mediated hyperphagic action. Although the possible antistress or anxiolytic actions of neurosteroids may confound the hyperphagia, behavioral effects observed were specific to food because the mice were adopted to the test environment and diet, and of a possible variation between various neurosteroids in the extent to which antistress or anxiolytic effect produced at hyperphagic doses. The hyperphagic effects of progesterone and 4'-chlordiazepam resembled that of neurosteroid allopregnanolone. Therefore, the effect of progesterone may be imputed to its metabolism to allopregnanolone, while the 4'-chlordiazepam-induced hyperphagic response is related to its MDR-stimulated neurosteroidogenesis and subsequent modulation of GABA-A receptors. The hypophagic response following DHEAS may, at least partly, involve an NMDA receptor mechanism. However, PS-induced hypophagia may be mediated by GABA-A or other receptor systems. These data suggest a pivotal role for GABA-A and mitochondrial DBI receptors in the hyperphagic effects of neurosteroids and reinforces a role for endogenous neurosteroids in regulating feeding behavior. Future studies may lead to the development of neurosteroid-based anorectic/hyperphagic agents for therapeutic use.

A novel positive allosteric modulator of the GABAA receptor: the action of (+)-ROD188

PubMed Central

Thomet, Urs; Baur, Roland; Razet, Rodolphe; Dodd, Robert H; Furtmüller, Roman; Sieghart, Werner; Sigel, Erwin

2000-01-01

(+)-ROD188 was synthesized in the search for novel ligands of the GABA binding site. It shares some structural similarity with bicuculline. (+)-ROD188 failed to displace [3H]-muscimol in binding studies and failed to induce channel opening in recombinant rat α1β2γ2 GABAA receptors functionally expressed in Xenopus oocytes. (+)-ROD188 allosterically stimulated GABA induced currents. Displacement of [3H]-Ro15-1788 indicated a low affinity action at the benzodiazepine binding site. In functional studies, stimulation by (+)-ROD188 was little sensitive to the presence of 1 μM of the benzodiazepine antagonist Ro 15-1788, and (+)-ROD188 also stimulated currents mediated by α1β2, indicating a major mechanism of action different from that of benzodiazepines. Allosteric stimulation by (+)-ROD188 was similar in α1β2N265S as in unmutated α1β2, while that by loreclezole was strongly reduced. (+)-ROD188 also strongly stimulated currents elicited by either pentobarbital or 5α-pregnan-3α-ol-20-one (3α-OH-DHP), in line with a mode of action different from that of barbiturates or neurosteroids as channel agonists. Stimulation by (+)-ROD188 was largest in α6β2γ2 (α6β2γ2>>α1β2γ2=α5β2γ2>α2β2γ2= α3β2γ2), indicating a unique subunit isoform specificity. Miniature inhibitory postsynaptic currents (mIPSC) in cultures of rat hippocampal neurons, caused by spontaneous release of GABA showed a prolonged decay time in the presence of 30 μM (+)-ROD188, indicating an enhanced synaptic inhibitory transmission. PMID:11030736

Non-neuronal, slow GABA signalling in the ventrobasal thalamus targets δ-subunit-containing GABAA receptors

PubMed Central

Jiménez-González, Cristina; Pirttimaki, Tiina; Cope, David W; Parri, H R

2011-01-01

The rodent ventrobasal (VB) thalamus contains a relatively uniform population of thalamocortical (TC) neurons that receive glutamatergic input from the vibrissae and the somatosensory cortex, and inhibitory input from the nucleus reticularis thalami (nRT). In this study we describe γ-aminobutyric acid (GABA)A receptor-dependent slow outward currents (SOCs) in TC neurons that are distinct from fast inhibitory postsynaptic currents (IPSCs) and tonic currents. SOCs occurred spontaneously or could be evoked by hypo-osmotic stimulus, and were not blocked by tetrodotoxin, removal of extracellular Ca2+ or bafilomycin A1, indicating a non-synaptic, non-vesicular GABA origin. SOCs were more common in TC neurons of the VB compared with the dorsal lateral geniculate nucleus, and were rarely observed in nRT neurons, whilst SOC frequency in the VB increased with age. Application of THIP, a selective agonist at δ-subunit-containing GABAA receptors, occluded SOCs, whereas the benzodiazepine site inverse agonist β-CCB had no effect, but did inhibit spontaneous and evoked IPSCs. In addition, the occurrence of SOCs was reduced in mice lacking the δ-subunit, and their kinetics were also altered. The anti-epileptic drug vigabatrin increased SOC frequency in a time-dependent manner, but this effect was not due to reversal of GABA transporters. Together, these data indicate that SOCs in TC neurons arise from astrocytic GABA release, and are mediated by δ-subunit-containing GABAA receptors. Furthermore, these findings suggest that the therapeutic action of vigabatrin may occur through the augmentation of this astrocyte–neuron interaction, and highlight the importance of glial cells in CNS (patho) physiology. PMID:21395866

Increased regional cerebral blood flow but normal distribution of GABAA receptor in the visual cortex of subjects with early-onset blindness.

PubMed

Mishina, Masahiro; Senda, Michio; Kiyosawa, Motohiro; Ishiwata, Kiichi; De Volder, Anne G; Nakano, Hideki; Toyama, Hinako; Oda, Kei-ichi; Kimura, Yuichi; Ishii, Kenji; Sasaki, Touru; Ohyama, Masashi; Komaba, Yuichi; Kobayashi, Shirou; Kitamura, Shin; Katayama, Yasuo

2003-05-01

Before the completion of visual development, visual deprivation impairs synaptic elimination in the visual cortex. The purpose of this study was to determine whether the distribution of central benzodiazepine receptor (BZR) is also altered in the visual cortex in subjects with early-onset blindness. Positron emission tomography was carried out with [(15)O]water and [(11)C]flumazenil on six blind subjects and seven sighted controls at rest. We found that the CBF was significantly higher in the visual cortex for the early-onset blind subjects than for the sighted control subjects. However, there was no significant difference in the BZR distribution in the visual cortex for the subject with early-onset blindness than for the sighted control subjects. These results demonstrated that early visual deprivation does not affect the distribution of GABA(A) receptors in the visual cortex with the sensitivity of our measurements. Synaptic elimination may be independent of visual experience in the GABAergic system of the human visual cortex during visual development.

Alcohol use disorders and current pharmacological therapies: the role of GABAA receptors

PubMed Central

Liang, Jing; Olsen, Richard W

2014-01-01

Alcohol use disorders (AUD) are defined as alcohol abuse and alcohol dependence, which create large problems both for society and for the drinkers themselves. To date, no therapeutic can effectively solve these problems. Understanding the underlying mechanisms leading to AUD is critically important for developing effective and safe pharmacological therapies. Benzodiazepines (BZs) are used to reduce the symptoms of alcohol withdrawal syndrome. However, frequent use of BZs causes cross-tolerance, dependence, and cross-addiction to alcohol. The FDA-approved naltrexone and acamprosate have shown mixed results in clinical trials. Naltrexone is effective to treat alcohol dependence (decreased length and frequency of drinking bouts), but its severe side effects, including withdrawal symptoms, are difficult to overcome. Acamprosate showed efficacy for treating alcohol dependence in European trials, but two large US trials have failed to confirm the efficacy. Another FDA-approved medication, disulfiram, does not diminish craving, and it causes a peripheral neuropathy. Kudzu is the only natural medication mentioned by the National Institute on Alcohol Abuse and Alcoholism, but its mechanisms of action are not yet established. It has been recently shown that dihydromyricetin, a flavonoid purified from Hovenia, has unique effects on GABAA receptors and blocks ethanol intoxication and withdrawal in alcoholic animal models. In this article, we review the role of GABAA receptors in the treatment of AUD and currently available and potentially novel pharmacological agents. PMID:25066321

Dual orexin receptor antagonists - promising agents in the treatment of sleep disorders.

PubMed

Pałasz, Artur; Lapray, Damien; Peyron, Christelle; Rojczyk-Gołębiewska, Ewa; Skowronek, Rafał; Markowski, Grzegorz; Czajkowska, Beata; Krzystanek, Marek; Wiaderkiewicz, Ryszard

2014-01-01

Insomnia is a serious medical and social problem, its prevalence in the general population ranges from 9 to 35% depending on the country and assessment method. Often, patients are subject to inappropriate and therefore dangerous pharmacotherapies that include prolonged administration of hypnotic drugs, benzodiazepines and other GABAA receptor modulators. This usually does not lead to a satisfactory improvement in patients' clinical states and may cause lifelong drug dependence. Brain state transitions require the coordinated activity of numerous neuronal pathways and brain structures. It is thought that orexin-expressing neurons play a crucial role in this process. Due to their interaction with the sleep-wake-regulating neuronal population, they can activate vigilance-promoting regions and prevent unwanted sleep intrusions. Understanding the multiple orexin modulatory effects is crucial in the context of pathogenesis of insomnia and should lead to the development of novel treatments. An important step in this process was the synthesis of dual antagonists of orexin receptors. Crucially, these drugs, as opposed to benzodiazepines, do not change the sleep architecture and have limited side-effects. This new pharmacological approach might be the most appropriate to treat insomnia.

ηηDiazepam-induced loss of inhibitory synapses mediated by PLCδ/ Ca 2+ /calcineurin signalling downstream of GABAA receptors.

PubMed

Nicholson, Martin W; Sweeney, Aaron; Pekle, Eva; Alam, Sabina; Ali, Afia B; Duchen, Michael; Jovanovic, Jasmina N

2018-06-14

Benzodiazepines facilitate the inhibitory actions of GABA by binding to γ-aminobutyric acid type A receptors (GABA A Rs), GABA-gated chloride/bicarbonate channels, which are the key mediators of transmission at inhibitory synapses in the brain. This activity underpins potent anxiolytic, anticonvulsant and hypnotic effects of benzodiazepines in patients. However, extended benzodiazepine treatments lead to development of tolerance, a process which, despite its important therapeutic implications, remains poorly characterised. Here we report that prolonged exposure to diazepam, the most widely used benzodiazepine in clinic, leads to a gradual disruption of neuronal inhibitory GABAergic synapses. The loss of synapses and the preceding, time- and dose-dependent decrease in surface levels of GABA A Rs, mediated by dynamin-dependent internalisation, were blocked by Ro 15-1788, a competitive benzodiazepine antagonist, and bicuculline, a competitive GABA antagonist, indicating that prolonged enhancement of GABA A R activity by diazepam is integral to the underlying molecular mechanism. Characterisation of this mechanism has revealed a metabotropic-type signalling downstream of GABA A Rs, involving mobilisation of Ca 2+ from the intracellular stores and activation of the Ca 2+ /calmodulin-dependent phosphatase calcineurin, which, in turn, dephosphorylates GABA A Rs and promotes their endocytosis, leading to disassembly of inhibitory synapses. Furthermore, functional coupling between GABA A Rs and Ca 2+ stores was sensitive to phospholipase C (PLC) inhibition by U73122, and regulated by PLCδ, a PLC isoform found in direct association with GABA A Rs. Thus, a PLCδ/Ca 2+ /calcineurin signalling cascade converts the initial enhancement of GABA A Rs by benzodiazepines to a long-term downregulation of GABAergic synapses, this potentially underpinning the development of pharmacological and behavioural tolerance to these widely prescribed drugs.

Topology characterization of a benzodiazepine-binding beta-rich domain of the GABAA receptor alpha1 subunit.

PubMed

Xu, Zhiwen; Fang, Shisong; Shi, Haifeng; Li, Hoiming; Deng, Yiqun; Liao, Yinglei; Wu, Jiun-Ming; Zheng, Hui; Zhu, Huaimin; Chen, Hueih-Min; Tsang, Shui Ying; Xue, Hong

2005-10-01

Structural investigation of GABAA receptors has been limited by difficulties imposed by its trans-membrane-complex nature. In the present study, the topology of a membrane-proximal beta-rich (MPB) domain in the C139-L269 segment of the receptor alpha1 subunit was probed by mapping the benzodiazepine (BZ)-binding and epitopic sites, as well as fluorescence resonance energy transfer (FRET) analysis. Ala-scanning and semiconservative substitutions within this segment revealed the contribution of the phenyl rings of Y160 and Y210, the hydroxy group of S186 and the positive charge on R187 to BZ-binding. FRET with the bound BZ ligand indicated the proximity of Y160, S186, R187, and S206 to the BZ-binding site. On the other hand, epitope-mapping using the monoclonal antibodies (mAbs) against the MPB domain established a clustering of T172, R173, E174, Q196, and T197. Based on the lack of FRET between Trp substitutionally placed at R173 or V198 and bound BZ, this epitope-mapped cluster is located on a separate end of the folded protein from the BZ-binding site. Mutations of the five conserved Cys and Trp residues in the MPB domain gave rise to synergistic and rescuing effects on protein secondary structures and unfolding stability that point to a CCWCW-pentad, reminiscent to the CWC-triad "pin" of immunoglobulin (Ig)-like domains, important for the structural maintenance. These findings, together with secondary structure and fold predictions suggest an anti-parallel beta-strand topology with resemblance to Ig-like fold, having the BZ-binding and the epitopic residues being clustered at two different ends of the fold.

Functional Characterization of the 1,5-Benzodiazepine Clobazam and Its Major Active Metabolite N-Desmethylclobazam at Human GABAA Receptors Expressed in Xenopus laevis Oocytes

PubMed Central

Hammer, Harriet; Ebert, Bjarke; Jensen, Henrik Sindal; Jensen, Anders A.

2015-01-01

The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences. The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABAAR) subtypes α1β2γ2S, α2β2γ2S, α3β2γ2S, α5β2γ2S and α6β2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited by the 1,4-benzodiazepine clonazepam. All three compounds potentiated GABA EC20-evoked responses through the α1,2,3,5β2γ2S GABAARs in a reversible and concentration-dependent manner, with each displaying similar EC50 values at the four subtypes. Furthermore, the degrees of potentiation of the GABA EC20 currents through the four receptors mediated by saturating modulator concentrations did not differ substantially for any of the three benzodiazepines. The three compounds were substantially less potent (200-3900 fold) as positive allosteric modulators at the α6β2δ GABAAR than at the α1,2,3,5β2γ2S receptors. Interestingly, however, clobazam and especially N-desmethylclobazam were highly efficacious potentiators of α6β2δ receptor signaling. Although this activity component is unlikely to contribute to the in vivo effects of clobazam/N-desmethylclobazam, the 1,5-benzodiazepine could constitute an interesting lead for novel modulators targeting this low-affinity binding site in GABAARs. In conclusion, the non-selective modulation exerted by clobazam, N-desmethylclobazam and clonazepam at the α1β2γ2S, α2β2γ2S, α3β2γ2S and α5β2γ2S GABAARs indicate that the observed clinical differences between clobazam and 1,4-benzodiazepines are likely to arise from factors other than their respective pharmacological properties at the GABAARs as investigated here. PMID:25798598

The GABAergic system contributes to the anxiolytic-like effect of essential oil from Cymbopogon citratus (lemongrass).

PubMed

Costa, Celso A Rodrigues de Almeida; Kohn, Daniele Oliveira; de Lima, Valéria Martins; Gargano, André Costa; Flório, Jorge Camilo; Costa, Mirtes

2011-09-01

The essential oil (EO) from Cymbopogon citratus (DC) Stapf is reported to have a wide range of biological activities and is widely used in traditional medicine as an infusion or decoction. However, despite this widely use, there are few controlled studies confirming its biological activity in central nervous system. The anxiolytic-like activity of the EO was investigated in light/dark box (LDB) and marble-burying test (MBT) and the antidepressant activity was investigated in forced-swimming test (FST) in mice. Flumazenil, a competitive antagonist of benzodiazepine binding and the selective 5-HT(1A) receptor antagonist WAY100635 was used in experimental procedures to determine the action mechanism of EO. To exclude any false positive results in experimental procedures, mice were submitted to the rota-rod test. We also quantified some neurotransmitters at specific brain regions after EO oral acute treatment. The present work found anxiolytic-like activity of the EO at the dose of 10mg/kg in a LDB. Flumazenil, but not WAY100635, was able to reverse the effect of the EO in the LDB, indicating that the EO activity occurs via the GABA(A) receptor-benzodiazepine complex. Only at higher doses did the EO potentiate diethyl-ether-induced sleeping time in mice. In the FST and MBT, EO showed no effect. Finally, the increase in time spent in the light chamber, demonstrated by concomitant treatment with ineffective doses of diazepam (DZP) and the EO, revealed a synergistic effect of the two compounds. The lack of activity after long-term treatment in the LDB test might be related to tolerance induction, even in the DZP-treated group. Furthermore, there were no significant differences between groups after either acute or repeated treatments with the EO in the rota-rod test. Neurochemical evaluation showed no amendments in neurotransmitter levels evaluated in cortex, striatum, pons, and hypothalamus. The results corroborate the use of Cymbopogon citratus in folk medicine and suggest that the anxiolytic-like effect of its EO is mediated by the GABA(A) receptor-benzodiazepine complex. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Residual effect of a 7-amino metabolite of clonazepam on GABAA receptor function in the nucleus reticularis thalami of the rat.

PubMed

Munakata, Mitsutoshi; Tsuchiya, Shigeru

2008-10-01

A considerable amount of 7-aminoclonazepam (ACZP), a major metabolite of clonazepam (CZP), is present in the brain during CZP treatment, yet the pharmacological properties of ACZP remain unknown. We investigated the effects of ACZP on the GABA(A) receptor-mediated currents (I(GABA)) in neurons from the nucleus reticularis thalami (NRT) of the rat, using a nystatin-perforated patch technique. Neurons in which CZP (10 nM) exerted prominent augmentation (>100% augmentation) of I(GABA), which comprised 32% of the neurons tested, were included for the analysis of ACZP. In these neurons, ACZP augmented I(GABA), which was blocked by 10 microM flumazenil, a benzodiazepine receptor (BZR) antagonist. The half-maximal effective concentration of ACZP was 124 nM, whereas that of CZP was 1.8 nM. The maximal enhancements induced by ACZP and CZP were 38% and 170%, respectively. In neurons from the ventrobasal complex of the thalamus, the effect of ACZP was negligible. Our results suggest that ACZP was a weak partial BZR agonist and that ACZP may competitively modify the effect of CZP, leading to clinical consequences for patients with high levels of ACZP.

Zolpidem reduces the blood oxygen level-dependent signal during visual system stimulation

PubMed Central

Licata, Stephanie C.; Lowen, Steven B.; Trksak, George H.; MacLean, Robert R.; Lukas, Scott E.

2011-01-01

Zolpidem is a short-acting imidazopyridine hypnotic that binds at the benzodiazepine binding site on specific GABAA receptors to enhance fast inhibitory neurotransmission. The behavioral and receptor pharmacology of zolpidem has been studied extensively, but little is known about its neuronal substrates in vivo. In the present within-subject, double-blind, and placebo-controlled study, blood oxygen level-dependent functional magnetic resonance imaging (BOLD fMRI) at 3 Tesla was used to assess the effects of zolpidem within the brain. Healthy participants (n=12) were scanned 60 minutes after acute oral administration of zolpidem (0, 5, 10, or 20 mg), and changes in BOLD signal were measured in the visual cortex during presentation of a flashing checkerboard. Heart rate and oxygen saturation were monitored continuously throughout the session. Zolpidem (10 and 20 mg) reduced the robust visual system activation produced by presentation of this stimulus, but had no effects on physiological activity during the fMRI scan. Zolpidem’s modulation of the BOLD signal within the visual cortex is consistent with the abundant distribution of GABAA receptors localized in this region, as well as previous studies showing a relationship between increased GABA-mediated neuronal inhibition and a reduction in BOLD activation. PMID:21640782

Evaluation of the anxiolytic and antidepressant effects of asiatic acid, a compound from Gotu kola or Centella asiatica, in the male Sprague Dawley rat.

PubMed

Ceremuga, Tomás Eduardo; Valdivieso, Debra; Kenner, Catherine; Lucia, Amy; Lathrop, Keith; Stailey, Owen; Bailey, Heather; Criss, Jonathan; Linton, Jessica; Fried, Jordan; Taylor, Andrew; Padron, Gina; Johnson, Arthur Don

2015-04-01

Herbal medication use continues to rise and interactions with existing medications propose risks and may have significant effects and consequences on the administration of anesthesia. The purpose of this study was to investigate the anxiolytic and antidepressant effects of asiatic acid and its potential modulation of the γ-aminobutyric acid (GABAA) receptor. Fifty-five male Sprague Dawley rats were divided into 5 groups: vehicle (DMSO), asiatic acid (AA), midazolam, or a combination of flumazenil + AA or midazolam + AA, and injected intraperitoneally 30 minutes prior to testing. The rats were tested on the Elevated Plus Maze (EPM) and the Forced Swim Test (FST). Data were analyzed using a two-tailed multivariate analysis of variance (MANOVA). Significance was found regarding the ratio of open arm time, maximum speed, and time spent mobile in the AA group and the midazolam + AA group (P < .05). Flumazenil decreased the anxiolytic effects, suggesting that AA modulates the benzodiazepine site on the GABAA receptor. Further studies are recommended to determine the efficacy of prolonged treatment for anxiety and depression.

Zolpidem, a clinical hypnotic that affects electronic transfer, alters synaptic activity through potential GABA receptors in the nervous system without significant free radical generation.

PubMed

Kovacic, Peter; Somanathan, Ratnasamy

2009-01-01

Zolpidem (trade name Ambien) has attracted much interest as a sleep-inducing agent and also in research. Attention has been centered mainly on receptor binding and electrochemistry in the central nervous system which are briefly addressed herein. A novel integrated approach to mode of action is presented. The pathways to be discussed involve basicity, reduction potential, electrostatics, cell signaling, GABA receptor binding, electron transfer (ET), pharmacodynamics, structure activity relationships (SAR) and side effects. The highly conjugated pyridinium salt formed by protonation of the amidine moiety is proposed to be the active form acting as an ET agent. Extrapolation of reduction potentials for related compounds supports the premise that zolpidem may act as an ET species in vivo. From recent literature reports, electrostatics is believed to play a significant role in drug action. The pyridinium cation displays molecular electrostatic potential which may well play a role energetically or as a bridging mechanism. An SAR analysis points to analogy with other physiologically active xenobiotics, namely benzodiazepines and paraquat in the conjugated iminium category. Inactivity of metabolites indicates that the parent is the active form of zolpidem. Absence of reactive oxygen species and oxidative stress is in line with minor side effects. In contrast, generally, the prior literature contains essentially no discussion of these fundamental biochemical relationships. Pharmacodynamics may play an important role. Concerning behavior at the blood-brain barrier, useful insight can be gained from investigations of the related cationic anesthetics that are structurally related to acetyl choline. Evidently, the neutral form of the drug penetrates the neuronal membrane, with the salt form operating at the receptor. The pathways of zolpidem have several clinical implications since the agent affects sedation, electroencephalographic activity, oxidative metabolites and receptors in the central nervous system. The drug acts at the GABA(A) receptor benzodiazepine site, displaying high and intermediate affinities to various receptor regions. Structural features for tight binding were determined. The sedative and anticonvulsant activities are due to its action on the alpha-1-GABA(A) receptors. One of the common adverse responses to zolpidem is hallucinations. Proposed mechanisms comprise changes in the GABA(A) receptor, pharmacodynamic interactions involving serotonin and neuronal-weak photon emission processes entailing redox phenomena. Reports cite cases of abuse with cravings based on anxiolytic and stimulating actions. It is important to recognize that insight concerning processes at the fundamental, molecular level can translate into beneficial results involving both positive and adverse side effects. In order for this to occur, interdisciplinary interaction is necessary. Suggestions are made for future research aimed at testing the various hypotheses.

Drug-induced modification of the system properties associated with spontaneous human electroencephalographic activity

NASA Astrophysics Data System (ADS)

Liley, David T.; Cadusch, Peter J.; Gray, Marcus; Nathan, Pradeep J.

2003-11-01

The benzodiazepine (BZ) class of minor tranquilizers are important modulators of the γ-amino butyric acid (GABAA)/BZ receptor complex that are well known to affect the spectral properties of spontaneous electroencephalographic activity. While it is experimentally well established that the BZs reduce total alpha band (8 13 Hz) power and increase total beta band (13 30 Hz) power, it is unclear what the physiological basis for this effect is. Based on a detailed theory of cortical electrorhythmogenesis it is conjectured that such an effect is explicable in terms of the modulation of GABAergic neurotransmission within locally connected populations of excitatory and inhibitory cortical neurons. Motivated by this theory, fixed order autoregressive moving average (ARMA) models were fitted to spontaneous eyes-closed electroencephalograms recorded from subjects before and approximately 2 h after the oral administration of a single 1 mg dose of the BZ alprazolam. Subsequent pole-zero analysis revealed that BZs significantly transform the dominant system pole such that its frequency and damping increase. Comparisons of ARMA derived power spectra with fast Fourier transform derived spectra indicate an enhanced ability to identify benzodiazepine induced electroencephalographic changes. This experimental result is in accord with the theoretical predictions implying that alprazolam enhances inhibition acting on inhibitory neurons more than inhibition acting on excitatory neurons. Further such a result is consistent with reported cortical neuronal distributions of the various GABAA receptor pharmacological subtypes. Therefore physiologically specified fixed order ARMA modeling is expected to become an important tool for the systematic investigation and modeling of a wide range of cortically acting compounds.

Inhibition of benzodiazepine binding in vitro by amentoflavone, a constituent of various species of Hypericum.

PubMed

Baureithel, K H; Büter, K B; Engesser, A; Burkard, W; Schaffner, W

1997-06-01

Flower extracts of Hypericum perforatum, Hypericum hirsutum, Hypericum patulum and Hypericum olympicum efficiently inhibited binding of [3H]flumazenil to rat brain benzodiazepine binding sites of the GABAA-receptor in vitro with IC50 values of 6.83, 6.97, 13.2 and 6.14 micrograms/ml, respectively. Single constituents of the extracts like hypericin, the flavones quercetin and luteolin, the glycosylated flavonoides rutin, hyperoside and quercitrin and the biflavone 13, II8-biapigenin did not inhibit binding up to concentrations of 1 microM. In contrast, amentoflavone revealed an IC50 = 14.9 +/- 1.9 nM on benzodiazepine binding in vitro. Comparative HPLC analyses of hypericin and amentoflavone in extracts of different Hypericum species revealed a possible correlation between the amentoflavone concentration and the inhibition of flumazenil binding. For hypericin no such correlation was observed. Our experimental data demonstrate that amentoflavone, in contrast to hypericin, presents a very active compound with regard to the inhibition of [3H]-flumazenil binding in vitro and thus might be involved in the antidepressant effects of Hypericum perforatum extracts.

Synthesis and Characterization of a Novel γ-Aminobutyric Acid Type A (GABAA) Receptor Ligand That Combines Outstanding Metabolic Stability, Pharmacokinetics, and Anxiolytic Efficacy.

PubMed

Poe, Michael M; Methuku, Kashi Reddy; Li, Guanguan; Verma, Ashwini R; Teske, Kelly A; Stafford, Douglas C; Arnold, Leggy A; Cramer, Jeffrey W; Jones, Timothy M; Cerne, Rok; Krambis, Michael J; Witkin, Jeffrey M; Jambrina, Enrique; Rehman, Sabah; Ernst, Margot; Cook, James M; Schkeryantz, Jeffrey M

2016-12-08

1,4-Benzodiazepines are used in the treatment of anxiety disorders but have limited long-term use due to adverse effects. HZ-166 (2) has been shown to have anxiolytic-like effects with reduced sedative/ataxic liabilities. A 1,3-oxazole KRM-II-81 (9) was discovered from a series of six bioisosteres with significantly improved pharmacokinetic and pharmacodynamic properties as compared to 2. Oxazole 9 was further characterized and exhibited improved anxiolytic-like effects in a mouse marble burying assay and a rat Vogel conflict test.

Anxiolytic-like actions of the hexane extract from leaves of Annona cherimolia in two anxiety paradigms: possible involvement of the GABA/benzodiazepine receptor complex.

PubMed

López-Rubalcava, C; Piña-Medina, B; Estrada-Reyes, R; Heinze, G; Martínez-Vázquez, M

2006-01-11

A hexane extract of leaves of Annona cherimolia produced anxiolytic-like actions when administered to mice and tested in two animal models of anxiety: the mouse avoidance exploratory behavior and the burying behavior tests. In order to discard unspecific drug-actions on general activity, all treatments studied in the anxiety paradigms were also analyzed in the open field test. Results showed that A. cherimolia induced anxiolytic-like actions at the doses of 6.25, 12.5, 25.0 and 50.0 mg/kg. Picrotoxin (0.25 mg/kg), a GABA-gated chloride ion channel blocker, antagonized the anxiolytic-like actions of A. cherimolia, while a sub-effective dose of muscimol (0.5 mg/kg), a selective GABA(A) receptor agonist, facilitated the effects of a sub-optimal dose of A. cherimolia (3.12 mg/kg). Thus, the involvement of the GABA(A) receptor complex in the anxiolytic-like actions of A. cherimolia hexane extract is suggested. In addition the extract was also able to enhance the duration of sodium pentobarbital induced sleeping time. Taken together, results indicate that the hexane extract of A. cherimolia has depressant activity on the Central Nervous System and could interact with the GABA(A) receptor complex. On the other hand, the chromatographic separation of this extract led to the isolation of palmitone, and beta-sitosterol as major constituents. In addition a GC-MS study of some fractions revealed the presence of several compounds such beta-cariophyllene, beta-selinene, alpha-cubebene, and linalool that have been reported to show effects on behavior that could explain some of the extract effects.

Clarified Açaí (Euterpe oleracea) Juice as an Anticonvulsant Agent: In Vitro Mechanistic Study of GABAergic Targets

PubMed Central

Arrifano, Gabriela P. F.; Lichtenstein, Mathieu P.; Souza-Monteiro, José Rogério; Rogez, Hervé

2018-01-01

Seizures affect about 50 million people around the world. Approximately 30% of seizures are refractory to the current pharmacological arsenal, so, the pursuit of new therapeutic alternatives is essential. Clarified Euterpe oleracea (EO) juice showed anticonvulsant properties similar to diazepam in an in vivo model with pentylenetetrazol, a GABAA receptor blocker. This study investigated the effects of EO on the main GABAergic targets for anticonvulsant drugs, analyzing the effect on the GABA receptor's benzodiazepine and picrotoxinin binding sites and the GABA uptake. Primary cultures of cortical neurons and astrocytes were treated with EO (0–25%) for up to 90 min. [3H]Flunitrazepam and [3H]TBOB binding, [3H]GABA uptake, cell viability, and morphology were assayed. Nonlethal concentrations of EO increased agonist binding and decreased antagonist binding in cortical neurons. Low concentrations significantly inhibited GABA uptake, especially in astrocytes, suggesting an accumulation of endogenous GABA in the synaptic cleft. The results demonstrate, for the first time, that EO can improve GABAergic neurotransmission via interactions with GABAA receptor and modulation of GABA uptake. Understanding these molecular mechanisms will help in the treatment of seizures and epilepsy, especially in developing countries where geographic isolation and low purchasing power are the main barriers to access to adequate treatment. PMID:29743978

Zolpidem reduces the blood oxygen level-dependent signal during visual system stimulation.

PubMed

Licata, Stephanie C; Lowen, Steven B; Trksak, George H; Maclean, Robert R; Lukas, Scott E

2011-08-15

Zolpidem is a short-acting imidazopyridine hypnotic that binds at the benzodiazepine binding site on specific GABA(A) receptors to enhance fast inhibitory neurotransmission. The behavioral and receptor pharmacology of zolpidem has been studied extensively, but little is known about its neuronal substrates in vivo. In the present within-subject, double-blind, and placebo-controlled study, blood oxygen level-dependent functional magnetic resonance imaging (BOLD fMRI) at 3 Tesla was used to assess the effects of zolpidem within the brain. Healthy participants (n=12) were scanned 60 min after acute oral administration of zolpidem (0, 5, 10, or 20mg), and changes in BOLD signal were measured in the visual cortex during presentation of a flashing checkerboard. Heart rate and oxygen saturation were monitored continuously throughout the session. Zolpidem (10 and 20mg) reduced the robust visual system activation produced by presentation of this stimulus, but had no effects on physiological activity during the fMRI scan. Zolpidem's modulation of the BOLD signal within the visual cortex is consistent with the abundant distribution of GABA(A) receptors localized in this region, as well as previous studies showing a relationship between increased GABA-mediated neuronal inhibition and a reduction in BOLD activation. Copyright © 2011 Elsevier Inc. All rights reserved.

BENZODIAZEPINE-INDUCED SPATIAL LEARNING DEFICITS IN RATS ARE REGULATED BY THE DEGREE OF MODULATION OF α1 GABAA RECEPTORS

PubMed Central

Joksimović, Srđan; Divljaković, Jovana; Van Linn, Michael L.; Varagic, Zdravko; Brajković, Gordana; Milinković, Marija M.; Yin, Wenyuan; Timić, Tamara; Sieghart, Werner; Cook, James M.; Savić, Miroslav M.

2012-01-01

Despite significant advances in understanding the role of benzodiazepine (BZ)-sensitive populations of GABAA receptors, containing the α1, α2, α3 or α5 subunit, factual substrates of BZ-induced learning and memory deficits are not yet fully elucidated. It was shown that α1-subunit affinity-selective antagonist β-CCt almost completely abolished spatial learning deficits induced by diazepam (DZP) in the Morris water maze. We examined a novel, highly (105 fold) α1-subunit selective ligand - WYS8 (0.2, 1 and 10 mg/kg), on its own and in combination with the non-selective agonist DZP (2 mg/kg) or β-CCt (5 mg/kg) in the water maze in rats. The in vitro efficacy study revealed that WYS8 acts as α1-subtype selective weak partial positive modulator (40% potentiation at 100 nM). Measurement of concentrations of WYS8 and DZP in rat serum and brain tissues suggested that they did not substantially cross-influence the respective disposition. In the water maze, DZP impaired spatial learning (acquisition trials) and memory (probe trial). WYS8 caused no effect per se, did not affect the overall influence of DZP on the water-maze performance and was devoid of any activity in this task when combined with β-CCt. Nonetheless, an additional analysis of the latency to reach the platform and the total distance swam suggested that WYS8 addition attenuated the run-down of the spatial impairment induced by DZP at the end of acquisition trials. These results demonstrate a clear difference in the influence of an α1 subtype-selective antagonist and a partial agonist on the effects of DZP on the water-maze acquisition. PMID:22633616

Seeking potential anticonvulsant agents that target GABAA receptors using experimental and theoretical procedures

NASA Astrophysics Data System (ADS)

Saavedra-Vélez, Margarita Virginia; Correa-Basurto, José; Matus, Myrna H.; Gasca-Pérez, Eloy; Bello, Martiniano; Cuevas-Hernández, Roberto; García-Rodríguez, Rosa Virginia; Trujillo-Ferrara, José; Ramos-Morales, Fernando Rafael

2014-12-01

The aim of this study was to identify compounds that possess anticonvulsant activity by using a pentylenetetrazol (PTZ)-induced seizure model. Theoretical studies of a set of ligands, explored the binding affinities of the ligands for the GABAA receptor (GABAAR), including some benzodiazepines. The ligands satisfy the Lipinski rules and contain a pharmacophore core that has been previously reported to be a GABAAR activator. To select the ligands with the best physicochemical properties, all of the compounds were analyzed by quantum mechanics and the energies of the highest occupied molecular orbital and lowest unoccupied molecular orbital were determined. Docking calculations between the ligands and the GABAAR were used to identify the complexes with the highest Gibbs binding energies. The identified compound D1 (dibenzo( b,f)(1,4)diazocine-6,11(5H,12H)-dione) was synthesized, experimentally tested, and the GABAAR-D1 complex was submitted to 12-ns-long molecular dynamics (MD) simulations to corroborate the binding conformation obtained by docking techniques. MD simulations were also used to analyze the decomposition of the Gibbs binding energy of the residues involved in the stabilization of the complex. To validate our theoretical results, molecular docking and MD simulations were also performed for three reference compounds that are currently in commercial use: clonazepam (CLZ), zolpidem and eszopiclone. The theoretical results show that the GABAAR-D1, and GABAAR-CLZ complexes bind to the benzodiazepine binding site, share a similar map of binding residues, and have similar Gibbs binding energies and entropic components. Experimental studies using a PTZ-induced seizure model showed that D1 possesses similar activity to CLZ, which corroborates the predicted binding free energy identified by theoretical calculations.

The novel antiepileptic drug imepitoin compares favourably to other GABA-mimetic drugs in a seizure threshold model in mice and dogs.

PubMed

Löscher, Wolfgang; Hoffmann, Katrin; Twele, Friederike; Potschka, Heidrun; Töllner, Kathrin

2013-11-01

Recently, the imidazolinone derivative imepitoin has been approved for treatment of canine epilepsy. Imepitoin acts as a low-affinity partial agonist at the benzodiazepine (BZD) site of the GABAA receptor and is the first compound with such mechanism that has been developed as an antiepileptic drug (AED). This mechanism offers several advantages compared to full agonists, including less severe adverse effects and a lack of tolerance and dependence liability, which has been demonstrated in rodents, dogs, and nonhuman primates. In clinical trials in epileptic dogs, imepitoin was shown to be an effective and safe AED. Recently, seizures in dogs have been proposed as a translational platform for human therapeutic trials on new epilepsy treatments. In the present study, we compared the anticonvulsant efficacy of imepitoin, phenobarbital and the high-affinity partial BZD agonist abecarnil in the timed i.v. pentylenetetrazole (PTZ) seizure threshold test in dogs and, for comparison, in mice. Furthermore, adverse effects of treatments were compared in both species. All drugs dose-dependently increased the PTZ threshold in both species, but anticonvulsant efficacy was higher in dogs than mice. At the doses selected for this study, imepitoin was slightly less potent than phenobarbital in increasing seizure threshold, but markedly more tolerable in both species. Effective doses of imepitoin in the PTZ seizure model were in the same range as those suppressing spontaneous recurrent seizures in epileptic dogs. The study demonstrates that low-affinity partial agonists at the benzodiazepine site of the GABAA receptor, such as imepitoin, offer advantages as a new category of AEDs. Copyright © 2013 Elsevier Ltd. All rights reserved.

Angiotensin II AT1 receptor blocker candesartan prevents the fast up-regulation of cerebrocortical benzodiazepine-1 receptors induced by acute inflammatory and restraint stress

PubMed Central

Sánchez-Lemus, Enrique; Honda, Masaru; Saavedra, Juan M.

2012-01-01

Centrally acting Angiotensin II AT1 receptor blockers (ARBs) protect from stress-induced disorders and decrease anxiety in a model of inflammatory stress, the systemic injection of bacterial endotoxin lipopolysaccharide (LPS). In order to better understand the anxiolytic effect of ARBs, we treated rats with LPS (50 µg/kg) with or without three days of pretreatment with the ARB candesartan (1 mg/kg/day), and studied cortical benzodiazepine (BZ) and corticotrophin-releasing factor (CRF) receptors. We compared the cortical BZ and CRF receptors expression pattern induced by LPS with that produced in restraint stress. Inflammation stress produced a generalized increase in cortical BZ1 receptors and reduced mRNA expression of the GABAA receptor γ2 subunit in cingulate cortex; changes were prevented by candesartan pretreatment. Moreover, restraint stress produced similar increases in cortical BZ1 receptor binding, and candesartan prevented these changes. Treatment with candesartan alone increased cortical BZ1 binding, and decreased γ2 subunit mRNA expression in the cingulate cortex. Conversely, we did not find changes in CRF1 receptor expression in any of the cortical areas studied, either after inflammation or restraint stress. Cortical CRF2 receptor binding was undetectable, but CRF2 mRNA expression was decreased by inflammation stress, a change prevented by candesartan. We conclude that stress promotes rapid and widespread changes in cortical BZ1 receptor expression; and that the stress-induced BZ1 receptor expression is under the control of AT1 receptor activity. The results suggest that the anti-anxiety effect of ARBs may be associated with their capacity to regulate stress-induced alterations in cortical BZ1 receptors. PMID:22503782

Antioxidant and neurosedative properties of polyphenols and iridoids from Lippia alba.

PubMed

Hennebelle, Thierry; Sahpaz, Sevser; Gressier, Bernard; Joseph, Henry; Bailleul, François

2008-02-01

The neurosedative and antioxidative properties of some major compounds isolated from a citral chemotype of Lippia alba were investigated. Binding assays were performed on two CNS inhibitory targets: benzodiazepine and GABA(A) receptors. The most active compound was luteolin-7-diglucuronide, with half maximal inhibitory concentrations (IC(50)) of 101 and 40 microm, respectively. Fifteen compounds isolated from Lippia alba were tested for their radical scavenging capacities against DPPH. Four of the major compounds (verbascoside, calceolarioside E, luteolin-7-diglucuronide and theveside) were also tested for their antioxidant activity against superoxide radical-anion in cell-free (hypoxanthine-xanthine oxidase) and cellular (PMA-stimulated neutrophil granulocytes) systems.

The neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one affects dopamine-mediated behavior in rodents.

PubMed

Khisti, Rahul T; Deshpande, Laxmikant S; Chopde, Chandrabhan T

2002-05-01

The neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) has been previously shown to induce catalepsy in mice that is modified by GABAergic, dopaminergic, adenosinergic and serotonergic agents. In light of the interaction of this endogenous neurosteroid with GABAergic and dopaminergic transmission, there is potential interest in the possible role of 3alpha,5alpha-THP in psychotic disorders. This study assessed the effect of 3alpha,5alpha-THP in certain dopamine-mediated behavioral paradigms that are widely used to predict antipsychotic-like activity. 3alpha,5alpha-THP (1-8 microg per animal, i.c.v.), the classic neuroleptic (dopamine receptor antagonist) haloperidol (0.25 mg/kg, i.p.), and the benzodiazepine diazepam (7 mg/kg, i.p.) were injected into different groups of animals, and their behavior was screened using the following animal tests: conditioned avoidance response, apomorphine-induced climbing, and amphetamine-induced motor hyperactivity. Separate groups of mice that received 3alpha,5alpha-THP (1-8 microg per animal, i.c.v.) were screened for catalepsy. Furthermore, the effect of a sub-cataleptic dose (0.1 microg per mouse, i.c.v.) of 3alpha,5alpha-THP, either alone or in combination with the GABA(A) receptor antagonist picrotoxin (0.8 mg/kg, i.p.) was measured on haloperidol-induced catalepsy. 3alpha,5alpha-THP like haloperidol reduced conditioned avoidance, apomorphine-induced cage climbing and amphetamine-induced motor hyperactivity. Diazepam only affected conditioned avoidance. 3alpha,5alpha-THP also induced dose-dependent catalepsy. Furthermore, sub-cataleptic doses of 3alpha,5alpha-THP potentiated haloperidol-induced catalepsy. This potentiation was blocked by prior treatment with the GABA(A) receptor antagonist picrotoxin. These findings suggest that 3alpha,5alpha-THP, by its action at the GABA(A) receptors, increases GABAergic tone leading to a behavioral profile similar to that of dopamine receptor antagonists.

Anxiolytic effect of Kami-Shoyo-San (TJ-24) in mice: possible mediation of neurosteroid synthesis.

PubMed

Mizowaki, M; Toriizuka, K; Hanawa, T

2001-09-21

We assessed the anxiolytic effect of Kami-Shoyo-San (Jia-wei-xiao-yao-san; TJ-24), one of a traditional Chinese herbal medicine used for the treatment of menopausal anxiety, by the social interaction (SI) test in male mice. Acute administration of TJ-24 (25-100 mg/kg, p.o.), as well as the gamma-amino-butyric acidA/benzodiazepine (GABA(A)/BZP) receptor agonist diazepam (1-3 mg/kg, i.p.), dose dependently increased the SI time, respectively. The GABA(A) receptor antagonist picrotoxin blocked the effects of TJ-24 and diazepam. TJ-24-induced SI behavior was significantly blocked by the GABA(A)/BZP receptor inverse agonist Ro 15-4513 and the GABA(A)/BZP receptor antagonist flumazenil. In addition, 5alpha-reductase inhibitor finasteride potently blocked the effect of TJ-24 without attenuating the basal level by itself. These findings suggest that TJ-24 shows the anxiolytic effect through the neurosteroid synthesis followed by GABA(A)/BDZ receptor stimulations.

The differential role of α1- and α5-containing GABAA receptors in mediating diazepam effects on spontaneous locomotor activity and water-maze learning and memory in rats

PubMed Central

Savić, Miroslav M.; Milinković, Marija M.; Rallapalli, Sundari; Clayton, Terry; Joksimović, Srðan; Van Linn, Michael; Cook, James M.

2009-01-01

The clinical use of benzodiazepines (BZs) is hampered by sedation and cognitive deterioration. Although genetic and pharmacological studies suggest that α1- and α5-containing GABAA receptors mediate and/or modulate these effects, their molecular substrate is not fully elucidated. By the use of two selective ligands : the α1-subunit affinity-selective antagonist β-CCt, and the α5-subunit affinity- and efficacy-selective antagonist XLi093, we examined the mechanisms of behavioural effects of diazepam in the tests of spontaneous locomotor activity and water-maze acquisition and recall, the two paradigms indicative of sedative- and cognition-impairing effects of BZs, respectively. The locomotor-activity decreasing propensity of diazepam (significant at 1.5 and 5 mg/kg) was antagonized by β-CCt (5 and 15 mg/kg), while it tended to be potentiated by XLi093 in doses of 10 mg/kg, and especially 20 mg/kg. Diazepam decreased acquisition and recall in the water maze, with a minimum effective dose of 1.5 mg/kg. Both antagonists reversed the thigmotaxis induced by 2 mg/kg diazepam throughout the test, suggesting that both GABAA receptor subtypes participate in BZ effects on the procedural component of the task. Diazepam-induced impairment in the declarative component of the task, as assessed by path efficiency, the latency and distance before finding the platform across acquisition trials, and also by the spatial parameters in the probe trial, was partially prevented by both, 15 mg/kg β-CCt and 10 mg/kg XLi093. Combining a BZ with β-CCt results in the near to control level of performance of a cognitive task, without sedation, and may be worth testing on human subjects. PMID:19265570

Women with premenstrual dysphoric disorder have altered sensitivity to allopregnanolone over the menstrual cycle compared to controls-a pilot study.

PubMed

Timby, Erika; Bäckström, Torbjörn; Nyberg, Sigrid; Stenlund, Hans; Wihlbäck, Anna-Carin N; Bixo, Marie

2016-06-01

In premenstrual dysphoric disorder (PMDD), a condition that afflicts 3-8 % of women in fertile ages, the cyclic recurrence of debilitating mood symptoms is restricted to the luteal phase of the menstrual cycle. The progesterone metabolite allopregnanolone is produced by the corpus luteum, and circulating levels are reflected in the brain. Allopregnanolone is a modulator of the GABAA receptor, enhancing the effect of γ-aminobutyric acid (GABA). Previous studies have demonstrated different sensitivity to other GABAA receptor agonists, i.e., benzodiazepines, alcohol, and pregnanolone, in PMDD patients compared to controls. This study aimed to investigate the sensitivity to intravenous allopregnanolone over the menstrual cycle in PMDD patients. Allopregnanolone, 0.05 mg/kg, was administered intravenously once in the mid-follicular and once in the luteal phase of the menstrual cycle to 10 PMDD patients and 10 control subjects. The saccadic eye velocity (SEV) was recorded by electrooculography as a measurement of functional GABAA receptor activity, at baseline and repeatedly after the injection. A mixed model was used to analyze data. There was a highly significant group × phase interaction in the SEV response to allopregnanolone (F(1,327.489) = 12.747, p < 0.001). In the PMDD group, the SEV response was decreased in the follicular phase compared to the luteal phase (F(1,168) = 7.776, p = 0.006), whereas in the control group, the difference was opposite during the menstrual cycle (F(1,158.45) = 5.70, p = 0.018). The effect of exogenous allopregnanolone is associated with menstrual cycle phase in PMDD patients and in controls. The results suggest an altered sensitivity to allopregnanolone in PMDD patients.

Genetic and radiation hybrid mapping of the hyperekplexia region on chromosome 5q.

PubMed Central

Ryan, S G; Dixon, M J; Nigro, M A; Kelts, K A; Markand, O N; Terry, J C; Shiang, R; Wasmuth, J J; O'Connell, P

1992-01-01

Hyperekplexia, or startle disease (STHE), is an autosomal dominant neurologic disorder characterized by muscular rigidity of central nervous system origin, particularly in the neonatal period, and by an exaggerated startle response to sudden, unexpected acoustic or tactile stimuli. STHE responds dramatically to the benzodiazepine drug clonazepam, which acts at gamma-aminobutyric acid type A (GABA-A) receptors. The STHE locus (STHE) was recently assigned to chromosome 5q, on the basis of tight linkage to the colony-stimulating factor 1-receptor (CSF1-R) locus in a single large family. We performed linkage analysis in the original and three additional STHE pedigrees with eight chromosome 5q microsatellite markers and placed several of the most closely linked markers on an existing radiation hybrid (RH) map of the region. The results provide strong evidence for genetic locus homogeneity and assign STHE to a 5.9-cM interval defined by CSF1-R and D5S379, which are separated by an RH map distance of 74 centirays (roughly 2.2-3.7 Mb). Two polymorphic markers (D5S119 and D5S209) lie within this region, but they could not be ordered with respect to STHE. RH mapping eliminated the candidate genes GABRA1 and GABRG2, which encode GABA-A receptor components, by showing that they are telomeric to the target region. PMID:1334371

Enhancement of GABA release through endogenous activation of axonal GABA(A) receptors in juvenile cerebellum.

PubMed

Trigo, Federico F; Chat, Mireille; Marty, Alain

2007-11-14

Recent evidence indicates the presence of presynaptic GABA(A) receptors (GABA(A)Rs) in the axon domain of several classes of central neurons, including cerebellar basket and stellate cells. Here, we investigate the possibility that these receptors could be activated in the absence of electrical or chemical stimulation. We find that low concentrations of GABA increase the frequency of miniature GABAergic synaptic currents. Submaximal concentrations of a GABA(A)R blocker, gabazine, decrease both the miniature current frequency and the probability of evoked GABA release. Zolpidem, an agonist of the benzodiazepine binding site, and NO-711 (1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride), a blocker of GABA uptake, both increase the frequency of miniature currents. These effects occur up to postnatal day 14, but not later. Immunohistochemistry indicates the presence of alpha1-containing GABA(A)Rs in interneuron presynaptic terminals with a similar age dependence. We conclude that, under resting conditions, axonal GABA(A)Rs are significantly activated, that this activation results in enhanced GABA release, and that it can be augmented by increasing the affinity of GABA(A)Rs or reducing GABA uptake. Our findings suggest the existence of a positive-feedback mechanism involving presynaptic GABA(A)Rs that maintains a high release rate and a high local GABA concentration in the immature cerebellar network.

GABAA-current rundown of temporal lobe epilepsy is associated with repetitive activation of GABAA “phasic” receptors

PubMed Central

Palma, Eleonora; Roseti, Cristina; Maiolino, Francesca; Fucile, Sergio; Martinello, Katiuscia; Mazzuferi, Manuela; Aronica, Eleonora; Manfredi, Mario; Esposito, Vincenzo; Cantore, Gianpaolo; Miledi, Ricardo; Simonato, Michele; Eusebi, Fabrizio

2007-01-01

A study was made of the “rundown” of GABAA receptors, microtransplanted to Xenopus oocytes from surgically resected brain tissues of patients afflicted with drug-resistant human mesial temporal lobe epilepsy (mTLE). Cell membranes, isolated from mTLE neocortex specimens, were injected into frog oocytes that rapidly incorporated functional GABAA receptors. Upon repetitive activation with GABA (1 mM), “epileptic” GABAA receptors exhibited a GABAA-current (IGABA) rundown that was significantly enhanced by Zn2+ (≤250 μM), and practically abolished by the high-affinity GABAA receptor inverse agonist SR95531 (gabazine; 2.5–25 μM). Conversely, IGABA generated by “control” GABAA receptors microtransplanted from nonepileptic temporal lobe, lesional TLE, or authoptic disease-free tissues remained stable during repetitive stimulation, even in oocytes treated with Zn2+. We conclude that rundown of mTLE epileptic receptors depends on the presence of “phasic GABAA receptors” that have low sensitivity to antagonism by Zn2+. Additionally, we found that GABAA receptors, microtransplanted from the cerebral cortex of adult rats exhibiting recurrent seizures, caused by pilocarpine-induced status epilepticus, showed greater rundown than control tissue, an event also occurring in patch-clamped rat pyramidal neurons. Rundown of epileptic rat receptors resembled that of human mTLE receptors, being enhanced by Zn2+ (40 μM) and sensitive to the antiepileptic agent levetiracetam, the neurotrophin brain-derived neurotrophic factor, and the phosphatase blocker okadaic acid. Our findings point to the rundown of GABAA receptors as a hallmark of TLE and suggest that modulating tonic and phasic mTLE GABAA receptor activity may represent a useful therapeutic approach to the disease. PMID:18083839

Regulated lysosomal trafficking as a mechanism for regulating GABAA receptor abundance at synapses in Caenorhabditis elegans.

PubMed

Davis, Kathleen M; Sturt, Brianne L; Friedmann, Andrew J; Richmond, Janet E; Bessereau, Jean-Louis; Grant, Barth D; Bamber, Bruce A

2010-08-01

GABA(A) receptor plasticity is important for both normal brain function and disease progression. We are studying GABA(A) receptor plasticity in Caenorhabditis elegans using a genetic approach. Acute exposure of worms to the GABA(A) agonist muscimol hyperpolarizes postsynaptic cells, causing paralysis. Worms adapt after several hours, but show uncoordinated locomotion consistent with decreased GABA signaling. Using patch-clamp and immunofluorescence approaches, we show that GABA(A) receptors are selectively removed from synapses during adaptation. Subunit mRNA levels were unchanged, suggesting a post-transcriptional mechanism. Mutants with defective lysosome function (cup-5) show elevated GABA(A) receptor levels at synapses prior to muscimol exposure. During adaptation, these receptors are removed more slowly, and accumulate in intracellular organelles positive for the late endosome marker GFP-RAB-7. These findings suggest that chronic agonist exposure increases endocytosis and lysosomal trafficking of GABA(A) receptors, leading to reduced levels of synaptic GABA(A) receptors and reduced postsynaptic GABA sensitivity.

Effects of phenazepam on the behavior of C57BL/6 and BALB/c mice in the open field test after naloxone pretreatment.

PubMed

Seredenin, S B; Nadorova, A V; Kolik, L G; Yarkova, M A

2013-07-01

We studied the effects of phenazepam (0.075 mg/kg) after pretreatment (5 minutes before) with naloxone (10 mg/kg) on open-field behavior of C57Bl/6 and BALB/c mice. In ex vivo experiments, we studied the effects of naloxone (1 and 10 mg/kg) on receptor binding of [(3)H]-flunitrazepam by membranes of brain fraction (P1+P2) of C57Bl/6 and BALB/c mice. It was shown that naloxone increased motor activity in the open field in BALB/c mice and decreased this parameter in C57Bl/6 mice. During combined treatment, naloxone potentiated the activating effects of phenazepam on the open-field behavior of BALB/c mice and slightly increased the sedative effect of this drug in C57Bl/6 mice. Naloxone stimulated reception of [(3)H]-flunitrazepam in BALB/c mice and slightly increased radioligand binding in C57Bl/6 mice. These data attest to enhanced reception in benzodiazepine site of GABAA-receptor under conditions of opioid receptor blockade, the presence of anxiolytic or sedative (depending on the phenotype of the response to emotional stress) effect of naloxone, and co-directed effects of naloxone and benzodiazepine tranquilizer on open-field behavior of C57Bl/6 and BALB/c mice.

Anxiety and Depression: Mouse Genetics and Pharmacological Approaches to the Role of GABAA Receptor Subtypes

PubMed Central

Smith, Kiersten S.; Rudolph, Uwe

2012-01-01

GABAA receptors mediate fast synaptic inhibitory neurotransmission throughout the central nervous system. Recent work indicates a role for GABAA receptors in physiologically modulating anxiety and depression levels. In this review, we summarize research that led to the identification of the essential role of GABAA receptors in counteracting trait anxiety and depression-related behaviors, and research aimed at identifying individual GABAA receptor subtypes involved in physiological and pharmacological modulation of emotions. PMID:21810433

Density and distribution of hippocampal neurotransmitter receptors in autism: an autoradiographic study.

PubMed

Blatt, G J; Fitzgerald, C M; Guptill, J T; Booker, A B; Kemper, T L; Bauman, M L

2001-12-01

Neuropathological studies in autistic brains have shown small neuronal size and increased cell packing density in a variety of limbic system structures including the hippocampus, a change consistent with curtailment of normal development. Based on these observations in the hippocampus, a series of quantitative receptor autoradiographic studies were undertaken to determine the density and distribution of eight types of neurotransmitter receptors from four neurotransmitter systems (GABAergic, serotoninergic [5-HT], cholinergic, and glutamatergic). Data from these single concentration ligand binding studies indicate that the GABAergic receptor system (3[H]-flunitrazepam labeled benzodiazepine binding sites and 3[H]-muscimol labeled GABA(A) receptors) is significantly reduced in high binding regions, marking for the first time an abnormality in the GABA system in autism. In contrast, the density and distribution of the other six receptors studied (3[H]-80H-DPAT labeled 5-HT1A receptors, 3[H]-ketanserin labeled 5-HT2 receptors, 3[H]-pirenzepine labled M1 receptors, 3[H]-hemicholinium labeled high affinity choline uptake sites, 3[H]-MK801 labeled NMDA receptors, and 3[H]-kainate labeled kainate receptors) in the hippocampus did not demonstrate any statistically significant differences in binding.

Association between alcoholism and the genetic polymorphisms of the GABAA receptor genes on chromosome 5q33-34 in Korean population.

PubMed

Park, Chul-Soo; Park, So-Young; Lee, Chul-Soon; Sohn, Jin-Wook; Hahn, Gyu-Hee; Kim, Bong-Jo

2006-06-01

Family, twin, and adoption studies have demonstrated that genes play an important role in the development of alcoholism. We investigated the association between alcoholism and the genetic polymorphisms of the GABAA receptor genes on chromosome 5q33-34 in Korean population. The genotype of the GABAA receptor gene polymorphisms were determined by performing polymerase chain reaction genotyping for 172 normal controls and 162 male alcoholics who are hospitalized in alcoholism treatment institute. We found a significant association between the genetic polymorphisms of the GABAA alpha1 and GABAA alpha6 receptor gene and alcoholism. The GG genotype of the GABAA alpha1 receptor gene was associated with the onset age of alcoholism and alcohol withdrawal symptoms, and a high score on the Korean version of the ADS. However, there was no association between the genetic polymorphisms of the GABAA beta2 and gamma2 receptor gene and alcoholisms. Our finding suggest that genetic polymorphisms of the GABAA alpha1 and GABAA alpha6 receptor gene may be associated with the development of alcoholism and that the GG genotype of the GABAA alpha1 receptor gene play an important role in the development of the early onset and the severe type of alcoholism.

Interneuron- and GABAA receptor-specific inhibitory synaptic plasticity in cerebellar Purkinje cells

NASA Astrophysics Data System (ADS)

He, Qionger; Duguid, Ian; Clark, Beverley; Panzanelli, Patrizia; Patel, Bijal; Thomas, Philip; Fritschy, Jean-Marc; Smart, Trevor G.

2015-07-01

Inhibitory synaptic plasticity is important for shaping both neuronal excitability and network activity. Here we investigate the input and GABAA receptor subunit specificity of inhibitory synaptic plasticity by studying cerebellar interneuron-Purkinje cell (PC) synapses. Depolarizing PCs initiated a long-lasting increase in GABA-mediated synaptic currents. By stimulating individual interneurons, this plasticity was observed at somatodendritic basket cell synapses, but not at distal dendritic stellate cell synapses. Basket cell synapses predominantly express β2-subunit-containing GABAA receptors; deletion of the β2-subunit ablates this plasticity, demonstrating its reliance on GABAA receptor subunit composition. The increase in synaptic currents is dependent upon an increase in newly synthesized cell surface synaptic GABAA receptors and is abolished by preventing CaMKII phosphorylation of GABAA receptors. Our results reveal a novel GABAA receptor subunit- and input-specific form of inhibitory synaptic plasticity that regulates the temporal firing pattern of the principal output cells of the cerebellum.

Tobacco smoking interferes with GABAA receptor neuroadaptations during prolonged alcohol withdrawal

PubMed Central

Cosgrove, Kelly P.; McKay, Reese; Esterlis, Irina; Kloczynski, Tracy; Perkins, Evgenia; Bois, Frederic; Pittman, Brian; Lancaster, Jack; Glahn, David C.; O’Malley, Stephanie; Carson, Richard E.; Krystal, John H.

2014-01-01

Understanding the effects of tobacco smoking on neuroadaptations in GABAA receptor levels over alcohol withdrawal will provide critical insights for the treatment of comorbid alcohol and nicotine dependence. We conducted parallel studies in human subjects and nonhuman primates to investigate the differential effects of tobacco smoking and nicotine on changes in GABAA receptor availability during acute and prolonged alcohol withdrawal. We report that alcohol withdrawal with or without concurrent tobacco smoking/nicotine consumption resulted in significant and robust elevations in GABAA receptor levels over the first week of withdrawal. Over prolonged withdrawal, GABAA receptors returned to control levels in alcohol-dependent nonsmokers, but alcohol-dependent smokers had significant and sustained elevations in GABAA receptors that were associated with craving for alcohol and cigarettes. In nonhuman primates, GABAA receptor levels normalized by 1 mo of abstinence in both groups—that is, those that consumed alcohol alone or the combination of alcohol and nicotine. These data suggest that constituents in tobacco smoke other than nicotine block the recovery of GABAA receptor systems during sustained alcohol abstinence, contributing to alcohol relapse and the perpetuation of smoking. PMID:25453062

Role of GABAA receptors in the physiology and pharmacology of sleep.

PubMed

Winsky-Sommerer, Raphaëlle

2009-05-01

Most sedative-hypnotics used in insomnia treatment target the gamma-aminobutyric acid (GABA)(A) receptors. A vast repertoire of GABA(A) receptor subtypes has been identified and displays specific electrophysiological and functional properties. GABA(A)-mediated inhibition traditionally refers to 'phasic' inhibition, arising from synaptic GABA(A) receptors which transiently inhibit neurons. However, there is growing evidence that peri- or extra-synaptic GABA(A) receptors are continuously activated by low GABA concentrations and mediate a 'tonic' conductance. This slower type of signaling appears to play a key role in controlling cell excitability. This review aims at summarizing recent knowledge on GABA transmission, including the emergence of tonic conductance, and highlighting the importance of GABA(A) receptor heterogeneity. The mechanism of action of sedative-hypnotic drugs and their effects on sleep and the electroencephalogram will be reported. Furthermore, studies using genetically engineered mice will be emphasized, providing insights into the role of GABA(A) receptors in mechanisms underlying physiological and pharmacological sleep. Finally, we will address the potential of GABA(A) receptor pharmacology for the treatment of insomnia.

Extrasynaptic GABAA receptors in rat pontine reticular formation increase wakefulness.

PubMed

Vanini, Giancarlo; Baghdoyan, Helen A

2013-03-01

Gamma-aminobutyric acid (GABA) causes phasic inhibition via synaptic GABAA receptors and tonic inhibition via extrasynaptic GABAA receptors. GABA levels in the extracellular space regulate arousal state and cognition by volume transmission via extrasynaptic GABAA receptors. GABAergic transmission in the pontine reticular formation promotes wakefulness. No previous studies have determined whether an agonist at extrasynaptic GABAA receptors administered into the pontine reticular formation alters sleep and wakefulness. Therefore, this study used gaboxadol (THIP; agonist at extrasynaptic GABAA receptors that contain a δ subunit) to test the hypothesis that extrasynaptic GABAA receptors within the pontine reticular formation modulate sleep and wakefulness. Within/between subjects. University of Michigan. Adult male Crl:CD*(SD) (Sprague-Dawley) rats (n = 10). Microinjection of gaboxadol, the nonsubtype selective GABAA receptor agonist muscimol (positive control), and saline (negative control) into the rostral pontine reticular formation. Gaboxadol significantly increased wakefulness and decreased both nonrapid eye movement sleep and rapid eye movement sleep in a concentration-dependent manner. Relative to saline, gaboxadol did not alter electroencephalogram power. Microinjection of muscimol into the pontine reticular formation of the same rats that received gaboxadol increased wakefulness and decreased sleep. Tonic inhibition via extrasynaptic GABAA receptors that contain a δ subunit may be one mechanism by which the extracellular pool of endogenous GABA in the rostral pontine reticular formation promotes wakefulness. Vanini G; Baghdoyan HA. Extrasynaptic GABAA receptors in rat pontine reticular formation increase wakefulness. SLEEP 2013;36(3):337-343.

Increased GABA-A receptor binding and reduced connectivity at the motor cortex in children with hemiplegic cerebral palsy: a multimodal investigation using 18F-fluoroflumazenil PET, immunohistochemistry, and MR imaging.

PubMed

Park, Hae-Jeong; Kim, Chul Hoon; Park, Eun Sook; Park, Bumhee; Oh, So Ra; Oh, Maeng-Keun; Park, Chang Il; Lee, Jong Doo

2013-08-01

γ-aminobutyric acid (GABA)-A receptor-mediated neural transmission is important to promote practice-dependent plasticity after brain injury. This study investigated alterations in GABA-A receptor binding and functional and anatomic connectivity within the motor cortex in children with cerebral palsy (CP). We conducted (18)F-fluoroflumazenil PET on children with hemiplegic CP to investigate whether in vivo GABA-A receptor binding is altered in the ipsilateral or contralateral hemisphere of the lesion site. To evaluate changes in the GABA-A receptor subunit after prenatal brain injury, we performed GABA-A receptor immunohistochemistry using rat pups with a diffuse hypoxic ischemic insult. We also performed diffusion tensor MR imaging and resting-state functional MR imaging on the same children with hemiplegic CP to investigate alterations in anatomic and functional connectivity at the motor cortex with increased GABA-A receptor binding. In children with hemiplegic CP, the (18)F-fluoroflumazenil binding potential was increased within the ipsilateral motor cortex. GABA-A receptors with the α1 subunit were highly expressed exclusively within cortical layers III, IV, and VI of the motor cortex in rat pups. The motor cortex with increased GABA-A receptor binding in children with hemiplegic CP had reduced thalamocortical and corticocortical connectivity, which might be linked to increased GABA-A receptor distribution in cortical layers in rats. Increased expression of the GABA-A receptor α1 subunit within the ipsilateral motor cortex may be an important adaptive mechanism after prenatal brain injury in children with CP but may be associated with improper functional connectivity after birth and have adverse effects on the development of motor plasticity.

Context-Dependent Modulation of GABAAR-Mediated Tonic Currents.

PubMed

Patel, Bijal; Bright, Damian P; Mortensen, Martin; Frølund, Bente; Smart, Trevor G

2016-01-13

Tonic GABA currents mediated by high-affinity extrasynaptic GABAA receptors, are increasingly recognized as important regulators of cell and neuronal network excitability. Dysfunctional GABAA receptor signaling that results in modified tonic GABA currents is associated with a number of neurological disorders. Consequently, developing compounds to selectively modulate the activity of extrasynaptic GABAA receptors underlying tonic inhibition is likely to prove therapeutically useful. Here, we examine the GABAA receptor subtype selectivity of the weak partial agonist, 5-(4-piperidyl)isoxazol-3-ol (4-PIOL), as a potential mechanism for modulating extrasynaptic GABAA receptor-mediated tonic currents. By using recombinant GABAA receptors expressed in HEK293 cells, and native GABAA receptors of cerebellar granule cells, hippocampal neurons, and thalamic relay neurons, 4-PIOL evidently displayed differential agonist and antagonist-type profiles, depending on the extrasynaptic GABAA receptor isoforms targeted. For neurons, this resulted in differential modulation of GABA tonic currents, depending on the cell type studied, their respective GABAA receptor subunit compositions, and critically, on the ambient GABA levels. Unexpectedly, 4-PIOL revealed a significant population of relatively low-affinity γ2 subunit-containing GABAA receptors in the thalamus, which can contribute to tonic inhibition under specific conditions when GABA levels are raised. Together, these data indicate that partial agonists, such as 4-PIOL, may be useful for modulating GABAA receptor-mediated tonic currents, but the direction and extent of this modulation is strongly dependent on relative expression levels of different extrasynaptic GABAA receptor subtypes, and on the ambient GABA levels. A background level of inhibition (tonic) is important in the brain for controlling neuronal excitability. Increased levels of tonic inhibition are associated with some neurological disorders but there are no specific ligands capable of selectively reducing tonic inhibition. Here we explore the use of a GABA partial agonist as a selective chemical tool in three different brain regions. We discover that the activity of a partial agonist is heavily dependent upon the GABAA receptor subunit composition underpinning tonic inhibition, and on the ambient levels of GABA in the brain. Copyright © 2016 Patel et al.

Context-Dependent Modulation of GABAAR-Mediated Tonic Currents

PubMed Central

Patel, Bijal; Bright, Damian P.; Mortensen, Martin; Frølund, Bente

2016-01-01

Tonic GABA currents mediated by high-affinity extrasynaptic GABAA receptors, are increasingly recognized as important regulators of cell and neuronal network excitability. Dysfunctional GABAA receptor signaling that results in modified tonic GABA currents is associated with a number of neurological disorders. Consequently, developing compounds to selectively modulate the activity of extrasynaptic GABAA receptors underlying tonic inhibition is likely to prove therapeutically useful. Here, we examine the GABAA receptor subtype selectivity of the weak partial agonist, 5-(4-piperidyl)isoxazol-3-ol (4-PIOL), as a potential mechanism for modulating extrasynaptic GABAA receptor-mediated tonic currents. By using recombinant GABAA receptors expressed in HEK293 cells, and native GABAA receptors of cerebellar granule cells, hippocampal neurons, and thalamic relay neurons, 4-PIOL evidently displayed differential agonist and antagonist-type profiles, depending on the extrasynaptic GABAA receptor isoforms targeted. For neurons, this resulted in differential modulation of GABA tonic currents, depending on the cell type studied, their respective GABAA receptor subunit compositions, and critically, on the ambient GABA levels. Unexpectedly, 4-PIOL revealed a significant population of relatively low-affinity γ2 subunit-containing GABAA receptors in the thalamus, which can contribute to tonic inhibition under specific conditions when GABA levels are raised. Together, these data indicate that partial agonists, such as 4-PIOL, may be useful for modulating GABAA receptor-mediated tonic currents, but the direction and extent of this modulation is strongly dependent on relative expression levels of different extrasynaptic GABAA receptor subtypes, and on the ambient GABA levels. SIGNIFICANCE STATEMENT A background level of inhibition (tonic) is important in the brain for controlling neuronal excitability. Increased levels of tonic inhibition are associated with some neurological disorders but there are no specific ligands capable of selectively reducing tonic inhibition. Here we explore the use of a GABA partial agonist as a selective chemical tool in three different brain regions. We discover that the activity of a partial agonist is heavily dependent upon the GABAA receptor subunit composition underpinning tonic inhibition, and on the ambient levels of GABA in the brain. PMID:26758848

Muscarinic Long-Term Enhancement of Tonic and Phasic GABAA Inhibition in Rat CA1 Pyramidal Neurons

PubMed Central

Domínguez, Soledad; Fernández de Sevilla, David; Buño, Washington

2016-01-01

Acetylcholine (ACh) regulates network operation in the hippocampus by controlling excitation and inhibition in rat CA1 pyramidal neurons (PCs), the latter through gamma-aminobutyric acid type-A receptors (GABAARs). Although, the enhancing effects of ACh on GABAARs have been reported (Dominguez et al., 2014, 2015), its role in regulating tonic GABAA inhibition has not been explored in depth. Therefore, we aimed at determining the effects of the activation of ACh receptors on responses mediated by synaptic and extrasynaptic GABAARs. Here, we show that under blockade of ionotropic glutamate receptors ACh, acting through muscarinic type 1 receptors, paired with post-synaptic depolarization induced a long-term enhancement of tonic GABAA currents (tGABAA) and puff-evoked GABAA currents (pGABAA). ACh combined with depolarization also potentiated IPSCs (i.e., phasic inhibition) in the same PCs, without signs of interactions of synaptic responses with pGABAA and tGABAA, suggesting the contribution of two different GABAA receptor pools. The long-term enhancement of GABAA currents and IPSCs reduced the excitability of PCs, possibly regulating plasticity and learning in behaving animals. PMID:27833531

GABA-BZD Receptor Modulating Mechanism of Panax quinquefolius against 72-h Sleep Deprivation Induced Anxiety like Behavior: Possible Roles of Oxidative Stress, Mitochondrial Dysfunction and Neuroinflammation

PubMed Central

Chanana, Priyanka; Kumar, Anil

2016-01-01

Rationale: Panax quinquefolius (American Ginseng) is known for its therapeutic potential against various neurological disorders, but its plausible mechanism of action still remains undeciphered. GABA (Gamma Amino Butyric Acid) plays an important role in sleep wake cycle homeostasis. Thus, there exists rationale in exploring the GABA-ergic potential of Panax quinquefolius as neuroprotective strategy in sleep deprivation induced secondary neurological problems. Objective: The present study was designed to explore the possible GABA-ergic mechanism in the neuro-protective effect of Panax quinquefolius against 72-h sleep deprivation induced anxiety like behavior, oxidative stress, mitochondrial dysfunction, HPA-axis activation and neuroinflammation. Materials and Methods: Male laca mice were sleep deprived for 72-h by using Grid suspended over water method. Panax quinquefolius (American Ginseng 50, 100, and 200 mg/kg) was administered alone and in combination with GABA modulators (GABA Cl− channel inhibitor, GABA-benzodiazepine receptor inhibitor and GABAA agonist) for 8 days, starting 5 days prior to 72-h sleep deprivation period. Various behavioral (locomotor activity, mirror chamber test), biochemical (lipid peroxidation, reduced glutathione, catalase, nitrite levels), mitochondrial complexes, neuroinflammation marker (Tumor Necrosis Factor, TNF-alpha), serum corticosterone, and histopathological sections of brains were assessed. Results: Seventy two hours sleep deprivation significantly impaired locomotor activity, caused anxiety-like behavior, conditions of oxidative stress, alterations in mitochondrial enzyme complex activities, raised serum corticosterone levels, brain TNFα levels and led to neuroinflammation like signs in discrete brain areas as compared to naive group. Panax quinquefolius (100 and 200 mg/kg) treatment restored the behavioral, biochemical, mitochondrial, molecular and histopathological alterations. Pre-treatment of GABA Cl− channel inhibitor as well as GABA-benzodiazepine receptor inhibitor, significantly reversed the protective effect of P. quinquefolius (100 mg/kg) in 72-h sleep deprived animals (P < 0.05). However, pretreatment with GABAA agonist, potentiated Panax quinquefolius's protective effect which was significant as compared to their effect per se (p < 0.05). Conclusion: GABA-ergic mechanism could be involved in the neuroprotective effect of P.quinquefolius against sleep deprivation induced anxiety-like behavior, oxidative stress, mitochondrial dysfunction, HPA axis activation and neuroinflammation. PMID:27013946

Extrasynaptic GABAA Receptors in Rat Pontine Reticular Formation Increase Wakefulness

PubMed Central

Vanini, Giancarlo; Baghdoyan, Helen A.

2013-01-01

Study Objectives: Gamma-aminobutyric acid (GABA) causes phasic inhibition via synaptic GABAA receptors and tonic inhibition via extrasynaptic GABAA receptors. GABA levels in the extracellular space regulate arousal state and cognition by volume transmission via extrasynaptic GABAA receptors. GABAergic transmission in the pontine reticular formation promotes wakefulness. No previous studies have determined whether an agonist at extrasynaptic GABAA receptors administered into the pontine reticular formation alters sleep and wakefulness. Therefore, this study used gaboxadol (THIP; agonist at extrasynaptic GABAA receptors that contain a δ subunit) to test the hypothesis that extrasynaptic GABAA receptors within the pontine reticular formation modulate sleep and wakefulness. Design: Within/between subjects. Setting: University of Michigan. Patients or Participants: Adult male Crl:CD*(SD) (Sprague-Dawley) rats (n = 10). Interventions: Microinjection of gaboxadol, the nonsubtype selective GABAA receptor agonist muscimol (positive control), and saline (negative control) into the rostral pontine reticular formation. Measurements and Results: Gaboxadol significantly increased wakefulness and decreased both nonrapid eye movement sleep and rapid eye movement sleep in a concentration-dependent manner. Relative to saline, gaboxadol did not alter electroencephalogram power. Microinjection of muscimol into the pontine reticular formation of the same rats that received gaboxadol increased wakefulness and decreased sleep. Conclusion: Tonic inhibition via extrasynaptic GABAA receptors that contain a δ subunit may be one mechanism by which the extracellular pool of endogenous GABA in the rostral pontine reticular formation promotes wakefulness. Citation: Vanini G; Baghdoyan HA. Extrasynaptic GABAA receptors in rat pontine reticular formation increase wakefulness. SLEEP 2013;36(3):337-343. PMID:23450652

The combination of huperzine A and imidazenil is an effective strategy to prevent diisopropyl fluorophosphate toxicity in mice.

PubMed

Pibiri, Fabio; Kozikowski, Alan P; Pinna, Graziano; Auta, James; Kadriu, Bashkim; Costa, Erminio; Guidotti, Alessandro

2008-09-16

Diisopropyl fluorophosphate (DFP) causes neurotoxicity related to an irreversible inhibition of acetylcholinesterase (AChE). Management of this intoxication includes: (i) pretreatment with reversible blockers of AChE, (ii) blockade of muscarinic receptors with atropine, and (iii) facilitation of GABA(A) receptor signal transduction by benzodiazepines. The major disadvantage associated with this treatment combination is that it must to be repeated frequently and, in some cases, protractedly. Also, the use of diazepam (DZP) and congeners includes unwanted side effects, including sedation, amnesia, cardiorespiratory depression, and anticonvulsive tolerance. To avoid these treatment complications but safely protect against DFP-induced seizures and other CNS toxicity, we adopted the strategy of administering mice with (i) small doses of huperzine A (HUP), a reversible and long-lasting (half-life approximately 5 h) inhibitor of AChE, and (ii) imidazenil (IMI), a potent positive allosteric modulator of GABA action selective for alpha(5)-containing GABA(A) receptors. Coadministration of HUP (50 microg/kg s.c., 15 min before DFP) with IMI (2 mg/kg s.c., 30 min before DFP) prevents DFP-induced convulsions and the associated neuronal damage and mortality, allowing complete recovery within 18-24 h. In HUP-pretreated mice, the ED(50) of IMI to block DFP-induced mortality is approximately 10 times lower than that of DZP and is devoid of sedation. Our data show that a combination of HUP with IMI is a prophylactic, potent, and safe therapeutic strategy to overcome DFP toxicity.

The combination of huperzine A and imidazenil is an effective strategy to prevent diisopropyl fluorophosphate toxicity in mice

PubMed Central

Pibiri, Fabio; Kozikowski, Alan P.; Pinna, Graziano; Auta, James; Kadriu, Bashkim; Costa, Erminio; Guidotti, Alessandro

2008-01-01

Diisopropyl fluorophosphate (DFP) causes neurotoxicity related to an irreversible inhibition of acetylcholinesterase (AChE). Management of this intoxication includes: (i) pretreatment with reversible blockers of AChE, (ii) blockade of muscarinic receptors with atropine, and (iii) facilitation of GABAA receptor signal transduction by benzodiazepines. The major disadvantage associated with this treatment combination is that it must to be repeated frequently and, in some cases, protractedly. Also, the use of diazepam (DZP) and congeners includes unwanted side effects, including sedation, amnesia, cardiorespiratory depression, and anticonvulsive tolerance. To avoid these treatment complications but safely protect against DFP-induced seizures and other CNS toxicity, we adopted the strategy of administering mice with (i) small doses of huperzine A (HUP), a reversible and long-lasting (half-life ≈5 h) inhibitor of AChE, and (ii) imidazenil (IMI), a potent positive allosteric modulator of GABA action selective for α5-containing GABAA receptors. Coadministration of HUP (50 μg/kg s.c., 15 min before DFP) with IMI (2 mg/kg s.c., 30 min before DFP) prevents DFP-induced convulsions and the associated neuronal damage and mortality, allowing complete recovery within 18–24 h. In HUP-pretreated mice, the ED50 of IMI to block DFP-induced mortality is ≈10 times lower than that of DZP and is devoid of sedation. Our data show that a combination of HUP with IMI is a prophylactic, potent, and safe therapeutic strategy to overcome DFP toxicity. PMID:18784370

The solvation structure of alprazolam.

PubMed

Sridhar, Akshay; Johnston, Andrew J; Varathan, Luxmmi; McLain, Sylvia E; Biggin, Philip C

2016-08-10

Alprazolam is a benzodiazepine that is commonly prescribed for the treatment of anxiety and other related disorders. Like other benzodiazepines, it is thought to exert its effect through interaction with GABAA receptors. However, it has also been described as a potent and selective protein interaction inhibitor of bromodomain and extra-terminal (BET) proteins. Indeed, the only crystal structure of alprazolam bound to a protein is a complex between alprazolam and the BRD4 bromodomain. The structure shows that the complex also involves many water interactions that mediate contacts between the drug and the protein, a scenario that exists in many drug-protein complexes. How such waters relate to solvation patterns of small molecules may improve our understanding of what dictates their appearance or absence in bridging positions within complexes and thus will be important in terms of future rational drug-design. Here, we use neutron diffraction in conjunction with molecular dynamics simulations to provide a detailed analysis of how water molecules interact with alprazolam in methanol/water mixtures. The agreement between the neutron diffraction and the molecular dynamics is extremely good. We discuss the results in the context of drug design.

Acutely increasing δGABAA receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus

PubMed Central

Whissell, Paul D.; Eng, Dave; Lecker, Irene; Martin, Loren J.; Wang, Dian-Shi; Orser, Beverley A.

2013-01-01

Extrasynaptic γ-aminobutyric acid type A (GABAA) receptors that contain the δ subunit (δGABAA receptors) are expressed in several brain regions including the dentate gyrus (DG) and CA1 subfields of the hippocampus. Drugs that increase δGABAA receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABAA receptor–preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing δGABAA receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p.) on memory performance in wild-type (WT) and δGABAA receptor null mutant (Gabrd−/−) mice. Additionally, the effects of THIP on long-term potentiation (LTP), a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd−/− mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd−/− mice, an effect that was blocked by GABAA receptor antagonist bicuculline. Thus, acutely increasing δGABAA receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing δGABAA receptor activity. PMID:24062648

The HIV antiretroviral drug efavirenz has LSD-like properties.

PubMed

Gatch, Michael B; Kozlenkov, Alexey; Huang, Ren-Qi; Yang, Wenjuan; Nguyen, Jacques D; González-Maeso, Javier; Rice, Kenner C; France, Charles P; Dillon, Glenn H; Forster, Michael J; Schetz, John A

2013-11-01

Anecdotal reports have surfaced concerning misuse of the HIV antiretroviral medication efavirenz ((4S)-6-chloro-4-(2-cyclopropylethynyl)-4-(trifluoromethyl)-2,4-dihydro-1H-3,1-benzoxazin-2-one) by HIV patients and non-infected teens who crush the pills and smoke the powder for its psychoactive effects. Molecular profiling of the receptor pharmacology of efavirenz pinpointed interactions with multiple established sites of action for other known drugs of abuse including catecholamine and indolamine transporters, and GABAA and 5-HT(2A) receptors. In rodents, interaction with the 5-HT(2A) receptor, a primary site of action of lysergic acid diethylamine (LSD), appears to dominate efavirenz's behavioral profile. Both LSD and efavirenz reduce ambulation in a novel open-field environment. Efavirenz occasions drug-lever responding in rats discriminating LSD from saline, and this effect is abolished by selective blockade of the 5-HT(2A) receptor. Similar to LSD, efavirenz induces head-twitch responses in wild-type, but not in 5-HT(2A)-knockout, mice. Despite having GABAA-potentiating effects (like benzodiazepines and barbiturates), and interactions with dopamine transporter, serotonin transporter, and vesicular monoamine transporter 2 (like cocaine and methamphetamine), efavirenz fails to maintain responding in rats that self-administer cocaine, and it fails to produce a conditioned place preference. Although its molecular pharmacology is multifarious, efavirenz's prevailing behavioral effect in rodents is consistent with LSD-like activity mediated via the 5-HT(2A) receptor. This finding correlates, in part, with the subjective experiences in humans who abuse efavirenz and with specific dose-dependent adverse neuropsychiatric events, such as hallucinations and night terrors, reported by HIV patients taking it as a medication.

The HIV Antiretroviral Drug Efavirenz has LSD-Like Properties

PubMed Central

Gatch, Michael B; Kozlenkov, Alexey; Huang, Ren-Qi; Yang, Wenjuan; Nguyen, Jacques D; González-Maeso, Javier; Rice, Kenner C; France, Charles P; Dillon, Glenn H; Forster, Michael J; Schetz, John A

2013-01-01

Anecdotal reports have surfaced concerning misuse of the HIV antiretroviral medication efavirenz ((4S)-6-chloro-4-(2-cyclopropylethynyl)-4-(trifluoromethyl)-2,4-dihydro-1H-3,1-benzoxazin-2-one) by HIV patients and non-infected teens who crush the pills and smoke the powder for its psychoactive effects. Molecular profiling of the receptor pharmacology of efavirenz pinpointed interactions with multiple established sites of action for other known drugs of abuse including catecholamine and indolamine transporters, and GABAA and 5-HT2A receptors. In rodents, interaction with the 5-HT2A receptor, a primary site of action of lysergic acid diethylamine (LSD), appears to dominate efavirenz's behavioral profile. Both LSD and efavirenz reduce ambulation in a novel open-field environment. Efavirenz occasions drug-lever responding in rats discriminating LSD from saline, and this effect is abolished by selective blockade of the 5-HT2A receptor. Similar to LSD, efavirenz induces head-twitch responses in wild-type, but not in 5-HT2A-knockout, mice. Despite having GABAA-potentiating effects (like benzodiazepines and barbiturates), and interactions with dopamine transporter, serotonin transporter, and vesicular monoamine transporter 2 (like cocaine and methamphetamine), efavirenz fails to maintain responding in rats that self-administer cocaine, and it fails to produce a conditioned place preference. Although its molecular pharmacology is multifarious, efavirenz's prevailing behavioral effect in rodents is consistent with LSD-like activity mediated via the 5-HT2A receptor. This finding correlates, in part, with the subjective experiences in humans who abuse efavirenz and with specific dose-dependent adverse neuropsychiatric events, such as hallucinations and night terrors, reported by HIV patients taking it as a medication. PMID:23702798

MmTX1 and MmTX2 from coral snake venom potently modulate GABAA receptor activity.

PubMed

Rosso, Jean-Pierre; Schwarz, Jürgen R; Diaz-Bustamante, Marcelo; Céard, Brigitte; Gutiérrez, José M; Kneussel, Matthias; Pongs, Olaf; Bosmans, Frank; Bougis, Pierre E

2015-02-24

GABAA receptors shape synaptic transmission by modulating Cl(-) conductance across the cell membrane. Remarkably, animal toxins that specifically target GABAA receptors have not been identified. Here, we report the discovery of micrurotoxin1 (MmTX1) and MmTX2, two toxins present in Costa Rican coral snake venom that tightly bind to GABAA receptors at subnanomolar concentrations. Studies with recombinant and synthetic toxin variants on hippocampal neurons and cells expressing common receptor compositions suggest that MmTX1 and MmTX2 allosterically increase GABAA receptor susceptibility to agonist, thereby potentiating receptor opening as well as desensitization, possibly by interacting with the α(+)/β(-) interface. Moreover, hippocampal neuron excitability measurements reveal toxin-induced transitory network inhibition, followed by an increase in spontaneous activity. In concert, toxin injections into mouse brain result in reduced basal activity between intense seizures. Altogether, we characterized two animal toxins that enhance GABAA receptor sensitivity to agonist, thereby establishing a previously unidentified class of tools to study this receptor family.

Neuroimaging studies of GABA in schizophrenia: a systematic review with meta-analysis

PubMed Central

Egerton, A; Modinos, G; Ferrera, D; McGuire, P

2017-01-01

Data from animal models and from postmortem studies suggest that schizophrenia is associated with brain GABAergic dysfunction. The extent to which this is reflected in data from in vivo studies of GABA function in schizophrenia is unclear. The Medline database was searched to identify articles published until 21 October 2016. The search terms included GABA, proton magnetic resonance spectroscopy (1H-MRS), positron emission tomography (PET), single photon emission computed tomography (SPECT), schizophrenia and psychosis. Sixteen GABA 1H-MRS studies (538 controls, 526 patients) and seven PET/SPECT studies of GABAA/benzodiazepine receptor (GABAA/BZR) availability (118 controls, 113 patients) were identified. Meta-analyses of 1H-MRS GABA in the medial prefrontal cortex (mPFC), parietal/occipital cortex (POC) and striatum did not show significant group differences (mFC: g=−0.3, 409 patients, 495 controls, 95% confidence interval (CI): −0.6 to 0.1; POC: g=−0.3, 139 patients, 111 controls, 95% CI: −0.9 to 0.3; striatum: g=−0.004, 123 patients, 95 controls, 95% CI: −0.7 to 0.7). Heterogeneity across studies was high (I2>50%), and this was not explained by subsequent moderator or meta-regression analyses. There were insufficient PET/SPECT receptor availability studies for meta-analyses, but a systematic review did not suggest replicable group differences in regional GABAA/BZR availability. The current literature does not reveal consistent alterations in in vivo GABA neuroimaging measures in schizophrenia, as might be hypothesized from animal models and postmortem data. The analysis highlights the need for further GABA neuroimaging studies with improved methodology and addressing potential sources of heterogeneity. PMID:28585933

Pharmacodynamic and pharmacokinetic effects of MK-0343, a GABA(A) alpha2,3 subtype selective agonist, compared to lorazepam and placebo in healthy male volunteers.

PubMed

de Haas, S L; de Visser, S J; van der Post, J P; Schoemaker, R C; van Dyck, K; Murphy, M G; de Smet, M; Vessey, L K; Ramakrishnan, R; Xue, L; Cohen, A F; van Gerven, J M A

2008-01-01

The use of non-selective gamma-aminobutyric acid (GABA) enhancers, such as benzodiazepines in the treatment of anxiety disorders is still widespread but hampered by unfavourable side effects. some of these may be associated with binding properties to certain subtypes of the GABA(A) receptor that are unnecessary for therapeutic effects. MK-0343 was designed to be a less sedating anxiolytic, based on reduced efficacy at the alpha1 subtype and significant efficacy at alpha2 and alpha3 subtypes of the GABA(A) receptor. This paper is a double-blind, four-way cross-over (n = 12) study to investigate the effects of MK-0343 (0.25 and 0.75 mg) in comparison to placebo and an anxiolytic dose (2 mg) of the non-selective agonist lorazepam. Effects were measured by eye movements, body sway, Visual Analogue scales (VAS) and memory tests. Lorazepam impaired saccadic peak velocity (SPV), VAs alertness scores, postural stability and memory and increased saccadic latency and inaccuracy. MK-0343 0.75 mg was equipotent with lorazepam as indicated by SPV (-42.4 deg/s), saccadic latency (0.02 s) and VAS alertness scores (1.50 ln mm), while effects on memory and postural stability were smaller. MK-0343 0.25 mg only affected postural stability to a similar extent as MK-0343 0.75 mg. The effect profile of MK-0343 0.75 mg is different from the full agonist lorazepam, which could reflect the selective actions of this compound. Although less effect on VAS alertness was expected, diminished effects on memory and postural stability were present. Clinical studies in anxiety patients should show whether this dose of MK-0343 is therapeutically effective with a different side-effect profile.

Early life stress is a risk factor for excessive alcohol drinking and impulsivity in adults and is mediated via a CRF/GABA(A) mechanism.

PubMed

Gondré-Lewis, Marjorie C; Warnock, Kaitlin T; Wang, Hong; June, Harry L; Bell, Kimberly A; Rabe, Holger; Tiruveedhula, Veera Venkata Naga Phani Babu; Cook, James; Lüddens, Hartmut; Aurelian, Laure; June, Harry L

2016-01-01

Childhood stress and trauma are associated with substance use disorders in adulthood, but the neurological changes that confer increased vulnerability are largely unknown. In this study, maternal separation (MS) stress, restricted to the pre-weaning period, was used as a model to study mechanisms of protracted effects of childhood stress/traumatic experiences on binge drinking and impulsivity. Using an operant self-administration model of binge drinking and a delay discounting assay to measure impulsive-like behavior, we report that early life stress due to MS facilitated acquisition of binge drinking and impulsivity during adulthood in rats. Previous studies have shown heightened levels of corticotropin releasing factor (CRF) after MS, and here, we add that MS increased expression levels of GABA(A) α2 subunit in central stress circuits. To investigate the precise role of these circuits in regulating impulsivity and binge drinking, the CRF1 receptor antagonist antalarmin and the novel GABA(A) α2 subunit ligand 3-PBC were infused into the central amygdala (CeA) and medial prefrontal cortex (mPFC). Antalarmin and 3-PBC at each site markedly reduced impulsivity and produced profound reductions on binge-motivated alcohol drinking, without altering responding for sucrose. Furthermore, whole-cell patch-clamp studies showed that low concentrations of 3-PBC directly reversed the effect of relatively high concentrations of ethanol on α2β3γ2 GABA(A) receptors, by a benzodiazepine site-independent mechanism. Together, our data provide strong evidence that maternal separation, i.e. early life stress, is a risk factor for binge drinking, and is linked to impulsivity, another key risk factor for excessive alcohol drinking. We further show that pharmacological manipulation of CRF and GABA receptor signaling is effective to reverse binge drinking and impulsive-like behavior in MS rats. These results provide novel insights into the role of the brain stress systems in the development of impulsivity and excessive alcohol consumption.

The α5 subunit-containing GABAA receptors contribute to chronic pain

PubMed Central

Bravo-Hernández, Mariana; Corleto, José A.; Barragán-Iglesias, Paulino; González-Ramírez, Ricardo; Pineda-Farias, Jorge B.; Felix, Ricardo; Calcutt, Nigel A.; Delgado-Lezama, Rodolfo; Marsala, Martin; Granados-Soto, Vinicio

2016-01-01

It has been recently proposed that α5-subunit containing GABAA receptors (α5-GABAA receptors) that mediate tonic inhibition might be involved in pain. The purpose of this study was to investigate the contribution of α5-GABAA receptors in the loss of GABAergic inhibition and in formalin-, Complete Freund’s adjuvant (CFA)- and L5/L6 spinal nerve ligation-induced long-lasting hypersensitivity. Formalin or CFA injection and L5/L6 spinal nerve ligation produced long-lasting allodynia and hyperalgesia. Moreover, formalin injection impaired the rate-dependent depression (RDD) of the Hofmann reflex. Peripheral and intrathecal pre-treatment or post-treatment with the α5-GABAA receptor antagonist, L-655,708 (0.15–15 nmol) prevented and reversed, respectively, these long-lasting behaviors. Formalin injection increased α5-GABAA receptors mRNA expression in the spinal cord and dorsal root ganglia (DRG) mainly at 3 days. α5-GABAA receptors were localized in the dorsal spinal cord and DRG co-labeling with NeuN, CGRP and IB4 suggesting their presence in peptidergic and non-peptidergic neurons. These receptors were found mainly in small- and medium-size neurons. Formalin injection enhanced α5-GABAA receptors fluorescence intensity in spinal cord and DRG at 3 and 6 days. Intrathecal administration of L-655,708 (15 nmol) prevented and reversed formalin-induced impairment of RDD. These results suggest that α5-GABAA receptors play a role in the loss of GABAergic inhibition and contribute to long-lasting secondary allodynia and hyperalgesia. PMID:26545088

An Allosteric Coagonist Model for Propofol Effects on α1β2γ2L γ-Aminobutyric Acid Type A Receptors

PubMed Central

Ruesch, Dirk; Neumann, Elena; Wulf, Hinnerk; Forman, Stuart A.

2011-01-01

Background Propofol produces its major actions via γ-aminobutyric acid type A (GABAA) receptors. At low concentrations, propofol enhances agonist-stimulated GABAA receptor activity, and high propofol concentrations directly activate receptors. Etomidate produces similar effects, and there is convincing evidence that a single class of etomidate sites mediate both agonist modulation and direct GABAA receptor activation. It is unknown if the propofol binding site(s) on GABAA receptors that modulate agonist-induced activity also mediate direct activation. Methods GABAA α1β2γ2L receptors were heterologously expressed in Xenopus oocytes and activity was quantified using voltage clamp electrophysiology. We tested whether propofol and etomidate display the same linkage between agonist modulation and direct activation of GABAA receptors by identifying equi-efficacious drug solutions for direct activation. We then determined whether these drug solutions produce equal modulation of GABA-induced receptor activity. We also measured propofol-dependent direct activation and modulation of low GABA responses. Allosteric coagonist models similar to that established for etomidate, but with variable numbers of propofol sites, were fitted to combined data. Results Solutions of 19 μM propofol and 10 μM etomidate were found to equally activate GABAA receptors. These two drug solutions also produced indistinguishable modulation of GABA-induced receptor activity. Combined electrophysiological data behaved in a manner consistent with allosteric co-agonist models with more than one propofol site. The best fit was observed when the model assumed three equivalent propofol sites. Conclusions Our results support the hypothesis that propofol, like etomidate, acts at GABAA receptor sites mediating both GABA modulation and direct activation. PMID:22104494

Benzodiazepine antagonism by harmane and other beta-carbolines in vitro and in vivo.

PubMed

Rommelspacher, H; Nanz, C; Borbe, H O; Fehske, K J; Müller, W E; Wollert, U

1981-03-26

Harmane and other related beta-carbolines are putative endogenous ligands of the benzodiazepine receptor. Since the compounds are potent convulsants they may have agonist activities at the benzodiazepine receptor while the benzodiazepines may be antagonists. This hypothesis was proved by comparing the in vivo and in vitro antagonism of benzodiazepines by harmane and other beta-carbolines. Harmane is clearly a competitive inhibitor of benzodiazepine receptor binding in vitro. Moreover, harmane-induced convulsions can be inhibited reversibly by diazepam in a manner which is consistent with the assumption of competitive antagonism in vivo. For some beta-carboline derivatives a correlation was found between the affinity for the benzodiazepine receptor in vitro and the convulsive potency in vivo. Thus, the data reported suggest that harmane or other related beta-carbolines are putative endogenous agonists of the benzodiazepine receptor. This suggestion is further supported by the observation that diazepam is equally potent in inhibiting harmane- or picrotoxin-induced convulsions, indicating a convulsive mechanism within the GABA receptor-benzodiazepine receptor system.

Pharmacological evaluation of the anxiolytic-like effects of EMD 386088, a partial 5-HT6 receptor agonist, in the rat elevated plus-maze and Vogel conflict tests.

PubMed

Jastrzębska-Więsek, Magdalena; Siwek, Agata; Partyka, Anna; Kubacka, Monika; Mogilski, Szczepan; Wasik, Anna; Kołaczkowski, Marcin; Wesołowska, Anna

2014-10-01

The 5-HT6 is one of the most recent additions to the 5-HT receptor family. Its pharmacological profile and anatomical distribution is suggestive of a putative role in mood disorders. Most of preclinical evidence suggests an anxiolytic-like action of 5-HT6 receptor antagonists. Evaluation the anxiolytic-like effects of EMD 386088, a partial 5-HT6receptor agonist, and its putative mechanism of action in rats. EMD 386088, administered intraperitoneally at a dose of 2.5 mg/kg evoked specific anxiolytic-like activity in the automated version of the conflict drinking Vogel and the elevated plus-maze tests visible by increasing all parameters indicating a potential anti-anxiety effect. Its activity was blocked by the selective 5-HT6 receptor antagonist SB 271046, but not by the selective GABAA/benzodiazepine receptor antagonist flumazenil. EMD 386088 did not intensify an anxiolytic-like effect produced by diazepam in the elevated plus-maze test. These findings suggest that EMD 386088, a 5-HT6 receptor agonist, produces anxiolytic-like activity after systemic administration which may result from direct stimulation of 5-HT6 receptors. Copyright © 2014 Elsevier Ltd. All rights reserved.

GABAa and GABAc receptor-mediated modulation of responses to color stimuli: electroretinographic study in the turtle Emys orbicularis.

PubMed

Kupenova, Petia; Vitanova, Lily; Popova, Elka

2010-04-01

GABAergic transmission is involved in color coding in the retina. The specific contribution of different GABA receptors to spectral sensitivity of the retinal responses is not well characterized. We studied GABAa and GABAc receptor-mediated effects on the intensity-response functions of the electroretinographic ON (b-wave) and OFF (d-wave) responses to color stimuli. For this purpose, we compared the effects of GABAa receptor blockade by bicuculline with the effects of GABAa + GABAc receptor blockade by picrotoxin. The blockade of both GABAa and GABAc receptors caused an amplitude increase of the electroretinographic responses, but the effects of the two blockades depended in a specific manner on stimulus intensity and wavelength. The effects of GABAa receptor blockade showed distinct color ON/OFF asymmetry. The absolute and relative sensitivities of the ON responses to blue stimuli and OFF responses to red stimuli were increased to the greatest degree while the sensitivity of the ON responses to red stimuli and OFF responses to blue stimuli was least increased. In contrast, color ON/OFF asymmetry was not typical of the effects of GABAc receptor blockade. The most prominent GABAc effect was the sensitivity increase of the ON and OFF responses to blue stimuli and, to some lesser extent, to green stimuli. The results of this study indicate a specific role of GABAa and GABAc receptor-mediated influences in processing of chromatic information in the distal retina.

Phosphatase inhibitors remove the run-down of γ-aminobutyric acid type A receptors in the human epileptic brain

PubMed Central

Palma, E.; Ragozzino, D. A.; Di Angelantonio, S.; Spinelli, G.; Trettel, F.; Martinez-Torres, A.; Torchia, G.; Arcella, A.; Di Gennaro, G.; Quarato, P. P.; Esposito, V.; Cantore, G.; Miledi, R.; Eusebi, F.

2004-01-01

The properties of γ-aminobutyric acid (GABA) type A receptors (GABAA receptors) microtransplanted from the human epileptic brain to the plasma membrane of Xenopus oocytes were compared with those recorded directly from neurons, or glial cells, in human brains slices. Cell membranes isolated from brain specimens, surgically obtained from six patients afflicted with drug-resistant temporal lobe epilepsy (TLE) were injected into frog oocytes. Within a few hours, these oocytes acquired GABAA receptors that generated GABA currents with an unusual run-down, which was inhibited by orthovanadate and okadaic acid. In contrast, receptors derived from membranes of a nonepileptic hippocampal uncus, membranes from mouse brain, or recombinant rat α1β2γ2-GABA receptors exhibited a much less pronounced GABA-current run-down. Moreover, the GABAA receptors of pyramidal neurons in temporal neocortex slices from the same six epileptic patients exhibited a stronger run-down than the receptors of rat pyramidal neurons. Interestingly, the GABAA receptors of neighboring glial cells remained substantially stable after repetitive activation. Therefore, the excessive GABA-current run-down observed in the membrane-injected oocytes recapitulates essentially what occurs in neurons, rather than in glial cells. Quantitative RT-PCR analyses from the same TLE neocortex specimens revealed that GABAA-receptor β1, β2, β3, and γ2 subunit mRNAs were significantly overexpressed (8- to 33-fold) compared with control autopsy tissues. Our results suggest that an abnormal GABA-receptor subunit transcription in the TLE brain leads to the expression of run-down-enhanced GABAA receptors. Blockage of phosphatases stabilizes the TLE GABAA receptors and strengthens GABAergic inhibition. It may be that this process can be targeted to develop new treatments for intractable epilepsy. PMID:15218107

Endogenous Positive Allosteric Modulation of GABAA Receptors by Diazepam binding inhibitor

PubMed Central

Christian, Catherine A.; Herbert, Anne G.; Holt, Rebecca L.; Peng, Kathy; Sherwood, Kyla D.; Pangratz-Fuehrer, Susanne; Rudolph, Uwe; Huguenard, John R.

2014-01-01

Summary Benzodiazepines (BZs) allosterically modulate γ-aminobutyric acid type-A receptors (GABAARs) to increase inhibitory synaptic strength. Diazepam binding inhibitor (DBI) protein is a BZ site ligand expressed endogenously in the brain, but functional evidence for BZ-mimicking positive modulatory actions has been elusive. We demonstrate an endogenous potentiation of GABAergic synaptic transmission and responses to GABA uncaging in the thalamic reticular nucleus (nRT) that is absent in both nm1054 mice, in which the Dbi gene is deleted, and mice in which BZ binding to α3 subunit-containing GABAARs is disrupted. Viral transduction of DBI into nRT is sufficient to rescue the endogenous potentiation of GABAergic transmission in nm1054 mice. Both mutations enhance thalamocortical spike-and-wave discharges characteristic of absence epilepsy. Together these results indicate that DBI mediates endogenous nucleus-specific BZ-mimicking (“endozepine”) roles to modulate nRT function and suppress thalamocortical oscillations. Enhanced DBI signaling might serve as a novel therapy for epilepsy and other neurological disorders. PMID:23727119

Frequency-dependent actions of benzodiazepines on GABAA receptors in cultured murine cerebellar granule cells.

PubMed Central

Mellor, J R; Randall, A D

1997-01-01

1. Miniature IPSCs recorded from cultured murine cerebellar granule cells increased in half-width and amplitude following application of the benzodiazepine (BDZ) Flunitrazepam (Flu, 1 microM). The increase in the half-width was much greater than that in the amplitude. 2. Five-millisecond applications of 1 mM GABA to nucleated outside-out patches elicited rapidly rising biexponentially decaying responses that resembled IPSCs. Flu had no effect on the amplitude of such responses, but consistently slowed their deactivation by approximately 50%. This effect was reversed by Flu washout or application of the BDZ antagonist Ro15-1788. The partial inverse agonist. Ro15-4513 speeded deactivation and depressed peak current amplitude by 23 +/- 12%. 3. The EC50 for GABA was between 45 and 50 microM. At submaximally effective agonist concentrations, Flu increased response amplitude and slowed response deactivation. Both effects were present in all cells taken from young cultures (4-7 days in vitro) but the latter was absent in 55% of the neurones obtained from older cultures (14-27 days in vitro). 4. With 120 ms applications of 20 microM GABA, responses activated monoexponentially (time constant, 39.8 +/- 2.8 ms) and deactivated biexponentially (time constants, 40.4 +/- 2.1 and 251 +/- 15 ms). Application of Flu slowed both activation and deactivation. The latter effect arose from an increased contribution of the slower component of decay. 5. Desensitization of responses to 1 mM GABA was biexponential, with time constants of 47 +/- 11 and 479 +/- 49 ms. Flu speeded desensitization by decreasing both fast and slow time constants. GABAA receptor desensitization consistently slowed subsequent deactivation. No significant relationship between the level of desensitization and the amount of slowing of deactivation produced by Flu was found. 6. Responses to paired 5 ms applications of 1 mM GABA indicated that the slowing of deactivation and the speeding of desensitization produced by Flu combine to generate a marked frequency dependence in the actions of this BDZ. Thus when compared with control responses, GABA-induced charge transfer was only enhanced by Flu during the first of two successive agonist applications. PMID:9306278

Regulation of α3-containing GABAA receptors in guinea-pig adrenal medullary cells by adrenal steroids.

PubMed

Inoue, M; Harada, K; Nakamura, J; Matsuoka, H

2013-12-03

GABA is thought to function as a paracrine factor in adrenal medullary (AM) cells. Thus, we electrophysiologically and immunologically examined the properties of GABAA receptors (GABAARs) in guinea-pig AM cells. Bath application of GABA produced an inward current at -60 mV in a dose-dependent manner with an EC50 of 32.3 μM. This GABA-induced current was enhanced by allopregnanolone at concentrations of 0.01 μM and more. A prior exposure to allopregnanolone resulted in a decrease in an EC50 for GABA in activating GABAARs. The GABA-induced current was suppressed by Zn(2+) in a dose-dependent manner with an IC50 of 18 μM, whereas it was enhanced by 100 μM La(3+). The benzodiazepine analog diazepam was three times more potent than zolpidem in enhancing the GABA current, and it was also augmented by L-838,417, which has no action on α1-containing GABAARs. The GABAAR α3, but not α1, and γ2 subunits were immunologically detected at the cell periphery. The expression of α3 subunits in PC12 cells was enhanced by glucocorticoid activity. The results indicated that GABAARs in guinea-pig AM cells mainly comprise α3, β, and γ2 subunits and are enhanced by allopreganalone and glucocorticoids may play a major role in the expression of α3 subunits. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Astrocytes potentiate GABAergic transmission in the thalamic reticular nucleus via endozepine signaling.

PubMed

Christian, Catherine A; Huguenard, John R

2013-12-10

Emerging evidence indicates that diazepam-binding inhibitor (DBI) mediates an endogenous benzodiazepine-mimicking (endozepine) effect on synaptic inhibition in the thalamic reticular nucleus (nRT). Here we demonstrate that DBI peptide colocalizes with both astrocytic and neuronal markers in mouse nRT, and investigate the role of astrocytic function in endozepine modulation in this nucleus by testing the effects of the gliotoxin fluorocitrate (FC) on synaptic inhibition and endozepine signaling in the nRT using patch-clamp recordings. FC treatment reduced the effective inhibitory charge of GABAA receptor (GABAAR)-mediated spontaneous inhibitory postsynaptic currents in WT mice, indicating that astrocytes enhance GABAAR responses in the nRT. This effect was abolished by both a point mutation that inhibits classical benzodiazepine binding to GABAARs containing the α3 subunit (predominant in the nRT) and a chromosomal deletion that removes the Dbi gene. Thus, astrocytes are required for positive allosteric modulation via the α3 subunit benzodiazepine-binding site by DBI peptide family endozepines. Outside-out sniffer patches pulled from neurons in the adjacent ventrobasal nucleus, which does not contain endozepines, show a potentiated response to laser photostimulation of caged GABA when placed in the nRT. FC treatment blocked the nRT-dependent potentiation of this response, as did the benzodiazepine site antagonist flumazenil. When sniffer patches were placed in the ventrobasal nucleus, however, subsequent treatment with FC led to potentiation of the uncaged GABA response, suggesting nucleus-specific roles for thalamic astrocytes in regulating inhibition. Taken together, these results suggest that astrocytes are required for endozepine actions in the nRT, and as such can be positive modulators of synaptic inhibition.

Astrocytes potentiate GABAergic transmission in the thalamic reticular nucleus via endozepine signaling

PubMed Central

Christian, Catherine A.; Huguenard, John R.

2013-01-01

Emerging evidence indicates that diazepam-binding inhibitor (DBI) mediates an endogenous benzodiazepine-mimicking (endozepine) effect on synaptic inhibition in the thalamic reticular nucleus (nRT). Here we demonstrate that DBI peptide colocalizes with both astrocytic and neuronal markers in mouse nRT, and investigate the role of astrocytic function in endozepine modulation in this nucleus by testing the effects of the gliotoxin fluorocitrate (FC) on synaptic inhibition and endozepine signaling in the nRT using patch-clamp recordings. FC treatment reduced the effective inhibitory charge of GABAA receptor (GABAAR)-mediated spontaneous inhibitory postsynaptic currents in WT mice, indicating that astrocytes enhance GABAAR responses in the nRT. This effect was abolished by both a point mutation that inhibits classical benzodiazepine binding to GABAARs containing the α3 subunit (predominant in the nRT) and a chromosomal deletion that removes the Dbi gene. Thus, astrocytes are required for positive allosteric modulation via the α3 subunit benzodiazepine-binding site by DBI peptide family endozepines. Outside-out sniffer patches pulled from neurons in the adjacent ventrobasal nucleus, which does not contain endozepines, show a potentiated response to laser photostimulation of caged GABA when placed in the nRT. FC treatment blocked the nRT-dependent potentiation of this response, as did the benzodiazepine site antagonist flumazenil. When sniffer patches were placed in the ventrobasal nucleus, however, subsequent treatment with FC led to potentiation of the uncaged GABA response, suggesting nucleus-specific roles for thalamic astrocytes in regulating inhibition. Taken together, these results suggest that astrocytes are required for endozepine actions in the nRT, and as such can be positive modulators of synaptic inhibition. PMID:24262146

Homocysteine alters cerebral microvascular integrity and causes remodeling by antagonizing GABA-A receptor*

PubMed Central

Lominadze, David; Tyagi, Neetu; Sen, Utpal; Ovechkin, Alexander; Tyagi, Suresh C.

2012-01-01

High levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), are associated with cerebrovascular diseases, such as vascualr dementia, stroke, and Alzheimer's disease. The -amino butyric acid (GABA) is a inhibitory neurotransmitter and a ligand of GABA-A receptor. By inhibiting excitatory response it may decrease complications associated with vascular dementia and stroke. Hcy specifically competes with the GABA-A receptors and acts as an excitotoxic neurotransmitter. Previously we have shown that Hcy increases levels of NADPH oxidase and reactive oxygen species (ROS), and decreases levels of thioredoxin and peroxiredoxin by antagonizing the GABA-A receptor. Hcy treatment leads to activation of matrix metalloproteinases (MMPs) in cerebral circualtion by inducing redox stress and ROS. The hypothesis is that Hcy induces MMPs and suppresses tissue inhibitors of metalloproteinase (TIMPs), in part, by inhibiting the GABA-A receptor. This leads to degradation of the matrix and disruption of the blood brain barrier. The brain cortex of transgenic mouse model of HHcy (cystathionine -synthase, CBS −/+) and GABA-A receptor null mice treated with and without muscimol (GABA-A receptor agonist) was analysed. The mRNA levels were measured by Q-RT-PCR. Levels of MMP-2, -9, -13, and TIMP-1, -2, -3, and -4 were evaluated by in situ labeling and PCR-gene arrays. Pial venular permeability to fluorescence-labeled albumin was assessed with intravital fluorescence microscopy. We found that Hcy increases metalloproteinase activity and decreases TIMP-4 by antagonizing the GABA-A receptor. The results demonstrate a novel mechanism in which brain microvascular permeability changes during HHcy and vascular dementias, and have therapeutic ramifications for microvascular disease in Alzheimer's patients. PMID:22886392

Building a bridge between neurobiology and mental illness.

PubMed

Costa, E

1992-10-01

GABA (gamma amino butyric acid) is the most abundant and important inhibitory transmitter in mammalian CNS. It counterbalances the glutamate mediated neuronal excitation. Abnormalities of the interaction of these two transmitters might change the mechanisms of neuronal group selection that according to Edelman [Neural Darwinism. Basic Books, New York] play a role in mediating several brain functions including cognition processes. Indeed imbalances in GABAergic functions were shown to elicit psychoses. They can be obtained by administration of drugs that affect synthesis, metabolism and uptake of GABA and thereby cause a persistent stimulation of GABAA receptors or perhaps by genetic abnormalities in DNA transcription, pre-mRNA splicing, mRNA translation and posttranslation modifications of GABAA receptor subunits. The complexities in the regulation of GABAA receptor subunit structure, synthesis, assembly and the brain location of specific mRNA encoding for these subunits are investigated with in situ mRNA hybridization specific for subunits of GABAA receptors. The role of the variability resulting from the complexities in the regulation of GABAA receptor allosteric modulation by drugs and putative endogenous allosteric modulators of GABA action at GABAA receptors is discussed. This discussion gives relevance to the possibility that genetic abnormalities in the expression of proteins participating in GABAergic function are to be considered as a possible target of the genetic defects operative in psychoses. In line with this thinking, it is suggested that partial allosteric modulators (partial agonists) of GABAA receptors and the phosphothioate or methylphosphonate analogs antisense to specific mRNA oligonucleotides that mediate the expression of genetic information concerning GABAA and glutamate receptor subunits may become valuable tools in psychiatric research. Perhaps in the future these studies might generate new ideas useful in the therapy of genetically determined psychiatric illness.

Surface active benzodiazepine-bromo-alkyl conjugate for potential GABAA-receptor purification.

PubMed

Turina, A V; Quinteros, G J; Caruso, B; Moyano, E L; Perillo, M A

2011-08-21

A conjugable analogue of the benzodiazepine 5-(2-hydroxyphenyl)-7-nitro-benzo[e][1,4]diazepin-2(3H)-one containing a bromide C(12)-aliphatic chain (BDC) at nitrogen N1 was synthesized. One-pot preparation of this benzodiazepine derivative was achieved using microwave irradiation giving 49% yield of the desired product. BDC inhibited FNZ binding to GABA(A)-R with an inhibition binding constant K(i) = 0.89 μM and expanded a model membrane packed up to 35 mN m(-1) when penetrating in it from the aqueous phase. BDC exhibited surface activity, with a collapse pressure π = 9.8 mN m(-1) and minimal molecular area A(min) = 52 Å(2)/molecule at the closest molecular packing, resulted fully and non-ideally mixed with a phospholipid in a monolayer up to a molar fraction x≅ 0.1. A geometrical-thermodynamic analysis along the π-A phase diagram predicted that at low x(BDC) (<0.1) and at all π, including the equilibrium surface pressures of bilayers, dpPC-BDC mixtures dispersed in water were compatible with the formation of planar-like structures. These findings suggest that, in a potential surface grafted BDC, this compound could be stabilize though London-type interactions within a phospholipidic coating layer and/or through halogen bonding with an electron-donor surface via its terminal bromine atom while GABA(A)-R might be recognized through the CNZ moiety.

Cognitive impairments of alcoholic cirrhotic patients: correlation with endogenous benzodiazepine receptor ligands and increased affinity of platelet receptors.

PubMed Central

Kapczinski, F; Curran, H V; Przemioslo, R; Williams, R; Fluck, E; Fernandes, C; File, S E

1996-01-01

OBJECTIVES--To determine whether differences in cognitive function between alcoholic and non-alcoholic cirrhotic patients relate to differences in endogenous ligands for the benzodiazepine receptor and/or benzodiazepine binding. METHODS--Seventeen grade-I hepatic encephalopathic patients (nine alcoholic, eight non-alcoholic) were compared with 10 matched controls on plasma concentrations of endogenous ligands for the neuronal benzodiazepine receptor, benzodiazepine binding in platelets, and performance on tests of cognitive function. RESULTS--Both groups of patients were impaired on verbal recall and on reaction time tasks compared with controls; alcoholic patients were also impaired on Reitan's trails test and digit cancellation. Four of the 17 patients had detectable concentrations of endogenous benzodiazepine ligands and they were more impaired than other patients on trails and cancellation tests. The groups did not differ in the density of benzodiazepine platelet receptors, but receptor affinity was higher in alcoholic patients than in controls; furthermore, receptor affinity correlated with the time to complete the cancellation task and with reaction time. CONCLUSION--Alcoholic cirrhotic patients may have enhanced concentrations of ligands for neuronal and peripheral benzodiazepine receptors and these may contribute to cognitive impairments in these patients. PMID:8648337

Micromolar-Affinity Benzodiazepine Receptors Regulate Voltage-Sensitive Calcium Channels in Nerve Terminal Preparations

NASA Astrophysics Data System (ADS)

Taft, William C.; Delorenzo, Robert J.

1984-05-01

Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.

Micromolar-affinity benzodiazepine receptors regulate voltage-sensitive calcium channels in nerve terminal preparations.

PubMed Central

Taft, W C; DeLorenzo, R J

1984-01-01

Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance. PMID:6328498

Modulation of GABAA receptors by valerian extracts is related to the content of valerenic acid.

PubMed

Trauner, Gabriele; Khom, Sophia; Baburin, Igor; Benedek, Birgit; Hering, Steffen; Kopp, Brigitte

2008-01-01

Valeriana Officinalis L . is a traditionally used sleep remedy, however, the mechanism of action and the substances responsible for its sedative and sleep-enhancing properties are not fully understood. As we previously identified valerenic acid as a subunit-specific allosteric modulator of GABAA receptors, we now investigated the relation between modulation of GABAA receptors by Valerian extracts of different polarity and the content of sesquiterpenic acids (valerenic acid, acetoxyvalerenic acid). All extracts were analysed by HPLC concerning the content of sesquiterpenic acids. GABAA receptors composed of alpha 1, beta 2 and gamma 2S subunits were expressed in Xenopus laevis oocytes and the modulation of chloride currents through GABAA receptors (IGABA) by Valerian extracts was investigated using the two-microelectrode voltage clamp technique. Apolar extracts induced a significant enhancement of IGABA, whereas polar extracts showed no effect. These results were confirmed by fractionating a highly active ethyl acetate extract: again fractions with high contents of valerenic acid exhibited strong receptor activation. In addition, removal of sesquiterpenic acids from the ethyl acetate extract led to a loss of I (GABA) enhancement. In conclusion, our data show that the extent of GABAA receptor modulation by Valerian extracts is related to the content of valerenic acid.

Participation of the GABAergic system in the anesthetic effect of Lippia alba (Mill.) N.E. Brown essential oil

PubMed Central

Heldwein, C.G.; Silva, L.L.; Reckziegel, P.; Barros, F.M.C.; Bürger, M.E.; Baldisserotto, B.; Mallmann, C.A.; Schmidt, D.; Caron, B.O.; Heinzmann, B.M.

2012-01-01

The objective of this study was to identify the possible involvement of the GABAergic system in the anesthetic effect of Lippia alba essential oil (EO). We propose a new animal model using silver catfish (Rhamdia quelen) exposed to an anesthetic bath to study the mechanism of action of EO. To observe the induction and potentiation of the anesthetic effect of EO, juvenile silver catfish (9.30 ± 1.85 g; 10.15 ± 0.95 cm; N = 6) were exposed to various concentrations of L. alba EO in the presence or absence of diazepam [an agonist of high-affinity binding sites for benzodiazepinic (BDZ) sites coupled to the GABAA receptor complex]. In another experiment, fish (N = 6) were initially anesthetized with the EO and then transferred to an anesthetic-free aquarium containing flumazenil (a selective antagonist of binding sites for BDZ coupled to the GABAA receptor complex) or water to assess recovery time from the anesthesia. In this case, flumazenil was used to observe the involvement of the GABA-BDZ receptor in the EO mechanism of action. The results showed that diazepam potentiates the anesthetic effect of EO at all concentrations tested. Fish exposed to diazepam and EO showed faster recovery from anesthesia when flumazenil was added to the recovery bath (12.0 ± 0.3 and 7.2 ± 0.7, respectively) than those exposed to water (9.2 ± 0.2 and 3.5 ± 0.3, respectively). In conclusion, the results demonstrated the involvement of the GABAergic system in the anesthetic effect of L. alba EO on silver catfish. PMID:22473320

Decreased hepatocyte membrane potential differences and GABAA-beta3 expression in human hepatocellular carcinoma.

PubMed

Minuk, Gerald Y; Zhang, Manna; Gong, Yuewen; Minuk, Leonard; Dienes, Hans; Pettigrew, Norman; Kew, Michael; Lipschitz, Jeremy; Sun, Dongfeng

2007-03-01

To determine whether hepatocyte membrane potential differences (PDs) are depolarized in human HCC and whether depolarization is associated with changes in GABAA receptor expression, hepatocyte PDs and gamma-aminobutyric acid (GABA)A receptor messenger RNA (mRNA) and protein expression were documented in HCC tissues via microelectrode impalement, real-time reverse-transcriptase polymerase chain reaction, and Western blot analysis, respectively. HCC tissues were significantly depolarized (-19.8+/-1.3 versus -25.9+/-3.2 mV, respectively [P<0.05]), and GABAA-beta3 expression was down-regulated (GABAA-beta3 mRNA and protein expression in HCC; 5,693+/-1,385 and 0.29+/-0.11 versus 11,046+/-4,979 copies/100 mg RNA and 0.62+/-0.16 optical density in adjacent tumor tissues, respectively [P=0.002 and P<0.0001, respectively]) when compared with adjacent nontumor tissues. To determine the physiological relevance of the down-regulation, human malignant hepatocytes deficient in GABAA-beta3 receptor expression (Huh-7 cells) were transfected with GABAA-beta3 complementary DNA (cDNA) or vector alone and injected into nu/nu nude mice (n=16-17 group). Tumors developed after a mean (+/-SD) of 51+/-6 days (range: 41-60 days) in 7/16 (44%) mice injected with vector-transfected cells and 70+/-12 days (range: 59-86 days) in 4/17 (24%) mice injected with GABAA-beta3 cDNA-transfected cells (P<0.005). The results of this study indicate that (1) human HCC tissues are depolarized compared with adjacent nontumor tissues, (2) hepatic GABAA-beta3 receptor expression is down-regulated in human HCC, and (3) restoration of GABAA-beta3 receptor expression results in attenuated in vivo tumor growth in nude mice.

Involvement of GABAA receptor in Bufo arenarum oocyte maturation.

PubMed

Toranzo, G Sánchez; Zelarayán, L; Bonilla, F; Oterino, J; Bühler, M I

2008-05-01

Amphibian oocytes meiotic arrest is released under the stimulus of progesterone; this hormone interacts with the oocyte surface and starts a cascade of events leading to the activation of a cytoplasmic maturation promoting factor (MPF) that induces germinal vesicle breakdown (GVBD), chromosome condensation and extrusion of the first polar body. The aim of this work was to determine whether the activation of a GABAA receptor is able to induce GVBD in fully grown denuded oocytes of Bufo arenarum and to analyse its possible participation in progesterone-induced maturation. We also evaluated the role of purines and phospholipids in the maturation process induced by a GABAA receptor agonist such as muscimol. Our results indicated that the activation of the GABAA receptor by muscimol induces maturation in a dose- and time-dependent manner and that this activation is a genuine maturation that enables oocytes to form pronuclei. Assays with a receptor antagonist, picrotoxine, showed that the maturation induced by muscimol was inhibited. Treatment with picrotoxine, however, shows that the participation of GABAA receptor in progesterone-induced maturation is not significant. In addition, our results indicate that high intracellular levels of purines obtained by the use of db-AMPc and theophylline or the inhibition of the phosphatidylinositol 4,5-bisphosphate (PIP2 hydrolysis by neomycin and PIP2 turn over by LiCl, respectively, inhibited the maturation induced by muscimol. Treatment with H-7 indicated, however, that PKC activation is not necessary for GVBD induced by the GABAA receptor agonist. Results suggest that the transduction pathway used by the GABAA receptor to induce maturation is different from those used by progesterone.

Low concentrations of ethanol do not affect radioligand binding to the delta-subunit-containing GABAA receptors in the rat brain.

PubMed

Mehta, Ashok K; Marutha Ravindran, C R; Ticku, Maharaj K

2007-08-24

In the present study, we investigated the co-localization pattern of the delta subunit with other subunits of GABA(A) receptors in the rat brain using immunoprecipitation and Western blotting techniques. Furthermore, we investigated whether low concentrations of ethanol affect the delta-subunit-containing GABA(A) receptor assemblies in the rat brain using radioligand binding to the rat brain membrane homogenates as well as to the immunoprecipitated receptor assemblies. Our results revealed that delta subunit is not co-localized with gamma(2) subunit but it is associated with the alpha(1), alpha(4) or alpha(6), beta(2) and/or beta(3) subunit(s) of GABA(A) receptors in the rat brain. Ethanol (1-50 mM) neither affected [(3)H]muscimol (3 nM) binding nor diazepam-insensitive [(3)H]Ro 15-4513 (2 nM) binding in the rat cerebellum and cerebral cortex membranes. However, a higher concentration of ethanol (500 mM) inhibited the binding of these radioligands to the GABA(A) receptors partially in the rat cerebellum and cerebral cortex. Similarly, ethanol (up to 50 mM) did not affect [(3)H]muscimol (15 nM) binding to the immunoprecipitated delta-subunit-containing GABA(A) receptor assemblies in the rat cerebellum and hippocampus but it inhibited the binding partially at a higher concentration (500 mM). These results suggest that the native delta-subunit-containing GABA(A) receptors do not play a major role in the pharmacology of clinically relevant low concentrations of ethanol.

Muscimol increases acetylcholine release by directly stimulating adult striatal cholinergic interneurons.

PubMed

Login, I S; Pal, S N; Adams, D T; Gold, P E

1998-01-01

Because GabaA ligands increase acetylcholine (ACh) release from adult striatal slices, we hypothesized that activation of GabaA receptors on striatal cholinergic interneurons directly stimulates ACh secretion. Fractional [3H]ACh release was recorded during perifusion of acutely dissociated, [3H]choline-labeled, adult male rat striata. The GabaA agonist, muscimol, immediately stimulated release maximally approximately 300% with EC50 = approximately 1 microM. This action was enhanced by the allosteric GabaA receptor modulators, diazepam and secobarbital, and inhibited by the GabaA antagonist, bicuculline, by ligands for D2 or muscarinic cholinergic receptors or by low calcium buffer, tetrodotoxin or vesamicol. Membrane depolarization inversely regulated muscimol-stimulated secretion. Release of endogenous and newly synthesized ACh was stimulated in parallel by muscimol without changing choline release. Muscimol pretreatment inhibited release evoked by K+ depolarization or by receptor-mediated stimulation with glutamate. Thus, GabaA receptors on adult striatal cholinergic interneurons directly stimulate voltage- and calcium-dependent exocytosis of ACh stored in vesamicol-sensitive synaptic vesicles. The action depends on the state of membrane polarization and apparently depolarizes the membrane in turn. This functional assay demonstrates that excitatory GabaA actions are not limited to neonatal tissues. GabaA-stimulated ACh release may be prevented in situ by normal tonic dopaminergic and muscarinic input to cholinergic neurons.

GABAA Receptor Regulation of Voluntary Ethanol Drinking Requires PKCε

PubMed Central

Besheer, Joyce; Lepoutre, Veronique; Mole, Beth; Hodge, Clyde W.

2010-01-01

Protein kinase C (PKC) regulates a variety of neural functions, including ion channel activity, neurotransmitter release, receptor desensitization and differentiation. We have shown previously that mice lacking the ε-isoform of PKC (PKCε) self-administer 75% less ethanol and exhibit supersensitivity to acute ethanol and allosteric positive modulators of GABAA receptors when compared with wild-type controls. The purpose of the present study was to examine involvement of PKCε in GABAA receptor regulation of voluntary ethanol drinking. To address this question, PKCε null-mutant and wild-type control mice were allowed to drink ethanol (10% v/v) vs. water on a two-bottle continuous access protocol. The effects of diazepam (nonselective GABAA BZ positive modulator), zolpidem (GABAA α1 agonist), L-655,708 (BZ-sensitive GABAA α5 inverse agonist), and flumazenil (BZ antagonist) were then tested on ethanol drinking. Ethanol intake (grams/kg/day) by wild-type mice decreased significantly after diazepam or zolpidem but increased after L-655,708 administration. Flumazenil antagonized diazepam-induced reductions in ethanol drinking in wild-type mice. However, ethanol intake by PKCε null mice was not altered by any of the GABAergic compounds even though effects were seen on water drinking in these mice. Increased acute sensitivity to ethanol and diazepam, which was previously reported, was confirmed in PKCε null mice. Thus, results of the present study show that PKCε null mice do not respond to doses of GABAA BZ receptor ligands that regulate ethanol drinking by wild-type control mice. This suggests that PKCε may be required for GABAA receptor regulation of chronic ethanol drinking. PMID:16881070

GABAA receptor activity modulating piperine analogs: In vitro metabolic stability, metabolite identification, CYP450 reaction phenotyping, and protein binding.

PubMed

Zabela, Volha; Hettich, Timm; Schlotterbeck, Götz; Wimmer, Laurin; Mihovilovic, Marko D; Guillet, Fabrice; Bouaita, Belkacem; Shevchenko, Bénédicte; Hamburger, Matthias; Oufir, Mouhssin

2018-01-01

In a screening of natural products for allosteric modulators of GABA A receptors (γ-aminobutyric acid type A receptor), piperine was identified as a compound targeting a benzodiazepine-independent binding site. Given that piperine is also an activator of TRPV1 (transient receptor potential vanilloid type 1) receptors involved in pain signaling and thermoregulation, a series of piperine analogs were prepared in several cycles of structural optimization, with the aim of separating GABA A and TRPV1 activating properties. We here investigated the metabolism of piperine and selected analogs in view of further cycles of lead optimization. Metabolic stability of the compounds was evaluated by incubation with pooled human liver microsomes, and metabolites were analyzed by UHPLC-Q-TOF-MS. CYP450 isoenzymes involved in metabolism of compounds were identified by reaction phenotyping with Silensomes™. Unbound fraction in whole blood was determined by rapid equilibrium dialysis. Piperine was the metabolically most stable compound. Aliphatic hydroxylation, and N- and O-dealkylation were the major routes of oxidative metabolism. Piperine was exclusively metabolized by CYP1A2, whereas CYP2C9 contributed significantly in the oxidative metabolism of all analogs. Extensive binding to blood constituents was observed for all compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

A network of autism linked genes stabilizes two pools of synaptic GABAA receptors

PubMed Central

Tong, Xia-Jing; Hu, Zhitao; Liu, Yu; Anderson, Dorian; Kaplan, Joshua M

2015-01-01

Changing receptor abundance at synapses is an important mechanism for regulating synaptic strength. Synapses contain two pools of receptors, immobilized and diffusing receptors, both of which are confined to post-synaptic elements. Here we show that immobile and diffusing GABAA receptors are stabilized by distinct synaptic scaffolds at C. elegans neuromuscular junctions. Immobilized GABAA receptors are stabilized by binding to FRM-3/EPB4.1 and LIN-2A/CASK. Diffusing GABAA receptors are stabilized by the synaptic adhesion molecules Neurexin and Neuroligin. Inhibitory post-synaptic currents are eliminated in double mutants lacking both scaffolds. Neurexin, Neuroligin, and CASK mutations are all linked to Autism Spectrum Disorders (ASD). Our results suggest that these mutations may directly alter inhibitory transmission, which could contribute to the developmental and cognitive deficits observed in ASD. DOI: http://dx.doi.org/10.7554/eLife.09648.001 PMID:26575289

Ester to amide substitution improves selectivity, efficacy and kinetic behavior of a benzodiazepine positive modulator of GABAA receptors containing the α5 subunit.

PubMed

Stamenić, Tamara Timić; Poe, Michael M; Rehman, Sabah; Santrač, Anja; Divović, Branka; Scholze, Petra; Ernst, Margot; Cook, James M; Savić, Miroslav M

2016-11-15

We have synthesized and characterized MP-III-022 ((R)-8-ethynyl-6-(2-fluorophenyl)-N,4-dimethyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxamide) in vitro and in vivo as a binding- and efficacy-selective positive allosteric modulator of GABA A receptors containing the α5 subunit (α5GABA A Rs). By approximation of the electrophysiological responses which the estimated free rat brain concentrations can induce, we demonstrated that convenient systemic administration of MP-III-022 in the dose range 1-10mg/kg may result in a selective potentiation, over a wide range from mild to moderate to strong, of α5βγ2 GABA A receptors. For eliciting a comparable range of potentiation, the widely studied parent ligand SH-053-2'F-R-CH3 containing an ester moiety needs to be administered over a much wider dose range (10-200mg/kg), but at the price of activating non-α5 GABA A Rs as well as the desired α5GABA A Rs at the highest dose. At the dose of 10mg/kg, which elicits a strong positive modulation of α5GABA A Rs, MP-III-022 caused mild, but significant muscle relaxation, while at doses 1-10mg/kg was devoid of ataxia, sedation or an influence on the anxiety level, characteristic for non-selective benzodiazepines. As an amide compound with improved stability and kinetic properties, MP-III-022 may represent an optimized tool to study the influence of α5GABA A Rs on the neuronal pathways related to CNS disorders such as schizophrenia, Alzheimer's disease, Down syndrome or autism. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of GABAA receptors in dorsal raphe nucleus in stress-induced reinstatement of morphine-conditioned place preference in rats.

PubMed

Li, Chen; Staub, Daniel R; Kirby, Lynn G

2013-12-01

The serotonin (5-hydroxytryptamine, 5-HT) system plays an important role in stress-related psychiatric disorders and substance abuse. Our data indicate that stress inhibits the dorsal raphe nucleus (DRN)-5-HT system via stimulation of GABA synaptic activity by the stress neurohormone corticotropin-releasing factor and, more recently, that morphine history sensitizes DRN-5-HT neurons to GABAergic inhibitory effects of stress. We tested the hypothesis that DRN GABAA receptors contribute to stress-induced reinstatement of morphine-conditioned place preference (CPP). First, we tested if activation of GABAA receptors in the DRN would reinstate morphine CPP. Second, we tested if blockade of GABAA receptors in the DRN would attenuate swim stress-induced reinstatement of morphine CPP. CPP was induced by morphine (5 mg/kg) in a 4-day conditioning phase followed by a conditioning test. Upon acquiring conditioning criteria, subjects underwent 4 days of extinction training followed by an extinction test. Upon acquiring extinction criteria, animals underwent a reinstatement test. For the first experiment, the GABAA receptor agonist muscimol (50 ng) or vehicle was injected into the DRN prior to the reinstatement test. For the second experiment, the GABAA receptor antagonist bicuculline (75 ng) or vehicle was injected into the DRN prior to a forced swim stress, and then, animals were tested for reinstatement of CPP. Intraraphe injection of muscimol reinstated morphine CPP, while intraraphe injection of bicuculline attenuated swim stress-induced reinstatement. These data provide evidence that GABAA receptor-mediated inhibition of the serotonergic DRN contributes to stress-induced reinstatement of morphine CPP.

Functional Validation of Virtual Screening for Novel Agents with General Anesthetic Action at Ligand-Gated Ion Channels

PubMed Central

Heusser, Stephanie A.; Howard, Rebecca J.; Borghese, Cecilia M.; Cullins, Madeline A.; Broemstrup, Torben; Lee, Ui S.; Lindahl, Erik; Carlsson, Jens

2013-01-01

GABAA receptors play a crucial role in the actions of general anesthetics. The recently published crystal structure of the general anesthetic propofol bound to Gloeobacter violaceus ligand-gated ion channel (GLIC), a bacterial homolog of GABAA receptors, provided an opportunity to explore structure-based ligand discovery for pentameric ligand-gated ion channels (pLGICs). We used molecular docking of 153,000 commercially available compounds to identify molecules that interact with the propofol binding site in GLIC. In total, 29 compounds were selected for functional testing on recombinant GLIC, and 16 of these compounds modulated GLIC function. Active compounds were also tested on recombinant GABAA receptors, and point mutations around the presumed binding pocket were introduced into GLIC and GABAA receptors to test for binding specificity. The potency of active compounds was only weakly correlated with properties such as lipophilicity or molecular weight. One compound was found to mimic the actions of propofol on GLIC and GABAA, and to be sensitive to mutations that reduce the action of propofol in both receptors. Mutant receptors also provided insight about the position of the binding sites and the relevance of the receptor’s conformation for anesthetic actions. Overall, the findings support the feasibility of the use of virtual screening to discover allosteric modulators of pLGICs, and suggest that GLIC is a valid model system to identify novel GABAA receptor ligands. PMID:23950219

Cyclohexanol analogues are positive modulators of GABAA receptor currents and act as general anaesthetics in vivo

USDA-ARS?s Scientific Manuscript database

GABAA receptors meet all the pharmacological criteria required to be considered important general anaesthetic targets. In the following study, the modulatory effects of various commercially available and novel cyclohexanol were investigated on recombinant human '-aminobutyric acid (GABAA, a1ß2'2s) r...

Modulation of neurosteroid potentiation by protein kinases at synaptic- and extrasynaptic-type GABAA receptors

PubMed Central

Adams, Joanna M.; Thomas, Philip; Smart, Trevor G.

2015-01-01

GABAA receptors are important for inhibition in the CNS where neurosteroids and protein kinases are potent endogenous modulators. Acting individually, these can either enhance or depress receptor function, dependent upon the type of neurosteroid or kinase and the receptor subunit combination. However, in vivo, these modulators probably act in concert to fine-tune GABAA receptor activity and thus inhibition, although how this is achieved remains unclear. Therefore, we investigated the relationship between these modulators at synaptic-type α1β3γ2L and extrasynaptic-type α4β3δ GABAA receptors using electrophysiology. For α1β3γ2L, potentiation of GABA responses by tetrahydro-deoxycorticosterone was reduced after inhibiting protein kinase C, and enhanced following its activation, suggesting this kinase regulates neurosteroid modulation. In comparison, neurosteroid potentiation was reduced at α1β3S408A,S409Aγ2L receptors, and unaltered by PKC inhibitors or activators, indicating that phosphorylation of β3 subunits is important for regulating neurosteroid activity. To determine whether extrasynaptic-type GABAA receptors were similarly modulated, α4β3δ and α4β3S408A,S409Aδ receptors were investigated. Neurosteroid potentiation was reduced at both receptors by the kinase inhibitor staurosporine. By contrast, neurosteroid-mediated potentiation at α4S443Aβ3S408A,S409Aδ receptors was unaffected by protein kinase inhibition, strongly suggesting that phosphorylation of α4 and β3 subunits is required for regulating neurosteroid activity at extrasynaptic receptors. Western blot analyses revealed that neurosteroids increased phosphorylation of β3S408,S409 implying that a reciprocal pathway exists for neurosteroids to modulate phosphorylation of GABAA receptors. Overall, these findings provide important insight into the regulation of GABAA receptors in vivo, and into the mechanisms by which GABAergic inhibitory transmission may be simultaneously tuned by two endogenous neuromodulators. This article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’. PMID:25278033

How theories evolved concerning the mechanism of action of barbiturates.

PubMed

Löscher, Wolfgang; Rogawski, Michael A

2012-12-01

The barbiturate phenobarbital has been in use in the treatment of epilepsy for 100 years. It has long been recognized that barbiturates act by prolonging and potentiating the action of γ-aminobutyric acid (GABA) on GABA(A) receptors and at higher concentrations directly activating the receptors. A large body of data supports the concept that GABA(A) receptors are the primary central nervous system target for barbiturates, including the finding that transgenic mice with a point mutation in the β3 GABA(A) -receptor subunit exhibit diminished sensitivity to the sedative and immobilizing actions of the anesthetic barbiturate pentobarbital. Although phenobarbital is only modestly less potent as a GABA(A) -receptor modulator than pentobarbital, phenobarbital is minimally sedating at effective anticonvulsant doses. Possible explanations for the reduced sedative effect of phenobarbital include more regionally restricted action; partial agonist activity; reduced propensity to directly activate GABA(A) receptors (possibly including extrasynaptic receptors containing δ subunits); and reduced activity at other ion channel targets, including voltage-gated calcium channels. In recent years, substantial progress has been made in defining the structural features of GABA(A) receptors responsible for gating and allosteric modulation by drugs. Although the precise sites of action of barbiturates have not yet been defined, the second and third transmembrane domains of the β subunit appear to be critical; binding may involve a pocket formed by β-subunit methionine 286 as well as α-subunit methionine 236. In addition to effects on GABA(A) receptors, barbiturates block AMPA/kainate receptors, and they inhibit glutamate release through an effect on P/Q-type high-voltage activated calcium channels. The combination of these various actions likely accounts for their diverse clinical activities. Despite the remarkable progress of the last century, there is still much to learn about the actions of barbiturates that can be applied to the discovery of new, more therapeutically useful agents. Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.

Mapping General Anesthetic Sites in Heteromeric γ-Aminobutyric Acid Type A Receptors Reveals a Potential For Targeting Receptor Subtypes.

PubMed

Forman, Stuart A; Miller, Keith W

2016-11-01

IV general anesthetics, including propofol, etomidate, alphaxalone, and barbiturates, produce important actions by enhancing γ-aminobutyric acid type A (GABAA) receptor activation. In this article, we review scientific studies that have located and mapped IV anesthetic sites using photoaffinity labeling and substituted cysteine modification protection. These anesthetics bind in transmembrane pockets between subunits of typical synaptic GABAA receptors, and drugs that display stereoselectivity also show remarkably selective interactions with distinct interfacial sites. These results suggest strategies for developing new drugs that selectively modulate distinct GABAA receptor subtypes.

The hypnotic zolpidem increases the synchrony of BOLD signal fluctuations in widespread brain networks during a resting paradigm

PubMed Central

Licata, Stephanie C.; Nickerson, Lisa D.; Lowen, Steven B.; Trksak, George H.; MacLean, Robert R.; Lukas, Scott E.

2013-01-01

Networks of brain regions having synchronized fluctuations of the blood oxygen level-dependent functional magnetic resonance imaging (BOLD fMRI) time-series at rest, or “resting state networks” (RSNs), are emerging as a basis for understanding intrinsic brain activity. RSNs are topographically consistent with activity-related networks subserving sensory, motor, and cognitive processes, and studying their spontaneous fluctuations following acute drug challenge may provide a way to understand better the neuroanatomical substrates of drug action. The present within-subject double-blind study used BOLD fMRI at 3T to investigate the functional networks influenced by the non-benzodiazepine hypnotic zolpidem (Ambien®). Zolpidem is a positive modulator of γ-aminobutyric acidA (GABAA) receptors, and engenders sedative effects that may be explained in part by how it modulates intrinsic brain activity. Healthy participants (n= 12) underwent fMRI scanning 45 min after acute oral administration of zolpidem (0, 5, 10, or 20 mg), and changes in BOLD signal were measured while participants gazed at a static fixation point (i.e., at rest). Data were analyzed using group independent component analysis (ICA) with dual regression and results indicated that compared to placebo, the highest dose of zolpidem increased functional connectivity within a number of sensory, motor, and limbic networks. These results are consistent with previous studies showing an increase in functional connectivity at rest following administration of the positive GABAA receptor modulators midazolam and alcohol, and suggest that investigating how zolpidem modulates intrinsic brain activity may have implications for understanding the etiology of its powerful sedative effects. PMID:23296183

The lack of bicuculline and picrotoxin influence on midazolam depressant action on brain oxygen consumption.

PubMed

Obradović, Dragan I; Savić, Miroslav M; Obradović, Miljana M; Ugresić, Nenad D; Bokonjić, Dubravko R

2006-04-24

In the previous study of the rat frontal cortex slices oxygen consumption (QO2), polarographically determined using the biological oxygen monitor, a moderate respiratory depressant action of midazolam ex vivo (1.0 mg/kg) has been observed. Antagonist of the benzodiazepine binding site, flumazenil, blocked the effect of the agonist. However, midazolam-gamma-aminobutyric acid (GABA) interactions pointed to the possibility that a part of midazolam action is independent of the classical GABA potentiation. To test this presumption, GABAA receptor antagonists bicuculline and picrotoxin were administered. Both blockers antagonized the QO2 reducing effect of the combination of per se effective doses of midazolam (1.0 mg/kg) and GABA (5 x 10(-4) mol/l), as well as of GABA (5 x 10(-4) mol/l) itself. However, neither effects of midazolam (1.0 mg/kg) on its own, nor those of midazolam in presence of the physiological, per se ineffective, concentration of GABA (10(-6) mol/l), were susceptible to antagonism. These results show that ex vivo influence of midazolam on cerebral metabolic activity should be partly ascribed to some of its cellular mechanisms probably associated to the GABA modulation, but distinct from the standard GABA-potentiating effects of benzodiazepines.

Involvement of the K+-Cl- co-transporter KCC2 in the sensitization to morphine-induced hyperlocomotion under chronic treatment with zolpidem in the mesolimbic system.

PubMed

Shibasaki, Masahiro; Masukawa, Daiki; Ishii, Kazunori; Yamagishi, Yui; Mori, Tomohisa; Suzuki, Tsutomu

2013-06-01

Benzodiazepines are commonly used as sedatives, sleeping aids, and anti-anxiety drugs. However, chronic treatment with benzodiazepines is known to induce dependence, which is considered related to neuroplastic changes in the mesolimbic system. This study investigated the involvement of K(+) -Cl(-) co-transporter 2 (KCC2) in the sensitization to morphine-induced hyperlocomotion after chronic treatment with zolpidem [a selective agonist of γ-aminobutyric acid A-type receptor (GABAA R) α1 subunit]. In this study, chronic treatment with zolpidem enhanced morphine-induced hyperlocomotion, which is accompanied by the up-regulation of KCC2 in the limbic forebrain. We also found that chronic treatment with zolpidem induced the down-regulation of protein phosphatase-1 (PP-1) as well as the up-regulation of phosphorylated protein kinase C γ (pPKCγ). Furthermore, PP-1 directly associated with KCC2 and pPKCγ, whereas pPKCγ did not associate with KCC2. On the other hand, pre-treatment with furosemide (a KCC2 inhibitor) suppressed the enhancing effects of zolpidem on morphine-induced hyperlocomotion. These results suggest that the mesolimbic dopaminergic system could be amenable to neuroplastic change through a pPKCγ-PP-1-KCC2 pathway by chronic treatment with zolpidem. © 2013 International Society for Neurochemistry.

Metabotropic Glutamate Receptors in the Trafficking of Ionotropic Glutamate and GABAA Receptors at Central Synapses

PubMed Central

Xiao, Min-Yi; Gustafsson, Bengt; Niu, Yin-Ping

2006-01-01

The trafficking of ionotropic glutamate (AMPA, NMDA and kainate) and GABAA receptors in and out of, or laterally along, the postsynaptic membrane has recently emerged as an important mechanism in the regulation of synaptic function, both under physiological and pathological conditions, such as information processing, learning and memory formation, neuronal development, and neurodegenerative diseases. Non-ionotropic glutamate receptors, primarily group I metabotropic glutamate receptors (mGluRs), co-exist with the postsynaptic ionotropic glutamate and GABAA receptors. The ability of mGluRs to regulate postsynaptic phosphorylation and Ca2+ concentration, as well as their interactions with postsynaptic scaffolding/signaling proteins, makes them well suited to influence the trafficking of ionotropic glutamate and GABAA receptors. Recent studies have provided insights into how mGluRs may impose such an influence at central synapses, and thus how they may affect synaptic signaling and the maintenance of long-term synaptic plasticity. In this review we will discuss some of the recent progress in this area: i) long-term synaptic plasticity and the involvement of mGluRs; ii) ionotropic glutamate receptor trafficking and long-term synaptic plasticity; iii) the involvement of postsynaptic group I mGluRs in regulating ionotropic glutamate receptor trafficking; iv) involvement of postsynaptic group I mGluRs in regulating GABAA receptor trafficking; v) and the trafficking of postsynaptic group I mGluRs themselves. PMID:18615134

Metabotropic glutamate receptors in the trafficking of ionotropic glutamate and GABA(A) receptors at central synapses.

PubMed

Xiao, Min-Yi; Gustafsson, Bengt; Niu, Yin-Ping

2006-01-01

The trafficking of ionotropic glutamate (AMPA, NMDA and kainate) and GABA(A) receptors in and out of, or laterally along, the postsynaptic membrane has recently emerged as an important mechanism in the regulation of synaptic function, both under physiological and pathological conditions, such as information processing, learning and memory formation, neuronal development, and neurodegenerative diseases. Non-ionotropic glutamate receptors, primarily group I metabotropic glutamate receptors (mGluRs), co-exist with the postsynaptic ionotropic glutamate and GABA(A) receptors. The ability of mGluRs to regulate postsynaptic phosphorylation and Ca(2+) concentration, as well as their interactions with postsynaptic scaffolding/signaling proteins, makes them well suited to influence the trafficking of ionotropic glutamate and GABA(A) receptors. Recent studies have provided insights into how mGluRs may impose such an influence at central synapses, and thus how they may affect synaptic signaling and the maintenance of long-term synaptic plasticity. In this review we will discuss some of the recent progress in this area: i) long-term synaptic plasticity and the involvement of mGluRs; ii) ionotropic glutamate receptor trafficking and long-term synaptic plasticity; iii) the involvement of postsynaptic group I mGluRs in regulating ionotropic glutamate receptor trafficking; iv) involvement of postsynaptic group I mGluRs in regulating GABA(A) receptor trafficking; v) and the trafficking of postsynaptic group I mGluRs themselves.

A Multifaceted GABAA Receptor Modulator: Functional Properties and Mechanism of Action of the Sedative-Hypnotic and Recreational Drug Methaqualone (Quaalude).

PubMed

Hammer, Harriet; Bader, Benjamin M; Ehnert, Corina; Bundgaard, Christoffer; Bunch, Lennart; Hoestgaard-Jensen, Kirsten; Schroeder, Olaf H-U; Bastlund, Jesper F; Gramowski-Voß, Alexandra; Jensen, Anders A

2015-08-01

In the present study, we have elucidated the functional characteristics and mechanism of action of methaqualone (2-methyl-3-o-tolyl-4(3H)-quinazolinone, Quaalude), an infamous sedative-hypnotic and recreational drug from the 1960s-1970s. Methaqualone was demonstrated to be a positive allosteric modulator at human α1,2,3,5β2,3γ2S GABAA receptors (GABAARs) expressed in Xenopus oocytes, whereas it displayed highly diverse functionalities at the α4,6β1,2,3δ GABAAR subtypes, ranging from inactivity (α4β1δ), through negative (α6β1δ) or positive allosteric modulation (α4β2δ, α6β2,3δ), to superagonism (α4β3δ). Methaqualone did not interact with the benzodiazepine, barbiturate, or neurosteroid binding sites in the GABAAR. Instead, the compound is proposed to act through the transmembrane β((+))/α((-)) subunit interface of the receptor, possibly targeting a site overlapping with that of the general anesthetic etomidate. The negligible activities displayed by methaqualone at numerous neurotransmitter receptors and transporters in an elaborate screening for additional putative central nervous system (CNS) targets suggest that it is a selective GABAAR modulator. The mode of action of methaqualone was further investigated in multichannel recordings from primary frontal cortex networks, where the overall activity changes induced by the compound at 1-100 μM concentrations were quite similar to those mediated by other CNS depressants. Finally, the free methaqualone concentrations in the mouse brain arising from doses producing significant in vivo effects in assays for locomotion and anticonvulsant activity correlated fairly well with its potencies as a modulator at the recombinant GABAARs. Hence, we propose that the multifaceted functional properties exhibited by methaqualone at GABAARs give rise to its effects as a therapeutic and recreational drug. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Weaker control of the electrical properties of cerebellar granule cells by tonically active GABAA receptors in the Ts65Dn mouse model of Down’s syndrome

PubMed Central

2013-01-01

Background Down’s syndrome (DS) is caused by triplication of all or part of human chromosome 21 and is characterized by a decrease in the overall size of the brain. One of the brain regions most affected is the cerebellum, in which the number of granule cells (GCs) is markedly decreased. GCs process sensory information entering the cerebellum via mossy fibres and pass it on to Purkinje cells and inhibitory interneurons. How GCs transform incoming signals depends on their input–output relationship, which is adjusted by tonically active GABAA receptor channels. Results We report that in the Ts65Dn mouse model of DS, in which cerebellar volume and GC number are decreased as in DS, the tonic GABAA receptor current in GCs is smaller than in wild-type mice and is less effective in moderating input resistance and raising the minimum current required for action potential firing. We also find that tonically active GABAA receptors curb the height and broaden the width of action potentials in wild-type GCs but not in Ts65Dn GCs. Single-cell real-time quantitative PCR reveals that these electrical differences are accompanied by decreased expression of the gene encoding the GABAA receptor β3 subunit but not genes coding for some of the other GABAA receptor subunits expressed in GCs (α1, α6, β2 and δ). Conclusions Weaker moderation of excitability and action potential waveform in GCs of the Ts65Dn mouse by tonically active GABAA receptors is likely to contribute to atypical transfer of information through the cerebellum. Similar changes may occur in DS. PMID:23870245

Grp94 Protein Delivers γ-Aminobutyric Acid Type A (GABAA) Receptors to Hrd1 Protein-mediated Endoplasmic Reticulum-associated Degradation.

PubMed

Di, Xiao-Jing; Wang, Ya-Juan; Han, Dong-Yun; Fu, Yan-Lin; Duerfeldt, Adam S; Blagg, Brian S J; Mu, Ting-Wei

2016-04-29

Proteostasis maintenance of γ-aminobutyric acid type A (GABAA) receptors dictates their function in controlling neuronal inhibition in mammalian central nervous systems. However, as a multisubunit, multispan, integral membrane protein, even wild type subunits of GABAA receptors fold and assemble inefficiently in the endoplasmic reticulum (ER). Unassembled and misfolded subunits undergo ER-associated degradation (ERAD), but this degradation process remains poorly understood for GABAA receptors. Here, using the α1 subunits of GABAA receptors as a model substrate, we demonstrated that Grp94, a metazoan-specific Hsp90 in the ER lumen, uses its middle domain to interact with the α1 subunits and positively regulates their ERAD. OS-9, an ER-resident lectin, acts downstream of Grp94 to further recognize misfolded α1 subunits in a glycan-dependent manner. This delivers misfolded α1 subunits to the Hrd1-mediated ubiquitination and the valosin-containing protein-mediated extraction pathway. Repressing the initial ERAD recognition step by inhibiting Grp94 enhances the functional surface expression of misfolding-prone α1(A322D) subunits, which causes autosomal dominant juvenile myoclonic epilepsy. This study clarifies a Grp94-mediated ERAD pathway for GABAA receptors, which provides a novel way to finely tune their function in physiological and pathophysiological conditions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

No association of the GABAA receptor genes on chromosome 5 with alcoholism in the collaborative study on the genetics of alcoholism sample.

PubMed

Dick, Danielle M; Edenberg, Howard J; Xuei, Xiaoling; Goate, Alison; Hesselbrock, Victor; Schuckit, Marc; Crowe, Raymond; Foroud, Tatiana

2005-01-05

A substantial body of literature suggests that gamma-aminobutyric acid (GABA) may be involved in the neurochemical pathways contributing to alcohol use and related disorders. Chromosome 5 contains a cluster of GABA(A) receptor genes, GABRA1, GABRA6, GABRB2, and GABRG2, which have been among the most extensively studied in relation to alcohol use. These studies have yielded mixed results. Using data from large, multiplex alcoholic families collected as part of the Collaborative Study on the Genetics of Alcoholism (COGA), we sought to provide more conclusive evidence regarding the role of the GABA(A) receptor genes on chromosome 5. Multiple single nucleotide polymorphisms (SNPs) were tested in each of the four chromosome 5q GABA(A) receptor genes, and we conducted both classic trio-based association analyzes and extended pedigree analyzes. We found no consistent evidence of association with alcohol dependence or alcohol dependence comorbid with antisocial personality disorder (ASPD) for any of the regions tested in the chromosome 5 GABA(A) receptor genes. These analyses suggest that the GABA(A) receptor genes on chromosome 5 do not play a strong role in alcohol dependence. Future studies are planned to test whether these genes are more important in influencing behavioral endophenotypes related to the risk of alcohol dependence. Copyright 2004 Wiley-Liss, Inc.

Prefrontal gamma-aminobutyric acid type A receptor insertion controls cue-induced relapse to nicotine seeking.

PubMed

Lubbers, Bart R; van Mourik, Yvar; Schetters, Dustin; Smit, August B; De Vries, Taco J; Spijker, Sabine

2014-11-01

Current smoking cessation therapies offer limited success, as relapse rates remain high. Nicotine, which is the major component of tobacco smoke, is thought to be primarily responsible for the addictive properties of tobacco. However, little is known about the molecular mechanisms underlying nicotine relapse, hampering development of more effective therapies. The objective of this study was to elucidate the role of medial prefrontal cortex (mPFC) glutamatergic and gamma-aminobutyric acid (GABA)ergic receptors in controlling relapse to nicotine seeking. Using an intravenous self-administration model, we studied glutamate and gamma-aminobutyric acid receptor regulation in the synaptic membrane fraction of the rat mPFC following extinction and cue-induced relapse to nicotine seeking. Subsequently, we locally intervened at the level of GABAergic signaling by using a mimetic peptide of the GABA receptor associated protein-interacting domain of GABA type A (GABAA) receptor subunit γ2 (TAT-GABAγ2) and muscimol, a GABAA receptor agonist. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and N-methyl-D-aspartate receptors were not regulated after the 30-min relapse test. However, GABAA receptor subunits α1 and γ2 were upregulated, and interference with GABAA receptor insertion in the cell membrane using the TAT-GABAγ2 peptide in the dorsal mPFC, but not the ventral mPFC, significantly increased responding during relapse. Increasing GABAA transmission with muscimol in the dorsal and ventral mPFC attenuated relapse. These data indicate that cue-induced relapse entails a GABAergic plasticity mechanism that limits nicotine seeking by restoring inhibitory control in the dorsal mPFC. GABAA receptor-mediated neurotransmission in the dorsal mPFC constitutes a possible future therapeutic target for maintaining smoking abstinence. Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Cell type specificity of GABA(A) receptor mediated signaling in the hippocampus.

PubMed

Semyanov, A

2003-08-01

Inhibitory signaling mediated by ionotropic GABA(1) receptors generally acts as a major brake against excessive excitability in the brain. This is especially relevant in epilepsy-prone structures such as the hippocampus, in which GABA(A) receptor mediated inhibition is critical in suppressing epileptiform activity. Indeed, potentiating GABA(A) receptor mediated signaling is an important target for antiepileptic drug therapy. GABA(A) receptor mediated inhibition has different roles in the network dependent on the target neuron. Inhibiting principal cells will thus reduce network excitability, whilst inhibiting interneurons will increase network excitability; GABAergic therapeutic agents do not distinguish between these two alternatives, which may explain why, on occasion, GABAergic antiepileptic drugs can be proconvulsant. The importance of the target-cell for the effect of neuroactive drugs has emerged from a number of recent studies. Immunocytochemical data have suggested non-uniform distribution of GABA(A) receptor subunits among hippocampal interneurons and pyramidal cells. This has been confirmed by subsequent electropharmacological data. These have demonstrated that compounds which act on GABA(A) receptors or the extracellular GABA concentration can have distinct effects in different neuronal populations. Recently, it has also been discovered that presynaptic glutamate heteroreceptors can modulate GABA release in the hippocampus in a postsynaptic cell-specific manner. Since systemically administrated drugs may act on different neuronal subtypes, they can exhibit paradoxical effects. Distinguishing compounds that have target specific effects on GABAergic signaling may lead to novel and more effective treatments against epilepsy.

Zolpidem dependence and withdrawal seizure--report of two cases.

PubMed

Wang, Liang-Jen; Ree, Shao-Chun; Chu, Chin-Lin; Juang, Yeong-Yuh

2011-03-01

Zolpidem is a non-benzodiazepine property which binds selectively to the ?1-GABAA receptors, and has been widely prescribed to patients suffering from insomnia. We report two cases of zolpidem dependence with withdrawal seizure in the Asian population. The first case is a 43-year-old woman who took zolpidem up to the dosage of 200 to 400 mg per night. The second case is a 35-year-old woman who even began to take zolpidem every 15 to 30 minutes to get euphoric and relaxed, and she gradually increased the dosage to 400 to 500mg per day. After abrupt discontinuation of zolpidem, both cases immediately developed anxiety, global insomnia, restlessness, and tonic seizure. The purpose of this case report is to suggest that clinicians should pay close attention to the potential of zolpidem tolerance, abuse and dependence. The possibility of withdrawal seizure cannot be excluded especially at high doses.

Modulation of neuronal and recombinant GABAA receptors by redox reagents

PubMed Central

Amato, Alessandra; Connolly, Christopher N; Moss, Stephen J; Smart, Trevor G

1999-01-01

The functional role played by the postulated disulphide bridge in γ-aminobutyric acid type A (GABAA) receptors and its susceptibility to oxidation and reduction were studied using recombinant (murine receptor subunits expressed in human embryonic kidney cells) and rat neuronal GABAA receptors in conjunction with whole-cell and single channel patch-clamp techniques. The reducing agent dithiothreitol (DTT) reversibly potentiated GABA-activated responses (IGABA) of α1β1 or α1β2 receptors while the oxidizing reagent 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) caused inhibition. Redox modulation of IGABA was independent of GABA concentration, membrane potential and the receptor agonist and did not affect the GABA EC50 or Hill coefficient. The endogenous antioxidant reduced glutathione (GSH) also potentiated IGABA in α1β2 receptors, while both the oxidized form of DTT and glutathione (GSSG) caused small inhibitory effects. Recombinant receptors composed of α1β1γ2S or α1β2γ2S were considerably less sensitive to DTT and DTNB. For neuronal GABAA receptors, IGABA was enhanced by flurazepam and relatively unaffected by redox reagents. However, in cultured sympathetic neurones, nicotinic acetylcholine-activated responses were inhibited by DTT whilst in cerebellar granule neurones, NMDA-activated currents were potentiated by DTT and inhibited by DTNB. Single GABA-activated ion channel currents exhibited a conductance of 16 pS for α1β1 constructs. DTT did not affect the conductance or individual open time constants determined from dwell time histograms, but increased the mean open time by affecting the channel open probability without increasing the number of cell surface receptors. A kinetic model of the effects of DTT and DTNB suggested that the receptor existed in equilibrium between oxidized and reduced forms. DTT increased the rate of entry into reduced receptor forms and also into desensitized states. DTNB reversed these kinetic effects. Our results indicate that GABAA receptors formed by α and β subunits are susceptible to regulation by redox agents. Inclusion of the γ2 subunit in the receptor, or recording from some neuronal GABAA receptors, resulted in reduced sensitivity to DTT and DTNB. Given the suggested existence of αβ subunit complexes in some areas of the central nervous system together with the generation and release of endogenous redox compounds, native GABAA receptors may be subject to regulation by redox mechanisms. PMID:10226147

GABAA Receptors, Anesthetics and Anticonvulsants in Brain Development

PubMed Central

Henschel, Oliver; Gipson, Keith E.; Bordey, Angelique

2008-01-01

GABA, acting via GABAA receptors, is well-accepted as the main inhibitory neurotransmitter of the mature brain, where it dampens neuronal excitability. The receptor's properties have been studied extensively, yielding important information about its structure, pharmacology, and regulation that are summarized in this review. Several GABAergic drugs have been commonly used as anesthetics, sedatives, and anticonvulsants for decades. However, findings that GABA has critical functions in brain development, in particular during the late embryonic and neonatal period, raise worthwhile questions regarding the side effects of GABAergic drugs that may lead to long-term cognitive deficits. Here, we will review some of these drugs in parallel with the control of CNS development that GABA exerts via activation of GABAA receptors. This review aims to provide a basic science and clinical perspective on the function of GABA and related pharmaceuticals acting at GABAA receptors. PMID:18537647

Dual Modulators of GABA-A and Alpha 7 Nicotinic Receptors for Treating Autism

DTIC Science & Technology

2015-10-01

AWARD NUMBER: W81XWH-13-1-0144 TITLE: Dual Modulators of GABA-A and Alpha 7 Nicotinic Receptors for Treating Autism PRINCIPAL INVESTIGATOR...SUBTITLE 5a. CONTRACT NUMBER Dual Modulators of GABA-A and Alpha 7 Nicotinic Receptors for Treating Autism 5b. GRANT NUMBER W81XWH-13-1-0144 5c...ABSTRACT Autism spectrum disorder (ASD) is a polygenic signaling disorder that may result, in part, from an imbalance in excitatory and inhibitory

Novel analogues of chlormethiazole are neuroprotective in four cellular models of neurodegeneration by a mechanism with variable dependence on GABAA receptor potentiation

PubMed Central

VandeVrede, Lawren; Tavassoli, Ehsan; Luo, Jia; Qin, Zhihui; Yue, Lan; Pepperberg, David R; Thatcher, Gregory R

2014-01-01

Background and Purpose: Chlormethiazole (CMZ), a clinical sedative/anxiolytic agent, did not reach clinical efficacy in stroke trials despite neuroprotection demonstrated in numerous animal models. Using CMZ as a lead compound, neuroprotective methiazole (MZ) analogues were developed, and neuroprotection and GABAA receptor dependence were studied. Experimental Approach: Eight MZs were selected from a novel library, of which two were studied in detail. Neuroprotection, glutamate release, intracellular calcium and response to GABA blockade by picrotoxin were measured in rat primary cortical cultures using four cellular models of neurodegeneration. GABA potentiation was assayed in oocytes expressing the α1β2γ2 GABAA receptor. Key Results: Neuroprotection against a range of insults was retained even with substantial chemical modification. Dependence on GABAA receptor activity was variable: at the extremes, neuroprotection by GN-28 was universally sensitive to picrotoxin, while GN-38 was largely insensitive. In parallel, effects on extracellular glutamate and intracellular calcium were associated with GABAA dependence. Consistent with these findings, GN-28 potentiated α1β2γ2 GABAA function, whereas GN-38 had a weak inhibitory effect. Neuroprotection against moderate dose oligomeric Aβ1–42 was also tolerant to structural changes. Conclusions and Implications: The results support the concept that CMZ does not contain a single pharmacophore, rather that broad-spectrum neuroprotection results from a GABAA-dependent mechanism represented by GN-28, combined with a mechanism represented in GN-38 that shows the least dependence on GABAA receptors. These findings allow further refinement of the neuroprotective pharmacophore and investigation into secondary mechanisms that will assist in identifying MZ-based compounds of use in treating neurodegeneration. PMID:24116891

Pharmacodynamic and pharmacokinetic effects of TPA023, a GABA(A) alpha(2,3) subtype-selective agonist, compared to lorazepam and placebo in healthy volunteers.

PubMed

de Haas, S L; de Visser, S J; van der Post, J P; de Smet, M; Schoemaker, R C; Rijnbeek, B; Cohen, A F; Vega, J M; Agrawal, N G B; Goel, T V; Simpson, R C; Pearson, L K; Li, S; Hesney, M; Murphy, M G; van Gerven, J M A

2007-06-01

TPA023, a GABA(A) alpha2,3 alphasubtype-selective partial agonist, is expected to have comparable anxiolytic efficacy as benzodiazepines with reduced sedating effects. The compound lacks efficacy at the alpha1 subtype, which is believed to mediate these effects. This study investigated the effects of 0.5 and 1.5 mg TPA023 and compared them with placebo and lorazepam 2 mg (therapeutic anxiolytic dose). Twelve healthy male volunteers participated in this placebo-controlled, double-blind, double-dummy, four-way, cross-over study. Saccadic eye movements and visual analogue scales (VAS) were used to assess the sedative properties of TPA023. The effects on posturaL stability and cognition were assessed using body sway and a standardized battery of neurophysiological memory tests. Lorazepam caused a significant reduction in saccadic peak velocity, the VAS alertness score and impairment of memory and body sway. TPA023 had significant dose dependent effects on saccadic peak velocity (85 deg/sec maximum reduction at the higher dose) that approximated the effects of lorazepam. In contrast to lorazepam, TPA023 had no detectabLe effects on saccadic latency or inaccuracy. Also unlike lorazepam, TPA023 did not affect VAS alertness, memory or body sway. These results show that the effect profile of TPA023 differs markedly from that of lorazepam, at doses that were equipotent with regard to effects on saccadic peak veLocity. Contrary to lorazepam, TPA023 caused no detectable memory impairment or postural imbalance. These differences reflect the selectivity of TPA023 for different GABA(A) receptor subtypes.

Aripiprazole Increases the PKA Signalling and Expression of the GABAA Receptor and CREB1 in the Nucleus Accumbens of Rats.

PubMed

Pan, Bo; Lian, Jiamei; Huang, Xu-Feng; Deng, Chao

2016-05-01

The GABAA receptor is implicated in the pathophysiology of schizophrenia and regulated by PKA signalling. Current antipsychotics bind with D2-like receptors, but not the GABAA receptor. The cAMP-responsive element-binding protein 1 (CREB1) is also associated with PKA signalling and may be related to the positive symptoms of schizophrenia. This study investigated the effects of antipsychotics in modulating D2-mediated PKA signalling and its downstream GABAA receptors and CREB1. Rats were treated orally with aripiprazole (0.75 mg/kg, ter in die (t.i.d.)), bifeprunox (0.8 mg/kg, t.i.d.), haloperidol (0.1 mg/kg, t.i.d.) or vehicle for 1 week. The levels of PKA-Cα and p-PKA in the prefrontal cortex (PFC), nucleus accumbens (NAc) and caudate putamen (CPu) were detected by Western blots. The mRNA levels of Gabrb1, Gabrb2, Gabrb3 and Creb1, and their protein expression were measured by qRT-PCR and Western blots, respectively. Aripiprazole elevated the levels of p-PKA and the ratio of p-PKA/PKA in the NAc, but not the PFC and CPu. Correlated with this elevated PKA signalling, aripiprazole elevated the mRNA and protein expression of the GABAA (β-1) receptor and CREB1 in the NAc. While haloperidol elevated the levels of p-PKA and the ratio of p-PKA/PKA in both NAc and CPu, it only tended to increase the expression of the GABAA (β-1) receptor and CREB1 in the NAc, but not the CPu. Bifeprunox had no effects on PKA signalling in these brain regions. These results suggest that aripiprazole has selective effects on upregulating the GABAA (β-1) receptor and CREB1 in the NAc, probably via activating PKA signalling.

Α2 GABAA receptor sub-units in the ventral hippocampus and α5 GABAA receptor sub-units in the dorsal hippocampus mediate anxiety and fear memory.

PubMed

McEown, K; Treit, D

2013-11-12

Temporary neuronal inactivation of the ventral hippocampus with the GABAA agonist muscimol suppresses unconditioned fear behavior (anxiety) but inactivation of the dorsal hippocampus does not. On the other hand, inactivating the dorsal hippocampus disrupts fear memory, while inactivating the ventral hippocampus does not. Here we investigate the roles of hippocampal GABAA receptor sub-units in mediating these anxiolytic and amnesic effects of GABAA receptor agonists. We microinfused TPA023 (α2 agonist) or TB-21007 (inverse α5 agonist) into the dorsal or ventral hippocampus prior to testing rats in two animal models of anxiety: the elevated plus-maze and shock-probe burying test. Twenty-four hours later rats were re-tested in the shock-probe chamber with a non-electrified probe to assess their memory of the initial shock-probe experience (i.e., fear memory). We found that TPA023 was anxiolytic in the plus-maze and shock-probe burying tests when microinfused into the ventral hippocampus. However, TPA023 did not affect anxiety-related behavior when infused into the dorsal hippocampus. Conversely, we found that the α5 sub-unit inverse agonist TB-21007 impaired rats' memory of the initial shock-probe experience when infused into the dorsal hippocampus, but not when infused into the ventral hippocampus. This double dissociation suggests that α2 GABAA receptor sub-units in the ventral hippocampus mediate unconditioned fear or anxiety, while α5 GABAA receptor sub-units in the dorsal hippocampus mediate conditioned fear memory. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Alteration of GABAergic synapses and gephyrin clusters in the thalamic reticular nucleus of GABAA receptor alpha3 subunit-null mice.

PubMed

Studer, Remo; von Boehmer, Lotta; Haenggi, Tatjana; Schweizer, Claude; Benke, Dietmar; Rudolph, Uwe; Fritschy, Jean-Marc

2006-09-01

Multiple GABAA-receptor subtypes are assembled from alpha, beta and gamma subunit variants. GABAA receptors containing the alpha3 subunit represent a minor population with a restricted distribution in the CNS. In addition, they predominate in monoaminergic neurons and in the nucleus reticularis thalami (nRT), suggesting a role in the regulation of cortical function and sleep. Mice with a targeted deletion of the alpha3 subunit gene (alpha3(0/0)) are viable and exhibit a subtle behavioural phenotype possibly related to dopaminergic hyperfunction. Here, we investigated immunohistochemically the consequences of the loss of alpha3 subunit for maturation of GABAA receptors and formation of GABAergic synapses in the nRT. Throughout postnatal development, the regional distribution of the alpha1, alpha2, or alpha5 subunit was unaltered in alpha3(0/0) mice and the prominent alpha3 subunit staining of nRT neurons in wildtype mice was not replaced. Subcellularly, as seen by double immunofluorescence, the alpha3 and gamma2 subunit were clustered at postsynaptic sites in the nRT of adult wildtype mice along with the scaffolding protein gephyrin. In alpha3(0/0) mice, gamma2 subunit clustering was disrupted and gephyrin formed large aggregates localized at the cell surface, but unrelated to postsynaptic sites, indicating that nRT neurons lack postsynaptic GABAA receptors in mutant mice. Furthermore, GABAergic terminals were enlarged and reduced in number, suggesting a partial deficit of GABAergic synapses. Therefore, GABAA receptors are required for gephyrin clustering and long-term synapse maintenance. The absence of GABAA-mediated transmission in the nRT may have a significant impact on the function of the thalamo-cortical loop of alpha3(0/0) mice.

Towards functional selectivity for α6β3γ2 GABAA receptors: a series of novel pyrazoloquinolinones

PubMed Central

Treven, Marco; Siebert, David C B; Holzinger, Raphael; Bampali, Konstantina; Fabjan, Jure; Varagic, Zdravko; Wimmer, Laurin; Steudle, Friederike; Scholze, Petra; Schnürch, Michael; Mihovilovic, Marko D

2017-01-01

Background and Purpose The GABAA receptors are ligand‐gated ion channels, which play an important role in neurotransmission. Their variety of binding sites serves as an appealing target for many clinically relevant drugs. Here, we explored the functional selectivity of modulatory effects at specific extracellular α+/β− interfaces, using a systematically varied series of pyrazoloquinolinones. Experimental Approach Recombinant GABAA receptors were expressed in Xenopus laevis oocytes and modulatory effects on GABA‐elicited currents by the newly synthesized and reference compounds were investigated by the two‐electrode voltage clamp method. Key Results We identified a new compound which, to the best of our knowledge, shows the highest functional selectivity for positive modulation at α6β3γ2 GABAA receptors with nearly no residual activity at the other αxβ3γ2 (x = 1–5) subtypes. This modulation was independent of affinity for α+/γ− interfaces. Furthermore, we demonstrated for the first time a compound that elicits a negative modulation at specific extracellular α+/β− interfaces. Conclusion and Implications These results constitute a major step towards a potential selective positive modulation of certain α6‐containing GABAA receptors, which might be useful to elicit their physiological role. Furthermore, these studies pave the way towards insights into molecular principles that drive positive versus negative allosteric modulation of specific GABAA receptor isoforms. PMID:29127702

Cyclodextrins sequester neuroactive steroids and differentiate mechanisms that rate limit steroid actions

PubMed Central

Shu, H-J; Zeng, C-M; Wang, C; Covey, D F; Zorumski, C F; Mennerick, S

2006-01-01

Background and purpose: Neuroactive steroids are potent modulators of GABAA receptors and are thus of interest for their sedative, anxiolytic, anticonvulsant and anaesthetic properties. Cyclodextrins may be useful tools to manipulate neuroactive effects of steroids on GABAA receptors because cyclodextrins form inclusion complexes with at least some steroids that are active at the GABAA receptor, such as (3α,5α)-3-hydroxypregnan-20-one (3α5αP, allopregnanolone). Experimental approach: To assess the versatility of cyclodextrins as steroid modulators, we investigated interactions between γ-cyclodextrin and neuroactive steroids of different structural classes. Key results: Both a bioassay based on electrophysiological assessment of GABAA receptor function and optical measurements of cellular accumulation of a fluorescent steroid analogue suggest that γ-cyclodextrin sequesters steroids rather than directly influencing GABAA receptor function. Neither a 5β-reduced A/B ring fusion nor a sulphate group at carbon 3 affected the presumed inclusion complex formation between steroid and γ-cyclodextrin. Apparent dissociation constants for interactions between natural steroids and γ-cyclodexrin ranged from 10-60 μM. Although γ-cyclodextrin accommodates a range of natural and synthetic steroids, C11 substitutions reduced inclusion complex formation. Using γ-cyclodextrin to remove steroid not directly bound to GABAA receptors, we found that cellular retention of receptor-unbound steroid rate limits potentiation by 3α- hydroxysteroids but not inhibition by sulphated steroids. Conclusions and implications: We conclude that γ-cyclodextrins can be useful, albeit non-specific, tools for terminating the actions of multiple classes of naturally occurring neuroactive steroids. PMID:17160009

α5-GABAA receptors negatively regulate MYC-amplified medulloblastoma growth

PubMed Central

Sengupta, Soma; Weeraratne, Shyamal Dilhan; Sun, Hongyu; Phallen, Jillian; Rallapalli, Sundari K.; Teider, Natalia; Kosaras, Bela; Amani, Vladimir; Pierre-Francois, Jessica; Tang, Yujie; Nguyen, Brian; Yu, Furong; Schubert, Simone; Balansay, Brianna; Mathios, Dimitris; Lechpammer, Mirna; Archer, Tenley C.; Tran, Phuoc; Reimer, Richard J.; Cook, James M.; Lim, Michael; Jensen, Frances E.; Pomeroy, Scott L.; Cho, Yoon-Jae

2013-01-01

Neural tumors often express neurotransmitter receptors as markers of their developmental lineage. Although these receptors have been well characterized in electrophysiological, developmental and pharmacological settings, their importance in the maintenance and progression of brain tumors, and importantly, the effect of their targeting in brain cancers remains obscure. Here, we demonstrate high levels of GABR5, which encodes the α-subunit of the GABAA receptor complex, in aggressive MYC-driven, “Group 3” medulloblastomas. We hypothesized that modulation of α-GABAA receptors alters medulloblastoma cell survival and monitored biological and electrophysiological responses of GABR5-expressing medulloblastoma cells upon pharmacological targeting of the GABAA receptor. While antagonists, inverse agonists and non-specific positive allosteric modulators had limited effects on medulloblastoma cells, a highly specific and potent α5-GABAA receptor agonist, QHii066, resulted in marked membrane depolarization and a significant decrease in cell survival. This effect was GABR5 dependent and mediated through the induction of apoptosis as well as accumulation of cells in S and G2 phases of the cell cycle. Chemical genomic profiling of QHii066-treated medulloblastoma cells confirmed inhibition of MYC-related transcriptional activity and revealed an enrichment of HOX5 target gene expression. siRNA-mediated knockdown of HOX5 markedly blunted the response of medulloblastoma cells to QHii066. Furthermore, QHii066 sensitized GABR5 positive medulloblastoma cells to radiation and chemotherapy consistent with the role of HOX5 in directly regulating p53 expression and inducing apoptosis. Thus, our results provide novel insights into the synthetic lethal nature of α5-GABAA receptor activation in MYC-driven/Group 3 medulloblastomas and propose its targeting as a novel strategy for the management of this highly aggressive tumor. PMID:24196163

Qualitative variation of photolabelled benzodiazepine receptors in different species.

PubMed

Hebebrand, J; Friedl, W; Lentes, K U; Propping, P

1986-01-01

In order to examine whether species differences of benzodiazepine receptor subunits exist, we compared the fluorographic pattern of photoaffinity labelled subunits after SDS-PAGE in five species: fish, frog, chicken, mouse and calf. Each species showed a distinct pattern of specifically labelled proteins. We conclude that species variation of benzodiazepine receptor does indeed exist.

Exploring Pharmacological Mechanisms of Lavender (Lavandula angustifolia) Essential Oil on Central Nervous System Targets

PubMed Central

López, Víctor; Nielsen, Birgitte; Solas, Maite; Ramírez, Maria J.; Jäger, Anna K.

2017-01-01

Lavender essential oil is traditionally used and approved by the European Medicines Agency (EMA) as herbal medicine to relieve stress and anxiety. Some animal and clinical studies reveal positive results in models of anxiety and depression although very little research has been done on molecular mechanisms. Our work consisted of evaluating the effects of lavender (Lavandula angustifolia) essential oil on central nervous system well-established targets, such as MAO-A, SERT, GABAAand NMDA receptors as well as in vitro models of neurotoxicity. The results showed that lavender essential oil and its main components exert affinity for the glutamate NMDA-receptor in a dose-dependent manner with an IC50 value of 0.04 μl/mL for lavender oil. In addition, lavender and linalool were also able to bind the serotonin transporter (SERT) whereas they did not show affinity for GABAA-benzodiazepine receptor. In three different models of neurotoxicity, lavender did not enhance the neurotoxic insult and improved viability of SH-SY5Y cells treated with hydrogen peroxide. According to our data, the anxiolytic and antidepressant-like effects attributed to lavender may be due to an antagonism on the NMDA-receptor and inhibition of SERT. This study suggests that lavender essential oil may exert pharmacological properties via modulating the NMDA receptor, the SERT as well as neurotoxicity induced by hydrogen peroxide. PMID:28579958

SB-205384 Is a Positive Allosteric Modulator of Recombinant GABAA Receptors Containing Rat α3, α5, or α6 Subunit Subtypes Coexpressed with β3 and γ2 Subunits

PubMed Central

Heidelberg, Laura S.; Warren, James W.

2013-01-01

Many drugs used to treat anxiety are positive modulators of GABAA receptors, which mediate fast inhibitory neurotransmission. The GABAA receptors can be assembled from a combination of at least 16 different subunits. The receptor’s subunit composition determines its pharmacologic and functional properties, and subunit expression varies throughout the brain. A primary goal for new treatments targeting GABAA receptors is the production of subunit-selective modulators acting upon a discrete population of receptors. The anxiolytic 4-amino-7-hydroxy-2-methyl-5,6,7,8,-tetrahydrobenzo[b]thieno[2,3-b]pyridine-3-carboxylic acid, but-2-ynyl ester (SB-205384) is widely considered to be selective for α3-containing GABAA receptors. However, it has been tested only on α1-, α2-, and α3-containing receptors. We examined the activity of SB-205384 at recombinant receptors containing the six different α subunits and found that receptors containing the α3, α5, and α6 subunits were potentiated by SB-205384, with the α6 subunit conferring the greatest responsiveness. Properties associated with chimeric α1/α6 subunits suggested that multiple structural domains influence sensitivity to SB-205384. Point mutations of residues within the extracellular N-terminal domain identified a leucine residue located in loop E of the agonist binding site as an important determinant of high sensitivity to modulation. In the α6 subunit the identity of this residue is species-dependent, with the leucine found in rat subunits but not in human. Our results indicate that SB-205384 is not an α3-selective modulator, and instead acts at several GABAA receptor isoforms. These findings have implications for the side-effect profile of this anxiolytic as well as for its use in neuronal and animal studies as a marker for contribution from α3-containing receptors. PMID:23902941

Modulatory Effects of Eschscholzia californica Alkaloids on Recombinant GABAA Receptors

PubMed Central

Fedurco, Milan; Gregorová, Jana; Šebrlová, Kristýna; Kantorová, Jana; Peš, Ondřej; Baur, Roland; Sigel, Erwin; Táborská, Eva

2015-01-01

The California poppy (Eschscholzia californica Cham.) contains a variety of natural compounds including several alkaloids found exclusively in this plant. Because of the sedative, anxiolytic, and analgesic effects, this herb is currently sold in pharmacies in many countries. However, our understanding of these biological effects at the molecular level is still lacking. Alkaloids detected in E. californica could be hypothesized to act at GABAA receptors, which are widely expressed in the brain mainly at the inhibitory interneurons. Electrophysiological studies on a recombinant α 1 β 2 γ 2 GABAA receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 μM. However, (S)-reticuline behaved as positive allosteric modulator at the α 3, α 5, and α 6 isoforms of GABAA receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by (S)-reticuline at the α 3 β 2 γ 2 and α 5 β 2 γ 2 GABAA receptors. Interestingly, α 1, α 3, and α 5 were not significantly affected by (R)-reticuline, 1,2-tetrahydroreticuline, codeine, and morphine—suspected (S)-reticuline metabolites in the rodent brain. PMID:26509084

Increased thermolability of benzodiazepine receptors in cerebral cortex of a baboon with spontaneous seizures: a case report.

PubMed

Squires, R; Naquet, R; Riche, D; Braestrup, C

1979-06-01

The benzodiazepine receptor in the cortex of 1 spontaneously epileptic baboon exhibited an increased rate of thermal inactivation at 65 degrees C when compared with those from 3 other baboons. In other respects (receptor concentration, affinities for flunitrazepam and diazepam, and response to changing pH), the benzodiazepine receptor from this animal was very similar to the receptors in the cortex of 3 other baboons. The 3H-QNB (muscarinic) and 3H-naloxone (opiate) binding sites in the brain of all 4 baboons appeared very similar with respect to all parameters studied (thermal stability, concentration, regional distribution, and affinities for respective ligands). An endogenous factor stabilizing the benzodiazepine receptor could be lacking in the spontaneously epileptic baboon.

Extrasynaptic GABAA receptors in the crosshairs of hormones and ethanol

PubMed Central

Mody, Istvan

2008-01-01

Gamma-aminobutyric acid (GABA) is the main chemical inhibitory neurotransmitter in the brain. In the central nervous system (CNS) it acts on two distinct types of receptor: an ion channel, i.e., an “ionotropic” receptor permeable to Cl− and HCO3− (GABAA receptors) and a G-protein coupled “metabotropic” receptor that is linked to various effector mechanisms (GABAB receptors). This review will summarize novel developments in the physiology and pharmacology of GABAA receptors (GABAARs), specifically those found outside synapses. The focus will be on a particular combination of GABAAR subunits sensitive to ovarian and adrenal cortical steroid hormone metabolites that are synthesized in the brain (neurosteroids) and to sobriety impairing concentrations of ethanol. These receptors may be the final common pathway for interactions between ethanol and ovarian and stress-related neurosteroids. PMID:17714830

Characterization of GABAA receptor ligands with automated patch-clamp using human neurons derived from pluripotent stem cells

PubMed Central

Yuan, Nina Y.; Poe, Michael M.; Witzigmann, Christopher; Cook, James M.; Stafford, Douglas; Arnold, Leggy A.

2016-01-01

Introduction Automated patch clamp is a recent but widely used technology to assess pre-clinical drug safety. With the availability of human neurons derived from pluripotent stem cells, this technology can be extended to determine CNS effects of drug candidates, especially those acting on the GABAA receptor. Methods iCell Neurons (Cellular Dynamics International, A Fujifilm Company) were cultured for ten days and analyzed by patch clamp in the presence of agonist GABA or in combination with positive allosteric GABAA receptor modulators. Both efficacy and affinity were determined. In addition, mRNA of GABAA receptor subunits were quantified by qRT-PCR. Results We have shown that iCell Neurons are compatible with the IonFlux microfluidic system of the automated patch clamp instrument. Resistance ranging from 15-25 MΩ was achieved for each trap channel of patch clamped cells in a 96-well plate format. GABA induced a robust change of current with an EC50 of 0.43 μM. Positive GABAA receptor modulators diazepam, HZ166, and CW-04-020 exhibited EC50 values of 0.42 μM, 1.56 μM, and 0.23 μM, respectively. The α2/α3/α5 selective compound HZ166-induced the highest potentiation (efficacy) of 810% of the current induced by 100 nM GABA. Quantification of GABAA receptor mRNA in iCell Neurons revealed high levels of α5 and β3 subunits and low levels of α1, which is similar to the configuration in human neonatal brain. Discussion iCell Neurons represent a new cellular model to characterize GABAergic compounds using automated patch clamp. These cells have excellent representation of cellular GABAA receptor distribution that enable determination of total small molecule efficacy and affinity as measured by cell membrane current change. PMID:27544543

Antianxiety effect of cannabis: involvement of central benzodiazepine receptors.

PubMed

Sethi, B B; Trivedi, J K; Kumar, P; Gulati, A; Agarwal, A K; Sethi, N

1986-01-01

The present work, involving clinical, behavioral, and biochemical studies, was undertaken to elucidate the probable mechanism of the observed antianxiety effects of cannabis. The population for the clinical study consisted of 50 male chronic cannabis users who were otherwise healthy and 50 matched controls. When evaluated on Taylor's Manifest Anxiety Scale (TMA), these subjects had low anxiety scores as compared with the controls. To explore the possible interaction of cannabis with the benzodiazepine receptors, behavioral and biochemical studies in mice were devised, involving acute and chronic cannabis administration. Behavioral study revealed that mice under chronic cannabis treatment scored significantly higher on foot shock-induced aggression, but this was significantly blocked by benzodiazepine receptor antagonist. Furthermore, chronic cannabis treatment significantly (p less than 0.001) increased the frequency of licking response periodically punished by shocks. This confirms the antianxiety effect of cannabis, which also appears to be mediated through a benzodiazepine receptor, as it was reduced significantly (p less than 0.001) by a benzodiazepine receptor blocker. Specific 3H-diazepam binding was carried out in frontal cortex to assess both the population and affinity of benzodiazepine receptors. Our results indicate that acute cannabis treatment has no significant effect, whereas chronic cannabis treatment significantly increased 3H-diazepam binding as compared with controls. Scatchard analysis further reveals that increased affinity is responsible for increased binding to these receptors. It is therefore our contention that the antianxiety effect of cannabis is mediated through central benzodiazepine receptors.

Lateral Orbitofrontal Cortical Modulation on the Medial Prefrontal Cortex-Amygdala Pathway: Differential Regulation of Intra-Amygdala GABAA and GABAB Receptors.

PubMed

Chang, Chun-Hui

2017-07-01

The basolateral complex of the amygdala receives inputs from neocortical areas, including the medial prefrontal cortex and lateral orbitofrontal cortex. Earlier studies have shown that lateral orbitofrontal cortex activation exerts an inhibitory gating on medial prefrontal cortex-amygdala information flow. Here we examined the individual role of GABAA and GABAB receptors in this process. In vivo extracellular single-unit recordings were done in anesthetized rats. We searched amygdala neurons that fire in response to medial prefrontal cortex activation, tested lateral orbitofrontal cortex gating at different delays (lateral orbitofrontal cortex-medial prefrontal cortex delays: 25, 50, 100, 250, 500, and 1000 milliseconds), and examined differential contribution of GABAA and GABAB receptors with iontophoresis. Relative to baseline, lateral orbitofrontal cortex stimulation exerted an inhibitory modulatory gating on the medial prefrontal cortex-amygdala pathway and was effective up to a long delay of 500 ms (long-delay latencies at 100, 250, and 500 milliseconds). Moreover, blockade of intra-amygdala GABAA receptors with bicuculline abolished the lateral orbitofrontal cortex inhibitory gating at both short- (25 milliseconds) and long-delay (100 milliseconds) intervals, while blockade of GABAB receptors with saclofen reversed the inhibitory gating at long delay (100 milliseconds) only. Among the majority of the neurons examined (8 of 9), inactivation of either GABAA or GABAB receptors during baseline did not change evoked probability per se, suggesting that local feed-forward inhibitory mechanism is pathway specific. Our results suggest that the effect of lateral orbitofrontal cortex inhibitory modulatory gating was effective up to 500 milliseconds and that intra-amygdala GABAA and GABAB receptors differentially modulate the short- and long-delay lateral orbitofrontal cortex inhibitory gating on the medial prefrontal cortex-amygdala pathway. © The Author 2017. Published by Oxford University Press on behalf of CINP.

GABRG2, rs211037 is associated with epilepsy susceptibility, but not with antiepileptic drug resistance and febrile seizures.

PubMed

Balan, Shabeesh; Sathyan, Sanish; Radha, Saradalekshmi K; Joseph, Vijai; Radhakrishnan, Kurupath; Banerjee, Moinak

2013-11-01

Several antiepileptic drugs (AEDs) are known to target the GABA(A) receptor through positive allosteric modulation of the receptors, thereby enhancing GABA(A) receptor-mediated inhibition. The large diversity of GABA(A) receptors has been reported in the central nervous system; some of these have been implicated in epilepsy susceptibility and AED resistance, which we aimed to examine. We investigated the association of single-nucleotide polymorphisms in GABA(A) receptor subunit subtype genes namely; rs2279020 (GABRA1), rs3219151 (GABRA6), rs2229944 (GABRB2), and rs211037 (GABRG2) with predisposition to epilepsy and AED resistance. This was assessed in three cohorts of ethnically matched South Indian ancestry: mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (prototype of AED-resistant epilepsy syndrome), juvenile myoclonic epilepsy (prototype of AED-responsive epilepsy syndrome), and nonepilepsy controls. A significant allelic (P=0.0006, odds ratio=1.6, 95% confidence interval=1.22-2.08) and genotypic (P=0.001) association of a synonymous variant in GABRG2, rs211037 (Asn196Asn) was observed with epilepsy irrespective of its phenotype, that is, MTLE-HS or juvenile myoclonic epilepsy. However, this association was not retained in epilepsy patients with a history of febrile seizures. The GABA(A) receptor subunit subtype genes were not found to have any association with AED resistance. In-silico analysis indicated that rs211037 plays a significant role in the transcriptional regulation and splicing regulation. We could substantiate that among the GABA(A) receptor subunit gene cluster polymorphisms, the GABRG2, rs211037 predisposes susceptibility to epilepsy, irrespective of its phenotype, but not to AED resistance.

Involvement of the CA1 GABAA receptors in MK-801-induced anxiolytic-like effects: an isobologram analysis.

PubMed

Naseri, Mohammad-Hasan; Hesami-Tackallou, Saeed; Torabi-Nami, Mohammad; Zarrindast, Mohammad-Reza; Nasehi, Mohammad

2014-06-01

There seems to be a close relationship between hippocampal N-methyl-D-aspartic acid (NMDA) and GABAA receptors with respect to the modulation of behavior that occurs in the CA1 region of the hippocampus. This study investigated the possible involvement of the CA1 GABAA receptors in anxiolytic-like effects induced by (+)-MK-801 (a noncompetitive antagonist of the NMDA subtype of the glutamate receptor). Male Wistar rats were subjected to the elevated plus-maze apparatus and open arm time (%OAT), and open arm entries (%OAE) for anxiety-related behaviors, and closed arm entries that correspond to the locomotor activity were assessed. An intra-CA1 injection of (+)-MK-801 (2 μg/rat) and muscimol (0.5 μg/rat; a GABAA receptor agonist) increased %OAT and %OAE by themselves while not altering the closed arm entries, indicating an anxiolytic-like effect of these drugs. Injection of bicuculline (0.1, 0.25, and 0.5 μg/rat; a GABAA receptor antagonist) did not alter any of the anxiety-related parameters. An intra-CA1 injection of a subthreshold dose of muscimol (0.1 μg/rat) or bicuculline (0.5 μg/rat), 5 min before injection of subthreshold and effective doses of (+)-MK-801 (0.5, 1 and 2 μg/rat), increased and decreased the anxiolytic-like effect of (+)-MK-801, respectively. The isobologram analysis of these findings suggested a synergistic anxiety-like effect of intra-CA1 (+)-MK-801 and muscimol. In conclusion, the CA1 GABAA receptors appear to be involved in anxiolytic-like behaviors induced by (+)-MK-801.

Perinatal intermittent hypoxia alters γ-aminobutyric acid: a receptor levels in rat cerebellum.

PubMed

Pae, Eung-Kwon; Yoon, Audrey J; Ahuja, Bhoomika; Lau, Gary W; Nguyen, Daniel D; Kim, Yong; Harper, Ronald M

2011-12-01

Perinatal hypoxia commonly causes brain injury in infants, but the time course and mechanisms underlying the preferential male injury are unclear. Intermittent hypoxia disturbs cerebellar γ-aminobutyric (GABA)-A receptor profiles during the perinatal period, possibly responding to transient excitatory processes associated with GABA(A) receptors. We examined whether hypoxic insults were particularly damaging to the male rodent cerebellum during a specific developmental time window. We evaluated cerebellar injury and GABA(A) receptor profiles following 5-h intermittent hypoxia (IH: 20.8% and 10.3% ambient oxygen, switched every 240s) or room-air control in groups of male and female rat pups on postnatal d 1-2, wk 1, or wk 3. The cerebella were harvested and compared between groups. The mRNA levels of GABA(A) receptors α6, normalized to a house-keeping gene GAPDH, and assessed using real-time reverse-transcriptase PCR assays were up-regulated by IH at wk 1, more extensively in male rats, with sex influencing the regulatory time-course. In contrast, GABA(A) α6 receptor protein expression levels, assessed using Western blot assays, reached a nadir at wk 1 in both male and female rats, possibly indicating involvement of a post-transcriptional mechanism. The extent of cerebellar damage and level of apoptosis, assessed by DNA fragmentation, were greatest in the wk 3 IH-exposed group. The findings suggest partial protection for female rats against early hypoxic insult in the cerebellum, and that down-regulation of GABA(A) receptors, rather than direct neural injury assessed by DNA fragmentation may modify cerebellar function, with potential later motor and other deficits. Copyright © 2011 ISDN. Published by Elsevier Ltd. All rights reserved.

GABA(A) receptors in visual and auditory cortex and neural activity changes during basic visual stimulation.

PubMed

Qin, Pengmin; Duncan, Niall W; Wiebking, Christine; Gravel, Paul; Lyttelton, Oliver; Hayes, Dave J; Verhaeghe, Jeroen; Kostikov, Alexey; Schirrmacher, Ralf; Reader, Andrew J; Northoff, Georg

2012-01-01

Recent imaging studies have demonstrated that levels of resting γ-aminobutyric acid (GABA) in the visual cortex predict the degree of stimulus-induced activity in the same region. These studies have used the presentation of discrete visual stimulus; the change from closed eyes to open also represents a simple visual stimulus, however, and has been shown to induce changes in local brain activity and in functional connectivity between regions. We thus aimed to investigate the role of the GABA system, specifically GABA(A) receptors, in the changes in brain activity between the eyes closed (EC) and eyes open (EO) state in order to provide detail at the receptor level to complement previous studies of GABA concentrations. We conducted an fMRI study involving two different modes of the change from EC to EO: an EO and EC block design, allowing the modeling of the haemodynamic response, followed by longer periods of EC and EO to allow the measuring of functional connectivity. The same subjects also underwent [(18)F]Flumazenil PET to measure GABA(A) receptor binding potentials. It was demonstrated that the local-to-global ratio of GABA(A) receptor binding potential in the visual cortex predicted the degree of changes in neural activity from EC to EO. This same relationship was also shown in the auditory cortex. Furthermore, the local-to-global ratio of GABA(A) receptor binding potential in the visual cortex also predicted the change in functional connectivity between the visual and auditory cortex from EC to EO. These findings contribute to our understanding of the role of GABA(A) receptors in stimulus-induced neural activity in local regions and in inter-regional functional connectivity.

A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of γ-Aminobutyric Acid, Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes.

PubMed

Woll, Kellie A; Murlidaran, Sruthi; Pinch, Benika J; Hénin, Jérôme; Wang, Xiaoshi; Salari, Reza; Covarrubias, Manuel; Dailey, William P; Brannigan, Grace; Garcia, Benjamin A; Eckenhoff, Roderic G

2016-09-23

Propofol, an intravenous anesthetic, is a positive modulator of the GABAA receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol, AziPm-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured ∼4% of the synaptosomal proteome, including the unbiased capture of five α or β GABAA receptor subunits. Lack of γ2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for α/β than γ-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABAA receptor subunit interfaces, with stable hydrogen bonds observed between propofol and α/β cavity residues but not γ cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABAA receptor subunit protection. This investigation demonstrates striking interfacial GABAA receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of γ-Aminobutyric Acid, Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes*

PubMed Central

Woll, Kellie A.; Murlidaran, Sruthi; Pinch, Benika J.; Hénin, Jérôme; Wang, Xiaoshi; Salari, Reza; Covarrubias, Manuel; Dailey, William P.; Brannigan, Grace; Garcia, Benjamin A.; Eckenhoff, Roderic G.

2016-01-01

Propofol, an intravenous anesthetic, is a positive modulator of the GABAA receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol, AziPm-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured ∼4% of the synaptosomal proteome, including the unbiased capture of five α or β GABAA receptor subunits. Lack of γ2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for α/β than γ-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABAA receptor subunit interfaces, with stable hydrogen bonds observed between propofol and α/β cavity residues but not γ cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABAA receptor subunit protection. This investigation demonstrates striking interfacial GABAA receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity. PMID:27462076

A therapeutic dose of zolpidem reduces thalamic GABA in healthy volunteers: A proton MRS study at 4 Tesla

PubMed Central

Licata, Stephanie C.; Jensen, J. Eric; Penetar, David M.; Prescot, Andrew P.; Lukas, scott E.; Renshaw, Perry F.

2009-01-01

Background Zolpidem is a non-benzodiazepine sedative/hypnotic that acts at GABAA receptors to influence inhibitory neurotransmission throughout the central nervous system. A great deal is known about the behavioral effects of this drug in humans and laboratory animals, but little is known about zolpidem’s specific effects on neurochemistry in vivo. Objectives We evaluated how acute administration of zolpidem affected levels of GABA, glutamate, glutamine, and other brain metabolites. Methods Proton magnetic resonance spectroscopy (1H MRS) at 4 Tesla was employed to measure the effects of zolpidem on brain chemistry in 19 healthy volunteers. Participants underwent scanning following acute oral administration of a therapeutic dose of zolpidem (10 mg) in a within-subject, single-blind, placebo-controlled, single-visit study. In addition to neurochemical measurements from single voxels within the anterior cingulate (ACC) and thalamus, a series of questionnaires were administered periodically throughout the experimental session to assess subjective mood states. Results Zolpidem reduced GABA levels in the thalamus, but not the ACC. There were no treatment effects with respect to other metabolite levels. Self-reported ratings of “dizzy”, “nauseous”, “confused”, and “bad effects” were increased relative to placebo, as were ratings on the sedation/intoxication (PCAG) and psychotomimetic/dysphoria (LSD) scales of the Addiction Research Center Inventory. Moreover, there was a significant correlation between the decrease in GABA and “dizzy”. Conclusions Zolpidem engendered primarily dysphoric-like effects and the correlation between reduced thalamic GABA and “dizzy” may be a function of zolpidem’s interaction with α1GABAA receptors in the cerebellum, projecting through the vestibular system to the thalamus. PMID:19125238

GABA-mediated changes in inter-hemispheric beta frequency activity in early-stage Parkinson’s disease

PubMed Central

Hall, S.D.; Prokic, E.J.; McAllister, C.J.; Ronnqvist, K.C.; Williams, A.C.; Yamawaki, N.; Witton, C.; Woodhall, G.L.; Stanford, I.M.

2014-01-01

In Parkinson’s disease (PD), elevated beta (15–35 Hz) power in subcortical motor networks is widely believed to promote aspects of PD symptomatology, moreover, a reduction in beta power and coherence accompanies symptomatic improvement following effective treatment with l-DOPA. Previous studies have reported symptomatic improvements that correlate with changes in cortical network activity following GABAA receptor modulation. In this study we have used whole-head magnetoencephalography to characterize neuronal network activity, at rest and during visually cued finger abductions, in unilaterally symptomatic PD and age-matched control participants. Recordings were then repeated following administration of sub-sedative doses of the hypnotic drug zolpidem (0.05 mg/kg), which binds to the benzodiazepine site of the GABAA receptor. A beamforming based ‘virtual electrode’ approach was used to reconstruct oscillatory power in the primary motor cortex (M1), contralateral and ipsilateral to symptom presentation in PD patients or dominant hand in control participants. In PD patients, contralateral M1 showed significantly greater beta power than ipsilateral M1. Following zolpidem administration contralateral beta power was significantly reduced while ipsilateral beta power was significantly increased resulting in a hemispheric power ratio that approached parity. Furthermore, there was highly significant correlation between hemispheric beta power ratio and Unified Parkinson’s Disease Rating Scale (UPDRS). The changes in contralateral and ipsilateral beta power were reflected in pre-movement beta desynchronization and the late post-movement beta rebound. However, the absolute level of movement-related beta desynchronization was not altered. These results show that low-dose zolpidem not only reduces contralateral beta but also increases ipsilateral beta, while rebalancing the dynamic range of M1 network oscillations between the two hemispheres. These changes appear to underlie the symptomatic improvements afforded by low-dose zolpidem. PMID:25261686

Expression of functional receptors by the human γ-aminobutyric acid A γ2 subunit

PubMed Central

Martínez-Torres, Ataúlfo; Miledi, Ricardo

2004-01-01

γ-Aminobutyric acid A (GABAA) receptors are heteromeric membrane proteins formed mainly by various combinations of α, β, and γ subunits; and it is commonly thought that the γ2 subunit alone does not form functional receptors. In contrast, we found that cDNA encoding the γ2L subunit of the human GABAA receptor, injected alone into Xenopus oocytes, expressed functional GABA receptors whose properties were investigated by using the two-microelectrode voltage-clamp technique. GABA elicited desensitizing membrane currents that recovered after a few minutes' wash. Repetitive applications of GABA induced a “run-up” of GABA currents that nearly doubled the amplitude of the first response. The GABA currents inverted direction at about -30 mV, indicating that they are carried mainly by Cl- ions. The homomeric γ2L receptors were also activated by β-alanine > taurine > glycine, and, like some types of heteromeric GABAA receptors, the γ2L receptors were blocked by bicuculline and were potentiated by pentobarbital and flunitrazepam. These results indicate that the human γ2L subunit is capable of forming fully functional GABA receptors by itself in Xenopus oocytes and suggest that the roles proposed for the various subunits that make up the heteromeric GABAA receptors in situ require further clarification. PMID:14981251

Behavioral differences between subgroups of rats with high and low threshold to clonic convulsions induced by DMCM, a benzodiazepine inverse agonist.

PubMed

Contó, Marcos Brandão; de Carvalho, José Gilberto Barbosa; Benedito, Marco Antonio Campana

2005-11-01

In epileptic patients, there is a high incidence of psychiatric comorbidities, such as anxiety. Gamma-aminobutyric acid (GABA) ionotropic receptor GABA(A)/benzodiazepine allosteric site is involved in both epilepsy and anxiety. This involvement is based on the fact that benzodiazepine allosteric site agonists are anticonvulsant and anxiolytic drugs; on the other hand, benzodiazepine inverse agonists are potent convulsant and anxiogenic drugs. The aim of this work was to determine if subgroups of rats selected according to their susceptibility to clonic convulsions induced by a convulsant dose 50% (CD50) of DMCM, a benzodiazepine inverse agonist, would differ in behavioral tests commonly used to measure anxiety (elevated plus-maze, open field) and depression (forced swimming test). In the first experiment, subgroups of adult male Wistar rats were selected after a single dose of DMCM and in the second experiment they were selected after two injections of DMCM given after an interval of 1 week. Those rats presenting full clonic convulsions were termed Low Threshold rats to DMCM-induced clonic convulsions (LTR) and those not having clonic convulsions High Threshold rats to DMCM-induced clonic convulsions (HTR). In both experiments, only those rats presenting full clonic convulsions induced by DMCM and those not showing any signs of motor disturbances were used in the behavioral tests. The results showed that the LTR subgroup selected after two injections of a CD50 of DMCM spent a significantly lower time in the open arms of the elevated plus-maze and in the off the walls area of the open field; moreover, this group also presented a higher number of rearings in the open field. There were no significant differences between HTR and LTR subgroups in the forced swimming test. LTR and HTR subgroups selected after only one injection of DMCM did not differ in the three behavioral tests. To verify if the behavioral differences between HTR and LTR subgroups of rats selected after two injections of DMCM were due to the clonic convulsion, another experiment was carried out in which subgroups of rats susceptible and nonsusceptible to clonic convulsions induced by a CD50 of picrotoxin, a GABA(A) receptor channel blocker, were selected and submitted to the elevated plus-maze and open field tests. The results obtained did not show any significant differences between these two subgroups in the elevated plus-maze and open field tests. In another approach to determine the relation between fear/anxiety and susceptibility to clonic convulsions, subgroups of rats were selected in the elevated plus-maze as more or less fearful/anxious. The CD50 for clonic convulsions induced by DMCM was determined for each of these two subgroups. The results showed a significantly lower CD50 for the more fearful/anxious subgroup, which means a higher susceptibility to clonic convulsions induced by DMCM. The present findings show a relation between susceptibility to clonic convulsions and fear/anxiety and vice versa which may be due to differences in the assembly of GABA(A)/allosteric benzodiazepine site receptors in regions of the brain.

Characterization of ( sup 3 H)alprazolam binding to central benzodiazepine receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCabe, R.T.; Mahan, D.R.; Smith, R.B.

1990-10-01

The binding of the triazolobenzodiazepine ({sup 3}H)alprazolam was studied to characterize the in vitro interactions with benzodiazepine receptors in membrane preparations of rat brain. Studies using nonequilibrium and equilibrium binding conditions for ({sup 3}H)alprazolam resulted in high specific to nonspecific (signal to noise) binding ratios. The binding of ({sup 3}H)alprazolam was saturable and specific with a low nanomolar affinity for benzodiazepine receptors in the rat brain. The Kd was 4.6 nM and the Bmax was 2.6 pmol/mg protein. GABA enhanced ({sup 3}H)alprazolam binding while several benzodiazepine receptor ligands were competitive inhibitors of this drug. Compounds that bind to other receptormore » sites had a very weak or negligible effect on ({sup 3}H)alprazolam binding. Alprazolam, an agent used as an anxiolytic and in the treatment of depression, acts in vitro as a selective and specific ligand for benzodiazepine receptors in the rat brain. The biochemical binding profile does not appear to account for the unique therapeutic properties which distinguish this compound from the other benzodiazepines in its class.« less

Gamma-Aminobutyric acid and benzodiazepine receptors in the kindling model of epilepsy: a quantitative radiohistochemical study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shin, C.; Pedersen, H.B.; McNamara, J.O.

1985-10-01

Quantitative radiohistochemistry was utilized to study alterations of gamma-aminobutyric acid (GABA) and benzodiazepine receptors in the kindling model of epilepsy. The radioligands used for GABA and benzodiazepine receptors were (TH) muscimol and (TH)flunitrazepam, respectively. GABA receptor binding was increased by 22% in fascia dentata of the hippocampal formation but not in neocortex or substantia nigra of kindled rats. Within fascia dentata, GABA receptor binding was increased to an equivalent extent in stratum granulosum and throughout stratum moleculare; no increase was found in dentate hilus or stratum lacunosummoleculare or stratum radiatum of CA1. The increased binding was present at 24 hrmore » but not at 28 days after the last kindled seizure. The direction, anatomic distribution, and time course of the increased GABA receptor binding were paralleled by increased benzodiazepine receptor binding. The anatomic distribution of the increased GABA receptor binding is consistent with a localization to somata and dendritic trees of dentate granule cells. The authors suggest that increased GABA and benzodiazepine receptor binding may contribute to enhanced inhibition of dentate granule cells demonstrated electrophysiologically in kindled animals.« less

Diversity in GABAergic signaling.

PubMed

Vogt, Kaspar

2015-01-01

GABA(A) receptor-mediated synaptic transmission is responsible for inhibitory control of neural function in the brain. Recent progress has shown that GABA(A) receptors also provide a wide range of additional functions beyond simple inhibition. This diversity of functions is mediated by a large variety of different interneuron classes acting on a diverse population of receptor subtypes. Here, I will focus on an additional source of GABAergic signaling diversity, caused by the highly variable ion signaling mechanism of GABA(A) receptors. In concert with the other two sources of GABAergic heterogeneity, this variability in signaling allows for a wide array of GABAergic effects that are crucial for the development of the brain and its function. © 2015 Elsevier Inc. All rights reserved.

The natural products magnolol and honokiol are positive allosteric modulators of both synaptic and extra-synaptic GABAA receptors

PubMed Central

Alexeev, Mikhail; Grosenbaugh, Denise K.; Mott, David D.; Fisher, Janet L.

2012-01-01

The National Center for Complementary and Alternative Medicine (NCCAM) estimates that nearly 40% of adults in the United States use alternative medicines, often in the form of an herbal supplement. Extracts from the tree bark of magnolia species have been used for centuries in traditional Chinese and Japanese medicines to treat a variety of neurological diseases, including anxiety, depression, and seizures. The active ingredients in the extracts have been identified as the bi-phenolic isomers magnolol and honokiol. These compounds were shown to enhance the activity of GABAA receptors, consistent with their biological effects. The GABAA receptors exhibit substantial subunit heterogeneity, which influences both their functional and pharmacological properties. We examined the activity of magnolol and honokiol at different populations of both neuronal and recombinant GABAA receptors to characterize their mechanism of action and to determine whether sensitivity to modulation was dependent upon the receptor’s subunit composition. We found that magnolol and honokiol enhanced both phasic and tonic GABAergic neurotransmission in hippocampal dentate granule neurons. In addition, all recombinant receptors examined were sensitive to modulation, regardless of the identity of the α, β, or γ subunit subtype, although the compounds showed particularly high efficacy at δ-containing receptors. This direct positive modulation of both synaptic and extra-synaptic populations of GABAA receptors suggests that supplements containing magnolol and/or honokiol would be effective anxiolytics, sedatives, and anti-convulsants. However, significant side-effects and risk of drug interactions would also be expected. PMID:22445602

Somato-synaptic variation of GABA(A) receptors in cultured murine cerebellar granule cells: investigation of the role of the alpha6 subunit.

PubMed

Mellor, J R; Wisden, W; Randall, A D

2000-07-10

Electrophysiological investigation of cultured cerebellar murine granule cells revealed differences between the GABA(A) receptors at inhibitory synapses and those on the cell body. Specifically, mIPSCs decayed more rapidly than cell body receptors deactivated, the mean single channel conductance at the synapse (32 pS) was greater than that at cell body (21 pS) and only cell body receptors were sensitive to Zn(2+) (150 microM), which depressed response amplitude by 82+/-5% and almost doubled the rate of channel deactivation. The GABA(A) receptor alpha6 subunit is selectively expressed in cerebellar granule cells. Although concentrated at synapses, it is also found on extrasynaptic membranes. Using a mouse line (Deltaalpha6lacZ) lacking this subunit, we investigated its role in the somato-synaptic differences in GABA(A) receptor function. All differences between cell body and synaptic GABA(A) receptors observed in wild-type (WT) granule cells persisted in Deltaalpha6lacZ cells, thus demonstrating that they are not specifically due to the cellular distribution of the alpha6 subunit. However, mIPSCs from WT and Deltaalpha6lacZ cells differed in both their kinetics (faster decay in WT cells) and underlying single channel conductance (32 pS WT, 25 pS Deltaalpha6lacZ). This provides good evidence for a functional contribution of the alpha6 subunit to postsynaptic GABA(A) receptors in these cells. Despite this, deactivation kinetics of mIPSCs in WT and Deltaalpha6lacZ granule cells exhibited similar benzodiazepene (BDZ) sensitivity. This suggests that the enhanced BDZ-induced ataxia seen in Deltaalpha6lacZ mice may reflect physiological activity at extrasynaptic receptors which, unlike those at synapses, display differential BDZ-sensitivity in WT and Deltaalpha6lacZ granule cells (Jones, A.M., Korpi, E.R., McKernan, R.M., Nusser, Z., Pelz, R., Makela, R., Mellor, J.R., Pollard, S., Bahn, S., Stephenson, F.A., Randall, A.D., Sieghart, W., Somogyi, P., Smith, A.J.H., Wisden, W., 1997. Ligand-gated ion channel partnerships: GABA(A) receptor alpha(6) subunit inactivation inhibits delta subunit expression. Journal of Neuroscience 17, 1350-1362).

Purification of a benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain.

PubMed Central

Sangameswaran, L; Fales, H M; Friedrich, P; De Blas, A L

1986-01-01

An endogenous brain substance that binds to the central-type benzodiazepine receptors with agonist properties is present in both rat and bovine brains. This substance has been purified to homogeneity from bovine brain by immunoaffinity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reversed-phase HPLC steps. The purified substance was characterized as the benzodiazepine N-desmethyldiazepam (nordiazepam). The techniques used for the identification were mass spectrometry, HPLC, spectrophotometry, benzodiazepine receptor binding, and immunological techniques. Benzodiazepine-like immunoreactivity was also found in all the human brains tested, including six brains that had been stored in paraffin since 1940, fifteen years before the first synthesis of benzodiazepines. These results show that benzodiazepine-like molecules of natural origin--and possibly benzodiazepines themselves--are present in human and other mammalian brains. Images PMID:3024172

Insights into GABA receptor signalling in TM3 Leydig cells.

PubMed

Doepner, Richard F G; Geigerseder, Christof; Frungieri, Monica B; Gonzalez-Calvar, Silvia I; Calandra, Ricardo S; Raemsch, Romi; Fohr, Karl; Kunz, Lars; Mayerhofer, Artur

2005-01-01

Gamma-aminobutyric acid (GABA) is an emerging signalling molecule in endocrine organs, since it is produced by endocrine cells and acts via GABA(A) receptors in a paracrine/autocrine fashion. Testicular Leydig cells are producers and targets for GABA. These cells express GABA(A) receptor subunits and in the murine Leydig cell line TM3 pharmacological activation leads to increased proliferation. The signalling pathway of GABA in these cells is not known in this study. We therefore attempted to elucidate details of GABA(A) signalling in TM3 and adult mouse Leydig cells using several experimental approaches. TM3 cells not only express GABA(A )receptor subunits, but also bind the GABA agonist [(3)H]muscimol with a binding affinity in the range reported for other endocrine cells (K(d) = 2.740 +/- 0.721 nM). However, they exhibit a low B(max) value of 28.08 fmol/mg protein. Typical GABA(A) receptor-associated events, including Cl(-) currents, changes in resting membrane potential, intracellular Ca(2+) or cAMP, were not measurable with the methods employed in TM3 cells, or, as studied in part, in primary mouse Leydig cells. GABA or GABA(A) agonist isoguvacine treatment resulted in increased or decreased levels of several mRNAs, including transcription factors (c-fos, hsf-1, egr-1) and cell cycle-associated genes (Cdk2, cyclin D1). In an attempt to verify the cDNA array results and because egr-1 was recently implied in Leydig cell development, we further studied this factor. RT-PCR and Western blotting confirmed a time-dependent regulation of egr-1 in TM3. In the postnatal testis egr-1 was seen in cytoplasmic and nuclear locations of developing Leydig cells, which bear GABA(A) receptors and correspond well to TM3 cells. Thus, GABA acts via an atypical novel signalling pathway in TM3 cells. Further details of this pathway remain to be elucidated. Copyright (c) 2005 S. Karger AG, Basel.

Increased GABA(A) inhibition of the RVLM after hindlimb unloading in rats

NASA Technical Reports Server (NTRS)

Moffitt, Julia A.; Heesch, Cheryl M.; Hasser, Eileen M.

2002-01-01

Attenuated baroreflex-mediated increases in renal sympathetic nerve activity (RSNA) in hindlimb unloaded (HU) rats apparently are due to changes within the central nervous system. We hypothesized that GABA(A) receptor-mediated inhibition of the rostral ventrolateral medulla (RVLM) is increased after hindlimb unloading. Responses to bilateral microinjection of the GABA(A) antagonist (-)-bicuculline methiodide (BIC) into the RVLM were examined before and during caudal ventrolateral medulla (CVLM) inhibition in Inactin-anesthetized control and HU rats. Increases in mean arterial pressure (MAP), heart rate (HR), and RSNA in response to BIC in the RVLM were significantly enhanced in HU rats. Responses to bilateral CVLM blockade were not different. When remaining GABA(A) inhibition in the RVLM was blocked by BIC during CVLM inhibition, the additional increases in MAP and RSNA were significantly greater in HU rats. These data indicate that GABA(A) receptor-mediated inhibition of RVLM neurons is augmented after hindlimb unloading. Effects of input from the CVLM were unaltered. Thus, after cardiovascular deconditioning in rodents, the attenuated increase in sympathetic nerve activity in response to hypotension is associated with greater GABA(A) receptor-mediated inhibition of RVLM neurons originating at least in part from sources other than the CVLM.

Kindling and withdrawal changes at the benzodiazepine receptor.

PubMed

Little, H J; Nutt, D J; Taylor, S C

1987-01-01

Drugs acting at benzodiazepine receptors can have two types of pharmacological profile: benzodiazepine agonists are anxiolytic, anticonvulsant and sedative, whilst benzo diazepine inverse agonists cause anxiety and convulsions. In 1982 we showed that a benzo diazepine antagonist, Ro 15-1788, prevented the effects of both types of compound at doses without intrinsic activity in the tests used. We put forward the hypothesis that the benzo diazepine receptor complex could undergo two possible conformational changes, resulting in increases (benzodiazepine agonists) or decreases (benzodiazepine inverse agonists) in the effects of the inhibitory transmitter γ-aminobutyric acid (GABA). This concept has been widely accepted. We have now studied the effects of inverse agonists after chronic treatment with inverse agonists themselves and with benzodiazepine agonists, in order to see if tolerance develops (as seen with the agonists) or whether an opposite change occurs.

[The role of gamma-aminobutyric acid in the mechanism of action of anticonvulsant drugs].

PubMed

Chmielewska, B

2000-01-01

Decreased activity of gamma-aminobutyric acid, the major inhibitory neurotransmitter in CNS can be epileptogenic. Manipulation of the GABA system has been a target for development of antiepileptic drugs. The different ways for augmenting gabaergic inhibition by conventional and new AEDs are presented in this paper. Among the I generation, barbiturates and benzodiazepines are potent anticonvulsants that act as GABA modulators in postsynaptic GABA-A receptor complex but their usefulness is limited by dependence and tolerance to antiseizure activity. The II generation drugs vigabatrin and tiagabine, and to some extent gabapentin have been developed by a rationale strategy and none of them exert direct action in GABA receptors. Only two former drugs exhibit selective, strictly defined activity: vigabatrine is an irreversible inhibitor of GABA-aminotransferase and tiagabine acts as a GABA-uptake inhibitor from synaptic cleft into neurons and glia. Gabapentin binds to a novel receptors in epileptogenic areas in CNS and enhances GABA turnover. Drugs with multiple mechanisms of action, felbamate and topiramate not only potentiate gabaergic inhibition in several ways but also diminish the activity of excitatory amino acids at their NMDA or AMPA receptors; the later mechanism seems to be essential for their potential neuroprotective activity in epileptogenesis. None of gabamimetic drugs provide optimal seizure control but better tolerability of newer ones and well-established mechanisms of action provide possible harmless therapy.

Estrous cycle variations in GABAA receptor phosphorylation enable rapid modulation by anabolic androgenic steroids in the medial preoptic area

PubMed Central

Oberlander, JG; Porter, DM; Onakomaiya, MM; Penatti, CAA; Vithlani, M; Moss, SJ; Clark, AS; Henderson, LP

2012-01-01

Anabolic androgenic steroids (AAS), synthetic testosterone derivatives that are used for ergogenic purposes, alter neurotransmission and behaviors mediated by GABAA receptors. Some of these effects may reflect direct and rapid action of these synthetic steroids at the receptor. The ability of other natural allosteric steroid modulators to alter GABAA receptor-mediated currents is dependent upon the phosphorylation state of the receptor complex. Here we show that phosphorylation of the GABAA receptor complex immunoprecipitated by β2/β3 subunit-specific antibodies from the medial preoptic area (mPOA) of the mouse varies across the estrous cycle; with levels being significantly lower in estrus. Acute exposure to the AAS, 17α-testosterone (17α-MeT), had no effect on the amplitude or kinetics of inhibitory postsynaptic currents in the mPOA of estrous mice when phosphorylation was low, but increased the amplitude of these currents from mice in diestrus, when it was high. Inclusion of the protein kinase C (PKC) inhibitor, calphostin, in the recording pipette eliminated the ability of 17α-MeT to enhance currents from diestrous animals, suggesting that PKC-receptor phosphorylation is critical for the allosteric modulation elicited by AAS during this phase. In addition, a single injection of 17α-MeT was found to impair an mPOA-mediated behavior (nest-building) in diestrus, but not in estrus. PKC is known to target specific serine residues in the β3 subunit of the GABAA receptor. Although phosphorylation of these β3 serine residues showed a similar profile across the cycle, as did phosphoserine in mPOA lysates immunoprecipitated with β2/β3 antibody (lower in estrus than in diestrus or proestrus), the differences were not significant. These data suggest that the phosphorylation state of the receptor complex regulates both the ability of AAS to modulate receptor function in the mPOA and the expression of a simple mPOA-dependent behavior through PKC-dependent mechanism that involves the β3 subunit and other sites within the GABAA receptor complex. PMID:22989919

INTERACTION BETWEEN DELTA OPIOID RECEPTORS AND BENZODIAZEPINES IN CO2- INDUCED RESPIRATORY RESPONSES IN MICE

PubMed Central

Borkowski, Anne H.; Barnes, Dylan C.; Blanchette, Derek R.; Castellanos, F. Xavier; Klein, Donald F.; Wilson, Donald A.

2011-01-01

The false-suffocation hypothesis of panic disorder (Klein, 1993) suggested δ-opioid receptors as a possible source of the respiratory dysfunction manifested in panic attacks occurring in panic disorder (Preter and Klein, 2008). This study sought to determine if a lack of δ-opioid receptors in a mouse model affects respiratory response to elevated CO2, and whether the response is modulated by benzodiazepines, which are widely used to treat panic disorder. In a whole-body plethysmograph, respiratory responses to 5% CO2 were compared between δ-opioid receptor knockout mice and wild-type mice after saline, diazepam (1 mg/kg), and alprazolam (0.3 mg/kg) injection. The results show that lack of δ-opioid receptors does not affect normal response to elevated CO2, but does prevent benzodiazepines from modulating that response. Thus, in the presence of benzodiazepine agonists, respiratory responses to elevated CO2 were enhanced in δ-opioid receptor knockout mice compared to wild-type mice. This suggests an interplay between benzodiazepine receptors and δ-opioid receptors in regulating the respiratory effects of elevated CO2, which might be related to CO2 induced panic. PMID:21561601

Improvement in verbal memory following SSRI augmentation of antipsychotic treatment is associated with changes in the expression of mRNA encoding for the GABA-A receptor and BDNF in PMC of schizophrenic patients.

PubMed

Silver, Henry; Mandiuk, Nina; Einoch, Reef; Susser, Ehud; Danovich, Lena; Bilker, Warren; Youdim, Moussa; Weinreb, Orly

2015-05-01

Verbal memory impairment in schizophrenia is associated with abnormalities in gamma-aminobutyric acid (GABA)-ergic and brain-derived neurotrophic factor (BDNF) systems. Recent evidence from animal and clinical studies that adding fluvoxamine to antipsychotics alters the expression of transcripts encoding for the GABA-A receptor and BDNF led us to postulate that fluvoxamine augmentation may improve memory in schizophrenia. To test this, we examined the effect of add-on fluvoxamine on verbal memory and other cognitive functions and related it to the expression of mRNA coding for the GABA-A receptor and BDNF in peripheral mononuclear cells (PMC) of schizophrenic patients. Twenty-nine patients completed a 6-week study in which fluvoxamine (100 mg/day) was added to ongoing antipsychotic treatment. Verbal memory, abstraction working memory, object and face recognition, and psychomotor speed and clinical symptoms were assessed at baseline and after 3 and 6 weeks of treatment. Blood samples were taken at baseline and weeks 1, 3, and 6 and PMC was assayed for the GABA-A beta3 receptor and BDNF mRNA by quantitative real-time reverse transcription-PCR. Associative and logical verbal memory improved significantly and showed a significant correlation with changes in PMC BDNF and GABA-A beta3 receptor mRNA, which increased during treatment. Abstraction and object recognition improved, but this did not correlate with PMC measures. Negative and positive symptoms improved significantly; the latter showed significant correlations with changes in PMC measures. Addition of fluvoxamine to antipsychotics improves verbal memory. It is postulated that the mechanism involves enhanced GABA-A receptor/BDNF-dependent synaptic plasticity in the hippocampus.

Altered GABAA receptor-mediated synaptic transmission disrupts the firing of gonadotropin-releasing hormone neurons in male mice under conditions that mimic steroid abuse

PubMed Central

Penatti, Carlos A A; Davis, Matthew C; Porter, Donna M; Henderson, Leslie P

2010-01-01

Gonadotropin–releasing hormone (GnRH) neurons are the central regulators of reproduction. GABAergic transmission plays a critical role in pubertal activation of pulsatile GnRH secretion. Self-administration of excessive doses of anabolic androgenic steroids (AAS) disrupts reproductive function and may have critical repercussions for pubertal onset in adolescent users. Here, we demonstrate that chronic treatment of adolescent male mice with the AAS, 17α-methyltestosterone (17αMT), significantly decreased action potential frequency in GnRH neurons, reduced the serum gonadotropin levels, and decreased testes mass. AAS treatment did not induce significant changes in GABAA receptor subunit mRNA levels or alter the amplitude or decay kinetics of GABAA receptor-mediated spontaneous postsynaptic currents (sPSC) or tonic currents in GnRH neurons. However, AAS treatment significantly increased action potential frequency in neighboring medial preoptic area (mPOA) neurons and GABAA receptor-mediated sPSC frequency in GnRH neurons. In addition, physical isolation of the more lateral aspects of the mPOA from the medially-localized GnRH neurons abrogated the AAS-induced increase in GABAA receptor-mediated sPSC frequency and the decrease in action potential firing in the GnRH cells. Our results indicate that AAS act predominantly on steroid-sensitive presynaptic neurons within the mPOA to impart significant increases in GABAA receptor-mediated inhibitory tone onto downstream GnRH neurons resulting in diminished activity of these pivotal mediators of reproductive function. These AAS-induced changes in central GABAergic circuits of the forebrain may significantly contribute to the disruptive actions of these drugs on pubertal maturation and the development of reproductive competence in male steroid abusers. PMID:20463213

Expression of GABA receptor subunits in the hippocampus and thalamus after experimental traumatic brain injury

PubMed Central

Drexel, Meinrad; Puhakka, Noora; Kirchmair, Elke; Hörtnagl, Heide; Pitkänen, Asla; Sperk, Günther

2015-01-01

Traumatic brain injury is a major cause of death and disability worldwide and often associated with post-traumatic epilepsy. We recently demonstrated that TBI induces acquired GABAA receptors channelopathy that associates with hyperexcitability in granule cell layer (GCL). We now assessed the expression of GABAA and GABAB receptor subunit mRNAs between 6 h and 6 months post-TBI in the hippocampus and thalamus. The expression of major GABAA receptor subunit mRNAs (α1, α2, α5, β2, β3, γ2 and δ) was, often bilaterally, down-regulated in the GCL and in the CA3 pyramidal cells. Instead, expression of α4 (GCL, CA3, CA1), α5 (CA1) and γ2 (GCL, CA3, CA1) mRNA was up-regulated after 10 d and/or 4 months. Many of these changes were reversible. In the thalamus, we found decreases in α1, α4, β2, γ2 and δ mRNAs in the laterodorsal thalamus and in the area combining the posterior thalamic nuclear group, ventroposterolateral and ventroposteromedial complex at 6 h to 4 months post-TBI. Unlike in the hippocampus, thalamic subunit down-regulations were irreversible and limited to the ipsilateral side. However, contralaterally there was up-regulation of the subunits δ and α4 6 h and 4 months after TBI, respectively. PCR array analysis suggested a mild long-lasting GABAA receptor channelopathy in the GCL and thalamus after TBI. Whereas TBI induces transient changes in the expression of GABAA receptor subunits in the hippocampus (presumably representing compensatory mechanisms), alterations of GABAA receptor subunit mRNAs in the thalamus are long-lasting and related to degeneration of receptor-containing neurons in thalamo-cortical relay nuclei. This article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’. PMID:25229716

Expression of GABA receptor subunits in the hippocampus and thalamus after experimental traumatic brain injury.

PubMed

Drexel, Meinrad; Puhakka, Noora; Kirchmair, Elke; Hörtnagl, Heide; Pitkänen, Asla; Sperk, Günther

2015-01-01

Traumatic brain injury is a major cause of death and disability worldwide and often associated with post-traumatic epilepsy. We recently demonstrated that TBI induces acquired GABAA receptors channelopathy that associates with hyperexcitability in granule cell layer (GCL). We now assessed the expression of GABAA and GABAB receptor subunit mRNAs between 6 h and 6 months post-TBI in the hippocampus and thalamus. The expression of major GABAA receptor subunit mRNAs (α1, α2, α5, β2, β3, γ2 and δ) was, often bilaterally, down-regulated in the GCL and in the CA3 pyramidal cells. Instead, expression of α4 (GCL, CA3, CA1), α5 (CA1) and γ2 (GCL, CA3, CA1) mRNA was up-regulated after 10 d and/or 4 months. Many of these changes were reversible. In the thalamus, we found decreases in α1, α4, β2, γ2 and δ mRNAs in the laterodorsal thalamus and in the area combining the posterior thalamic nuclear group, ventroposterolateral and ventroposteromedial complex at 6 h to 4 months post-TBI. Unlike in the hippocampus, thalamic subunit down-regulations were irreversible and limited to the ipsilateral side. However, contralaterally there was up-regulation of the subunits δ and α4 6 h and 4 months after TBI, respectively. PCR array analysis suggested a mild long-lasting GABAA receptor channelopathy in the GCL and thalamus after TBI. Whereas TBI induces transient changes in the expression of GABAA receptor subunits in the hippocampus (presumably representing compensatory mechanisms), alterations of GABAA receptor subunit mRNAs in the thalamus are long-lasting and related to degeneration of receptor-containing neurons in thalamo-cortical relay nuclei. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Proton modulation of recombinant GABAA receptors: influence of GABA concentration and the β subunit TM2–TM3 domain

PubMed Central

Wilkins, Megan E; Hosie, Alastair M; Smart, Trevor G

2005-01-01

Regulation of GABAA receptors by extracellular pH exhibits a dependence on the receptor subunit composition. To date, the molecular mechanism responsible for the modulation of GABAA receptors at alkaline pH has remained elusive. We report here that the GABA-activated current can be potentiated at pH 8.4 for both αβ and αβγ subunit-containing receptors, but only at GABA concentrations below the EC40. Site-specific mutagenesis revealed that a single lysine residue, K279 in the β subunit TM2–TM3 linker, was critically important for alkaline pH to modulate the function of both α1β2 and α1β2γ2 receptors. The ability of low concentrations of GABA to reveal different pH titration profiles for GABAA receptors was also examined at acidic pH. At pH 6.4, GABA activation of αβγ receptors was enhanced at low GABA concentrations. This effect was ablated by the mutation H267A in the β subunit. Decreasing the pH further to 5.4 inhibited GABA responses via αβγ receptors, whereas those responses recorded from αβ receptors were potentiated. Inserting homologous β subunit residues into the γ2 subunit to recreate, in αβγ receptors, the proton modulatory profile of αβ receptors, established that in the presence of β2H267, the mutation γ2T294K was necessary to potentiate the GABA response at pH 5.4. This residue, T294, is homologous to K279 in the β subunit and suggests that a lysine at this position is an important residue for mediating the allosteric effects of both acidic and alkaline pH changes, rather than forming a direct site for protonation within the GABAA receptor. PMID:15946973

Atomic force microscopy of ionotropic receptors bearing subunit-specific tags provides a method for determining receptor architecture

NASA Astrophysics Data System (ADS)

Neish, Calum S.; Martin, Ian L.; Davies, Martin; Henderson, Robert M.; Edwardson, J. Michael

2003-08-01

We have developed an atomic force microscopy (AFM)-based method for the determination of the subunit architecture of ionotropic receptors, and tested the method using the GABAA receptor as a model system. The most common form of the GABAA receptor probably consists of 2alpha1-, 2beta2- and 1gamma2-subunits. We show here that the arrangement of subunits around the central Cl- ion channel can be deduced by AFM of receptors tagged with subunit-specific antibodies. Transfection of cells with DNA encoding alpha1-, beta2- and gamma2-subunits resulted in the production of receptors containing all three subunits, as judged by both immunoblot analysis and the binding of [3H]-Ro15-1788, a specific radioligand for the GABAA receptor. A His6-tag on the alpha1-subunit was used to purify the receptor from membrane fractions of transfected cells. After incubation with anti-His6 immunoglobulin G, some receptors became tagged with either one or two antibody molecules. AFM analysis of complexes containing two bound antibodies showed that the most common angle between the two tags was 135°, close to the value of 144° expected if the two alpha-subunits are separated by a third subunit. This method is applicable to the complete elucidation of the subunit arrangement around the GABAA receptor rosette, and can also be applied to other ionotropic receptors.

Treatment of Fragile X Syndrome with a Neuroactive Steroid

DTIC Science & Technology

2015-08-01

in the fragile X mouse model and the Drosophila (fruit fly) models of FXS that the GABAA system, including multiple receptors, is dramatically down... Drosophila (fruit fly) models of FXS that the GABAA system, including multiple receptors, is dramatically down-regulated. Ganaxolone is a drug that

Dynamic differentiation of GABAA-sensitive influences on orientation selectivity of complex cells in the cat striate cortex.

PubMed

Pfleger, B; Bonds, A B

1995-01-01

The influence of GABAA receptors on orientation selectivity of cat complex cells was tested by iontophoresis of the GABAA receptor blockers bicuculline and N-methyl-bicuculline while stimulating with drifting sinusoidal gratings. Reduction of orientation tuning was markedly less than reported in previous studies that used drifting bars as visual stimuli. Only 3/31 cells lost orientation selectivity, with an average increase in bandwidth of 33%, as opposed to half the cells losing selectivity and a bandwidth increase for the remainder of 47% as reported previously. Infusion of GABAA blockers revealed a prominent stimulus onset transient response, lasting about 120 ms, that showed a broadening of orientation selectivity comparable to that found using drifting bars under similar circumstances. We believe that drifting gratings emphasize a steady-state response component that retains, in the presence of GABAA blockers, significant orientation selectivity. Because the onset transient is initially unselective for orientation, we suggest that the steady-state, orientation-selective response component develops from an alternate inhibitory mechanism, possibly mediated by GABAB receptors.

Synthesis and biological evaluation of novel 2,3-disubstituted benzofuran analogues of GABA as neurotropic agents.

PubMed

Coaviche-Yoval, Arturo; Luna, Hector; Tovar-Miranda, Ricardo; Soriano-Ursua, Marvin Antonio; Trujillo-Ferrara, Jose G

2018-05-23

Benzofurans are heterocyclic compounds with neurotropic activity. Some have been developed for the treatment of acute and degenerative neuronal injuries. To evaluate the in silico binding of some promising benzofurans on the GABA receptors, and the in vivo neurotropic activity of benzofuran analogues (BZF 6-10) of gamma-aminobutyric acid (GABA) on a seizure model. The ligands with the best physicochemical attributes were docked on two GABA receptors (the alpha-1 subunit of GABAA-R and GBR1 subunit of GABAB-R). Selected benzofuran derivatives were synthesized by a multistep procedure and characterized. To examine the neurotropic effects, mice were pretreated with different concentrations of the compounds prior to PTZ- or 4-AP-induced seizures. We assessed acute toxicity, motor behavior, and the effects on seizures. The tested ligands that complied with Lipinski's rule of five were tested in silico with GABAA-R (ΔG = -5.51 to -5.84 kcal/mol) at the allosteric site for benzodiazepines. They bound to a similar cluster of residues as the reference compound (gaboxadol, ΔG = -5.51 kcal/mol). Synthesis was achieved with good overall yields (42-9.7%). Two compounds were selected for biological tests (BZF-7 and rac-BZF-10) on a mouse model of seizures, induced by pentylenetetrazol (PTZ) or 4-aminopyridine (4-AP). PTZ-induced seizures are associated with GABA receptors, and those 4-AP-induced with the blockage of the delayed rectifier-type potassium channel, which promotes the release of the NMDA-sensitive glutamatergic ionotropic receptor and other neurotransmitters. The biological assays demonstrated that BZF-7 and rac-BZF-10 do not protect against seizures. Indeed, BZF-7 increased the number of PTZ-induced seizures and decreased latency time. The 4-AP model apparently showed a potentiation of seizure effects after administration of the BZF-analogues, evidenced by the incidence and severity of the seizures and reduced latency time. The results suggest that the test compounds are GABAergic antagonists with stimulatory activity on the CNS. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Metabotropic glutamate receptor 5 upregulation in children with autism is associated with underexpression of both Fragile X mental retardation protein and GABAA receptor beta 3 in adults with autism

PubMed Central

Fatemi, S. Hossein; Folsom, Timothy D.; Kneeland, Rachel E.; Liesch, Stephanie B.

2011-01-01

Recent work has demonstrated the impact of dysfunction of the GABAergic signaling system in brain and the resultant behavioral pathologies in subjects with autism. In animal models, altered expression of Fragile X mental retardation protein (FMRP) has been linked to downregulation of GABA receptors. Interestingly, the autistic phenotype is also observed in individuals with Fragile X syndrome. This study was undertaken to test previous theories relating abnormalities in levels of FMRP to GABAA receptor underexpression. We observed a significant reduction in levels of FMRP in the vermis of adults with autism. Additionally, we found that levels of metabotropic glutamate receptor 5 (mGluR5) protein were significantly increased in vermis of children with autism vs. age and postmortem interval (PMI) matched controls. There was also a significant decrease in level of GABAA receptor beta 3 (GABRβ3) protein in vermis of adult subjects with autism. Finally, we found significant increases in glial fibrillary acidic protein (GFAP) in vermis of both children and adults with autism when compared with controls. Taken together, our results provide further evidence that altered FMRP expression and increased mGluR5 protein production potentially leads to altered expression of GABAA receptors. PMID:21901840

Acute Human Self-Poisoning with the N-Phenylpyrazole Insecticide Fipronil –A GABAA-Gated Chloride Channel Blocker

PubMed Central

Mohamed, Fahim; Senarathna, Lalith; Percy, Adrian; Abeyewardene, Manel; Eaglesham, Geoffrey; Cheng, Ron; Azher, Shifa; Hittarage, Ariyasena; Dissanayake, Wasantha; Sheriff, MH Rezvi; Davies, Willie; Buckley, Nick; Eddleston, Michael

2005-01-01

Objective Fipronil, a broad spectrum N-phenylpyrazole insecticide that inhibits GABAA-gated chloride channels, has been in use since the mid-1990s. A high affinity for insect compared to mammalian GABA receptors results in lower animal toxicity than other insecticides blocking this channel. To date, only two accidental cases of fipronil poisoning in humans have been published. Case series We report seven patients with fipronil self-poisoning seen prospectively in Sri Lanka together with pharmacokinetics for four patients. Non-sustained generalized tonic-clonic seizures were seen in two patients (peak measured plasma fipronil concentrations 1600 and 3744 μg/L); both were managed with diazepam without complications. A patient with a peak measured plasma concentration of 1040 μg/L was asymptomatic throughout his stay. Plasma concentration was still high at discharge 3–4 days post-ingestion when the patients were well. Retrospective review of >1000 pesticide poisoning deaths since 1995 found only one death from fipronil-based products. In contrast to the good outcome of the above cases, this patient required intubation and ventilation and had continuous fits despite therapy with barbiturates and benzodiazepines. Conclusions Our experience with prospectively observed patients suggests that fipronil poisoning is characterized by vomiting, agitation, and seizures, and normally has a favorable outcome. Management should concentrate on supportive care and early treatment of seizures. However, further experience is needed to determine whether increased susceptibility to fipronil or larger doses can produce status epilepticus. PMID:15641641

Mechanism of RDX-Induced Seizures in Rats

DTIC Science & Technology

2009-09-01

acetylcholine receptors , the glycine receptor , the site 2 sodium channel, and the family of GABAA ligand sites, as well as several others. A complete list...acetylchohnesterase was also measured. Also. RDX was screened for affinity to a library of brain receptors to determine if RDX affected any seizure-related...site on the GABAa receptor with an IC 50 of 22 uM. The mechanism of RDX-induced seizure is likely due to dis-inhibition of excitatory neuioas by

Chronic administration of aripiprazole activates GSK3β-dependent signalling pathways, and up-regulates GABAA receptor expression and CREB1 activity in rats.

PubMed

Pan, Bo; Huang, Xu-Feng; Deng, Chao

2016-07-20

Aripiprazole is a D2-like receptor (D2R) partial agonist with a favourable clinical profile. Previous investigations indicated that acute and short-term administration of aripiprazole had effects on PKA activity, GSK3β-dependent pathways, GABAA receptors, NMDA receptor and CREB1 in the brain. Since antipsychotics are used chronically in clinics, the present study investigated the long-term effects of chronic oral aripiprazole treatment on these cellular signalling pathways, in comparison with haloperidol (a D2R antagonist) and bifeprunox (a potent D2R partial agonist). We found that the Akt-GSK3β pathway was activated by aripiprazole and bifeprunox in the prefrontal cortex; NMDA NR2A levels were reduced by aripiprazole and haloperidol. In the nucleus accumbens, all three drugs increased Akt-GSK3β signalling; in addition, both aripiprazole and haloperidol, but not bifeprunox, increased the expression of Dvl-3, β-catenin and GABAA receptors, NMDA receptor subunits, as well as CREB1 phosphorylation levels. The results suggest that chronic oral administration of aripiprazole affects schizophrenia-related cellular signalling pathways and markers (including Akt-GSK3β signalling, Dvl-GSK3β-β-catenin signalling, GABAA receptor, NMDA receptor and CREB1) in a brain-region-dependent manner; the selective effects of aripiprazole on these signalling pathways might be associated with its unique clinical effects.

Inhaled Anesthetic Responses of Recombinant Receptors and Knockin Mice Harboring α2(S270H/L277A) GABAA Receptor Subunits That Are Resistant to Isoflurane

PubMed Central

Werner, D. F.; Swihart, A.; Rau, V.; Jia, F.; Borghese, C. M.; McCracken, M. L.; Iyer, S.; Fanselow, M. S.; Oh, I.; Sonner, J. M.; Eger, E. I.; Harrison, N. L.; Harris, R. A.

2011-01-01

The mechanism by which the inhaled anesthetic isoflurane produces amnesia and immobility is not understood. Isoflurane modulates GABAA receptors (GABAA-Rs) in a manner that makes them plausible targets. We asked whether GABAA-R α2 subunits contribute to a site of anesthetic action in vivo. Previous studies demonstrated that Ser270 in the second transmembrane domain is involved in the modulation of GABAA-Rs by volatile anesthetics and alcohol, either as a binding site or a critical allosteric residue. We engineered GABAA-Rs with two mutations in the α2 subunit, changing Ser270 to His and Leu277 to Ala. Recombinant receptors with these mutations demonstrated normal affinity for GABA, but substantially reduced responses to isoflurane. We then produced mutant (knockin) mice in which this mutated subunit replaced the wild-type α2 subunit. The adult mutant mice were overtly normal, although there was evidence of enhanced neonatal mortality and fear conditioning. Electrophysiological recordings from dentate granule neurons in brain slices confirmed the decreased actions of isoflurane on mutant receptors contributing to inhibitory synaptic currents. The loss of righting reflex EC50 for isoflurane did not differ between genotypes, but time to regain the righting reflex was increased in N2 generation knockins. This effect was not observed at the N4 generation. Isoflurane produced immobility (as measured by tail clamp) and amnesia (as measured by fear conditioning) in both wild-type and mutant mice, and potencies (EC50) did not differ between the strains for these actions of isoflurane. Thus, immobility or amnesia does not require isoflurane potentiation of the α2 subunit. PMID:20807777

GABAA receptor-expressing neurons promote consumption in Drosophila melanogaster.

PubMed

Cheung, Samantha K; Scott, Kristin

2017-01-01

Feeding decisions are highly plastic and bidirectionally regulated by neurons that either promote or inhibit feeding. In Drosophila melanogaster, recent studies have identified four GABAergic interneurons that act as critical brakes to prevent incessant feeding. These GABAergic neurons may inhibit target neurons that drive consumption. Here, we tested this hypothesis by examining GABA receptors and neurons that promote consumption. We find that Resistance to dieldrin (RDL), a GABAA type receptor, is required for proper control of ingestion. Knockdown of Rdl in a subset of neurons causes overconsumption of tastants. Acute activation of these neurons is sufficient to drive consumption of appetitive substances and non-appetitive substances and acute silencing of these neurons decreases consumption. Taken together, these studies identify GABAA receptor-expressing neurons that promote Drosophila ingestive behavior and provide insight into feeding regulation.

HPLC-based activity profiling for GABAA receptor modulators from the traditional Chinese herbal drug Kushen (Sophora flavescens root)

PubMed Central

2011-01-01

An EtOAc extract from the roots of Sophora flavescens (Kushen) potentiated γ -aminobutyric acid (GABA)-induced chloride influx in Xenopus oocytes transiently expressing GABAA receptors with subunit composition, α1β2γ2S. HPLC-based activity profiling of the extract led to the identification of 8-lavandulyl flavonoids, kushenol I, sophoraflavanone G, (–)-kurarinone, and kuraridine as GABAA receptor modulators. In addition, a series of inactive structurally related flavonoids were characterized. Among these, kushenol Y (4) was identified as a new natural product. The 8-lavandulyl flavonoids are first representatives of a novel scaffold for the target. PMID:21207144

Metabolic products of linalool and modulation of GABAA receptors

NASA Astrophysics Data System (ADS)

Milanos, Sinem; Elsharif, Shaimaa A.; Janzen, Dieter; Buettner, Andrea; Villmann, Carmen

2017-06-01

Terpenoids are major subcomponents in aroma substances which harbor sedative physiological potential. We have demonstrated that various monoterpenoids such as the acyclic linalool enhance GABAergic currents in an allosteric manner in vitro upon overexpression of inhibitory a1b2 GABAA receptors in various expression systems. However, in plants or humans, i.e. following intake via inhalation or ingestion, linalool undergoes metabolic modifications including oxygenation and acetylation, which may affect the modulatory efficacy of the generated linalool derivatives. Here, we analyzed the modulatory potential of linalool derivatives at a1b2g2 GABAA receptors upon transient overexpression. Following receptor expression control, electrophysiological recordings in a whole cell configuration were used to determine the chloride influx upon co-application of GABA EC5-10 together with the modulatory substance. Our results show that only oxygenated linalool metabolites at carbon 8 positively affect GABAergic currents whereas derivatives hydroxylated or carboxylated at carbon 8 were rather ineffective. Acetylated linalool derivatives resulted in non-significant changes of GABAergic currents. We can conclude that metabolism of linalool reduces its positive allosteric potential at GABAA receptors compared to the significant potentiation effects of the parent molecule linalool itself.

1-Methyl-beta-carboline (harmane), a potent endogenous inhibitor of benzodiazepine receptor binding.

PubMed

Rommelspacher, H; Nanz, C; Borbe, H O; Fehske, K J; Müller, W E; Wollert, U

1980-10-01

The interaction of several beta-carbolines with specific [3H]-flunitrazepam binding to benzodiazepine receptors in rat brain membranes was investigated. Out of the investigated compounds, harmane and norharmane were the most potent inhibitors of specific [3H]-flunitrazepam binding, with IC50-values in the micromolar range. All other derivatives, including harmine, harmaline, and several tetrahydroderivatives were at least ten times less potent. Harmane has been previously found in rat brain and human urine, so it is the most potent endogenous inhibitor of specific [3H]-flunitrazepam binding known so far, with a several fold higher affinity for the benzodiazepine receptor than inosine and hypoxanthine. Thus, we suggest that harmane or other related beta-carbolines could be potential candidates as endogenous ligands of the benzodiazepine receptor.

The metabotropic glutamate receptors: structure, activation mechanism and pharmacology.

PubMed

Pin, Jean-Philippe; Acher, Francine

2002-06-01

The metabotropic glutamate receptors are G-protein coupled receptors (GPCR) involved in the regulation of many synapses, including most glutamatergic fast excitatory synapses. Eight subtypes have been identified that can be classified into three groups. The molecular characterization of these receptors revealed proteins much more complex than any other GPCRs. They are composed of a Venus Flytrap (VFT) module where glutamate binds, connected to a heptahelical domain responsible for G-protein coupling. Recent data including the structure of the VFT module determined with and without glutamate, indicate that these receptors function as dimers. Moreover a number of intracellular proteins can regulate their targeting and transduction mechanism. Such structural features of mGlu receptors offer multiple possibilities for synthetic compounds to modulate their activity. In addition to agonists and competitive antagonists acting at the glutamate binding site, a number of non-competitive antagonists with inverse agonist activity, and positive allosteric modulators have been discovered. These later compounds share specific properties that make them good candidates for therapeutic applications. First, their non-amino acid structure makes them pass more easily the blood brain barrier. Second, they are much more selective than any other compound identified so far, being the first subtype selective molecules. Third, for the negative modulators, their non competitive mechanism of action makes them relatively unaffected by high concentrations of glutamate that may be present in disease states (e.g. stroke, epilepsy, neuropathic pain, etc.). Fourth, like the benzodiazepines acting at the GABA(A) receptors, the positive modulators offer a new way to increase the activity of these receptors in vivo, with a low risk of inducing their desensitization. The present review article focuses on the specific structural features of these receptors and highlights the various possibilities these offer for drug development.

Effects of Antecedent GABAA Activation With Alprazolam on Counterregulatory Responses to Hypoglycemia in Healthy Humans

PubMed Central

Hedrington, Maka S.; Farmerie, Stephnie; Ertl, Andrew C.; Wang, Zhihui; Tate, Donna B.; Davis, Stephen N.

2010-01-01

OBJECTIVE To date, there are no data investigating the effects of GABAA activation on counterregulatory responses during repeated hypoglycemia in humans. The aim of this study was to determine the effects of prior GABAA activation using the benzodiazepine alprazolam on the neuroendocrine and autonomic nervous system (ANS) and metabolic counterregulatory responses during next-day hypoglycemia in healthy humans. RESEARCH DESIGN AND METHODS Twenty-eight healthy individuals (14 male and 14 female, age 27 ± 6 years, BMI 24 ± 3 kg/m2, and A1C 5.2 ± 0.1%) participated in four randomized, double-blind, 2-day studies. Day 1 consisted of either morning and afternoon 2-h hyperinsulinemic euglycemia or 2-h hyperinsulinemic hypoglycemia (2.9 mmol/l) with either 1 mg alprazolam or placebo administered 30 min before the start of each clamp. Day 2 consisted of a single-step hyperinsulinemic-hypoglycemic clamp of 2.9 mmol/l. RESULTS Despite similar hypoglycemia (2.9 ± 1 mmol/l) and insulinemia (672 ± 108 pmol/l) during day 2 studies, GABAA activation with alprazolam during day 1 euglycemia resulted in significant blunting (P < 0.05) of ANS (epinephrine, norepinephrine, muscle sympathetic nerve activity, and pancreatic polypeptide), neuroendocrine (glucagon and growth hormone), and metabolic (glucose kinetics, lipolysis, and glycogenolysis) counterregulatory responses. GABAA activation with alprazolam during prior hypoglycemia caused further significant (P < 0.05) decrements in subsequent glucagon, growth hormone, pancreatic polypeptide, and muscle sympathetic nerve activity counterregulatory responses. CONCLUSIONS Alprazolam activation of GABAA pathways during day 1 hypoglycemia can play an important role in regulating a spectrum of key physiologic responses during subsequent (day 2) hypoglycemia in healthy man. PMID:20086227

Effects of antecedent GABAA activation with alprazolam on counterregulatory responses to hypoglycemia in healthy humans.

PubMed

Hedrington, Maka S; Farmerie, Stephnie; Ertl, Andrew C; Wang, Zhihui; Tate, Donna B; Davis, Stephen N

2010-04-01

To date, there are no data investigating the effects of GABA(A) activation on counterregulatory responses during repeated hypoglycemia in humans. The aim of this study was to determine the effects of prior GABA(A) activation using the benzodiazepine alprazolam on the neuroendocrine and autonomic nervous system (ANS) and metabolic counterregulatory responses during next-day hypoglycemia in healthy humans. Twenty-eight healthy individuals (14 male and 14 female, age 27 +/- 6 years, BMI 24 +/- 3 kg/m(2), and A1C 5.2 +/- 0.1%) participated in four randomized, double-blind, 2-day studies. Day 1 consisted of either morning and afternoon 2-h hyperinsulinemic euglycemia or 2-h hyperinsulinemic hypoglycemia (2.9 mmol/l) with either 1 mg alprazolam or placebo administered 30 min before the start of each clamp. Day 2 consisted of a single-step hyperinsulinemic-hypoglycemic clamp of 2.9 mmol/l. Despite similar hypoglycemia (2.9 +/- 1 mmol/l) and insulinemia (672 +/- 108 pmol/l) during day 2 studies, GABA(A) activation with alprazolam during day 1 euglycemia resulted in significant blunting (P < 0.05) of ANS (epinephrine, norepinephrine, muscle sympathetic nerve activity, and pancreatic polypeptide), neuroendocrine (glucagon and growth hormone), and metabolic (glucose kinetics, lipolysis, and glycogenolysis) counterregulatory responses. GABA(A) activation with alprazolam during prior hypoglycemia caused further significant (P < 0.05) decrements in subsequent glucagon, growth hormone, pancreatic polypeptide, and muscle sympathetic nerve activity counterregulatory responses. Alprazolam activation of GABA(A) pathways during day 1 hypoglycemia can play an important role in regulating a spectrum of key physiologic responses during subsequent (day 2) hypoglycemia in healthy man.

Effects of central histamine receptors blockade on GABA(A) agonist-induced food intake in broiler cockerels.

PubMed

Morteza, Zendehdel; Vahhab, Babapour; Hossein, Jonaidi

2008-02-01

In this study, the effect of intracerebroventricular (i.c.v) injection of H1, H2 and H3 antagonists on feed intake induced by GABA(A) agonist was evaluated. In Experiment 1, the animals received chloropheniramine, a H1 antagonist and then muscimol, a GABA(A) agonist. In Experiment 2, chickens received famotidine, a H2 receptor antagonist, prior to injection of muscimol. Finally in Experiment 3, the birds were injected with thioperamide, a H3 receptor antagonist and muscimol. Cumulative food intake was measured 15, 30, 45, 60, 90, 120, 150 and 180 min after injections. The results of this study indicated that effects of muscimol on food intake inhibited by pretreatment with chloropheneramine maleate (p < or = 0.05), significantly, while the famotidine and thioperamide were ineffective. These results suggest the existence of H1-receptor mediated histamine-GABA(A) receptor interaction on food intake in broiler cockerels.

Rescue of deficient amygdala tonic γ-aminobutyric acidergic currents in the Fmr-/y mouse model of fragile X syndrome by a novel γ-aminobutyric acid type A receptor-positive allosteric modulator.

PubMed

Martin, Brandon S; Martinez-Botella, Gabriel; Loya, Carlos M; Salituro, Francesco G; Robichaud, Albert J; Huntsman, Molly M; Ackley, Mike A; Doherty, James J; Corbin, Joshua G

2016-06-01

Alterations in the ratio of excitatory to inhibitory transmission are emerging as a common component of many nervous system disorders, including autism spectrum disorders (ASDs). Tonic γ-aminobutyric acidergic (GABAergic) transmission provided by peri- and extrasynaptic GABA type A (GABAA ) receptors powerfully controls neuronal excitability and plasticity and, therefore, provides a rational therapeutic target for normalizing hyperexcitable networks across a variety of disorders, including ASDs. Our previous studies revealed tonic GABAergic deficits in principal excitatory neurons in the basolateral amygdala (BLA) in the Fmr1(-/y) knockout (KO) mouse model fragile X syndrome. To correct amygdala deficits in tonic GABAergic neurotransmission in Fmr1(-/y) KO mice, we developed a novel positive allosteric modulator of GABAA receptors, SGE-872, based on endogenously active neurosteroids. This study shows that SGE-872 is nearly as potent and twice as efficacious for positively modulating GABAA receptors as its parent molecule, allopregnanolone. Furthermore, at submicromolar concentrations (≤1 μM), SGE-872 is selective for tonic, extrasynaptic α4β3δ-containing GABAA receptors over typical synaptic α1β2γ2 receptors. We further find that SGE-872 strikingly rescues the tonic GABAergic transmission deficit in principal excitatory neurons in the Fmr1(-/y) KO BLA, a structure heavily implicated in the neuropathology of ASDs. Therefore, the potent and selective action of SGE-872 on tonic GABAA receptors containing α4 subunits may represent a novel and highly useful therapeutic avenue for ASDs and related disorders involving hyperexcitability of neuronal networks. © 2015 Wiley Periodicals, Inc.

Laser photolysis of DPNI-GABA, a tool for investigating the properties and distribution of GABA receptors and for silencing neurons in situ.

PubMed

Trigo, Federico F; Papageorgiou, George; Corrie, John E T; Ogden, David

2009-07-30

Laser photolysis to release GABA at precisely defined times and locations permits investigation of the distribution of functional GABA(A) receptors in neuronal compartments, the activation kinetics and pharmacology of GABA(A) receptors in situ, and the role of individual neurons in neural circuits by selective silencing with low GABA concentrations. We describe the experimental evaluation and applications of a new nitroindoline-caged GABA, DPNI-GABA, modified to minimize the pharmacological interference commonly found with caged GABA reagents, but retaining the advantages of nitroindoline cages. Unlike the 5-methoxycarbonylmethyl-7-nitroindolinyl-GABA tested previously, DPNI-GABA inhibited GABA(A) receptors with much lower affinity, reducing peak GABA-evoked responses with an IC(50) of approximately 0.5 mM. Most importantly, the kinetics of receptor activation, determined as 10-90% rise-times, were comparable to synaptic events and were little affected by DPNI-GABA present at 1mM concentration, permitting photolysis of DPNI-GABA to mimic synaptic activation of GABA(A) receptors. With a laser spot of 1 microm applied to cerebellar molecular layer interneurons, the spatial resolution of uncaging DPNI-GABA in dendrites was estimated as 2 microm laterally and 7.5 microm focally. Finally, at low DPNI-GABA concentration, photorelease restricted to the area of the soma suppressed spiking in single Purkinje neurons or molecular layer interneurons for periods controlled by the flash intensity and duration. DPNI-GABA has properties better adapted for fast kinetic studies with laser photolysis at GABA(A) receptors than previously reported caged GABA reagents, and can be used in experiments where spatial resolution is determined by the dimensions of the laser light spot.

The effects of serotonin1A receptor on female mice body weight and food intake are associated with the differential expression of hypothalamic neuropeptides and the GABAA receptor.

PubMed

Butt, Isma; Hong, Andrew; Di, Jing; Aracena, Sonia; Banerjee, Probal; Shen, Chang-Hui

2014-10-01

Both common eating disorders anorexia nervosa and bulimia nervosa are characteristically diseases of women. To characterize the role of the 5-HT1A receptor (5-HT1A-R) in these eating disorders in females, we investigated the effect of saline or 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) treatment on feeding behavior and body weight in adult WT female mice and in adult 5-HT1A-R knockout (KO) female mice. Our results showed that KO female mice have lower food intake and body weight than WT female mice. Administration of 8-OH-DPAT decreased food intake but not body weight in WT female mice. Furthermore, qRT-PCR was employed to analyze the expression levels of neuropeptides, γ-aminobutyric acid A receptor subunit β (GABAA β subunits) and glutamic acid decarboxylase in the hypothalamic area. The results showed the difference in food intake between WT and KO mice was accompanied by differential expression of POMC, CART and GABAA β2, and the difference in body weight between WT and KO mice was associated with significantly different expression levels of CART and GABAA β2. As such, our data provide new insight into the role of 5-HT1A-R in both feeding behavior and the associated expression of neuropeptides and the GABAA receptor. Copyright © 2014 Elsevier Ltd. All rights reserved.

Assessment of Homology Templates and an Anesthetic Binding Site within the γ-Aminobutyric Acid Receptor

PubMed Central

Bertaccini, Edward J.; Yoluk, Ozge; Lindahl, Erik R.; Trudell, James R.

2013-01-01

Background Anesthetics mediate portions of their activity via modulation of the γ-aminobutyric acid receptor (GABAaR). While its molecular structure remains unknown, significant progress has been made towards understanding its interactions with anesthetics via molecular modeling. Methods The structure of the torpedo acetylcholine receptor (nAChRα), the structures of the α4 and β2 subunits of the human nAChR, the structures of the eukaryotic glutamate-gated chloride channel (GluCl), and the prokaryotic pH sensing channels, from Gloeobacter violaceus and Erwinia chrysanthemi, were aligned with the SAlign and 3DMA algorithms. A multiple sequence alignment from these structures and those of the GABAaR was performed with ClustalW. The Modeler and Rosetta algorithms independently created three-dimensional constructs of the GABAaR from the GluCl template. The CDocker algorithm docked a congeneric series of propofol derivatives into the binding pocket and scored calculated binding affinities for correlation with known GABAaR potentiation EC50’s. Results Multiple structure alignments of templates revealed a clear consensus of residue locations relevant to anesthetic effects except for torpedo nAChR. Within the GABAaR models generated from GluCl, the residues notable for modulating anesthetic action within transmembrane segments 1, 2, and 3 converged on the intersubunit interface between alpha and beta subunits. Docking scores of a propofol derivative series into this binding site showed strong linear correlation with GABAaR potentiation EC50. Conclusion Consensus structural alignment based on homologous templates revealed an intersubunit anesthetic binding cavity within the transmembrane domain of the GABAaR, which showed correlation of ligand docking scores with experimentally measured GABAaR potentiation. PMID:23770602

Assessment of homology templates and an anesthetic binding site within the γ-aminobutyric acid receptor.

PubMed

Bertaccini, Edward J; Yoluk, Ozge; Lindahl, Erik R; Trudell, James R

2013-11-01

Anesthetics mediate portions of their activity via modulation of the γ-aminobutyric acid receptor (GABAaR). Although its molecular structure remains unknown, significant progress has been made toward understanding its interactions with anesthetics via molecular modeling. The structure of the torpedo acetylcholine receptor (nAChRα), the structures of the α4 and β2 subunits of the human nAChR, the structures of the eukaryotic glutamate-gated chloride channel (GluCl), and the prokaryotic pH-sensing channels, from Gloeobacter violaceus and Erwinia chrysanthemi, were aligned with the SAlign and 3DMA algorithms. A multiple sequence alignment from these structures and those of the GABAaR was performed with ClustalW. The Modeler and Rosetta algorithms independently created three-dimensional constructs of the GABAaR from the GluCl template. The CDocker algorithm docked a congeneric series of propofol derivatives into the binding pocket and scored calculated binding affinities for correlation with known GABAaR potentiation EC50s. Multiple structure alignments of templates revealed a clear consensus of residue locations relevant to anesthetic effects except for torpedo nAChR. Within the GABAaR models generated from GluCl, the residues notable for modulating anesthetic action within transmembrane segments 1, 2, and 3 converged on the intersubunit interface between α and β subunits. Docking scores of a propofol derivative series into this binding site showed strong linear correlation with GABAaR potentiation EC50. Consensus structural alignment based on homologous templates revealed an intersubunit anesthetic binding cavity within the transmembrane domain of the GABAaR, which showed a correlation of ligand docking scores with experimentally measured GABAaR potentiation.

Responses to GABA(A) receptor activation are altered in NTS neurons isolated from renal-wrap hypertensive rats.

PubMed

Tolstykh, Gleb; Belugin, Sergei; Tolstykh, Olga; Mifflin, Steve

2003-10-01

The inhibitory amino acid GABA is a potent modulator of the spontaneous discharge and the responses to afferent inputs of neurons in the nucleus of the solitary tract (NTS). To determine if responses to activation of GABA(A) receptors are altered in hypertension, GABA(A) receptor-evoked whole cell currents were measured in enzymatically dispersed NTS neurons from 33 normotensive (NT, 109+/-4 mm Hg, n=7) and 24 hypertensive (HT, 167+/-5 mm Hg, n=24) rats. GABA(A) receptor-evoked currents reversed at the calculated equilibrium potential for chloride and were blocked by bicuculline (n=6). Membrane capacitance was the same in neurons from NT (7.5+/-0.6 pF, n=62) and HT (6.8+/-0.6 pF, n=51) rats. The EC50 for peak GABA-evoked currents cells was significantly greater in neurons from HT (21.0+/-2.6 micromol/L, n=16) compared with NT rats (13.0+/-1.8 micromol/L, n=14, P=0.01). The EC50 of neurons exhibiting DiA labeling of presumptive aortic nerve terminals was no different than that observed in the nonlabeled cells (19.0+/-4.9 micromol/L, n=4). The time constant for desensitization of GABA(A)-evoked currents was the same in neurons from HT (4.5+/-0.3 seconds, n=17) and NT rats (3.8+/-0.3 seconds, n=17, P>0.05). Repetitive pulse application of GABA revealed a more rapid decline in the evoked current in neurons from HT compared with NT rats. The amplitude of the 5th pulse of GABA (5-second duration, 2-second interval) was 21+/-2% the amplitude of the 1st pulse in NT rats (n=10) and 14+/-2% in HT rats (n=11, P<0.05). These alterations in GABAA-receptor evoked currents could render the neurons less sensitive to GABA(A) receptor inhibition and influence afferent integration by NTS neurons in HT.

Multiple functions of GABA A and GABA B receptors during pattern processing in the zebrafish olfactory bulb.

PubMed

Tabor, Rico; Yaksi, Emre; Friedrich, Rainer W

2008-07-01

gamma-Aminobutyric acid (GABA)ergic synapses are thought to play pivotal roles in the processing of activity patterns in the olfactory bulb (OB), but their functions have been difficult to study during odor responses in the intact system. We pharmacologically manipulated GABA(A) and GABA(B) receptors in the OB of zebrafish and analysed the effects on odor responses of the output neurons, the mitral cells (MCs), by electrophysiological recordings and temporally deconvolved two-photon Ca2+ imaging. The blockade of GABA(B) receptors enhanced presynaptic Ca2+ influx into afferent axon terminals, and changed the amplitude and time course of a subset of MC responses, indicating that GABA(B) receptors have a modulatory influence on OB output activity. The blockade of GABA(A) receptors induced epileptiform firing, enhanced excitatory responses and abolished fast oscillations in the local field potential. Moreover, the topological reorganization and decorrelation of MC activity patterns during the initial phase of the response was perturbed. These results indicate that GABA(A) receptor-containing circuits participate in the balance of excitation and inhibition, the regulation of total OB output activity, the synchronization of odor-dependent neuronal ensembles, and the reorganization of odor-encoding activity patterns. GABA(A) and GABA(B) receptors are therefore differentially involved in multiple functions of neuronal circuits in the OB.

Antidepressant and anxiolytic properties of the methanolic extract of Momordica charantia Linn (Cucurbitaceae) and its mechanism of action.

PubMed

Ishola, I O; Akinyede, A A; Sholarin, A M

2014-07-01

The whole plant of Momordica charantia Linn (Cucurbitaceae) is used in traditional African medicine in the management of depressive illness. Momordica charantia (MC) (50-400 mg/kg, p.o.) was administered 1 h before behavioural studies using the forced swimming test (FST) and tail suspension test (TST) to investigate antidepressant-like effect while the anxiolytic-like effect was evaluated with elevated plus maze test (EPM), hole-board test (HBT), and light-dark test (LDT). Acute treatment with MC (50-400 mg/kg) significantly increased swimming time (86.51%) and reduced the duration of immobility (52.35%) in FST and TST with peak effects observed at 200 mg/kg, respectively, in comparison to control. The pretreatment of mice with either sulpiride (dopamine D2 receptor antagonist), or metergoline (5-HT2 receptor antagonist), or cyproheptadine (5-HT2 receptor antagonist), or prazosin (α1-adrenoceptor antagonist), or yohimbine (α2-adrenoceptor antagonist), and atropine (muscarinic cholinergic receptor antagonist) 15 min before oral administration of MC (200 mg/kg) significantly blocked its anti-immobility effect. Similarly, MC (200 mg/kg) significantly reduced anxiety by increasing the open arm exploration (64.27%) in EPM, number of head-dips in HBT (34.38%), and time spent in light compartment (29.38%) in the LDT. However, pretreatment with flumazenil (GABAA receptor antagonist) 15 min before MC (200 mg/kg) significantly blocked (54.76%) its anxiolytic effect. The findings in this study showed that MC possesses antidepressant-like effect that is dependent on the serotonergic (5-HT2 receptor), noradrenergic (α1- and α2-adrenoceptors), dopaminergic (D2 receptor), and muscarinic cholinergic systems and an anxiolytic-like effect that might involve an action on benzodiazepine-type receptor. © Georg Thieme Verlag KG Stuttgart · New York.

Kinetic modeling of benzodiazepine receptor binding with PET and high specific activity [(11)C]Iomazenil in healthy human subjects.

PubMed

Bremner, J D; Horti, A; Staib, L H; Zea-Ponce, Y; Soufer, R; Charney, D S; Baldwin, R

2000-01-01

Quantitation of the PET benzodiazepine receptor antagonist, [(11)C]Iomazenil, using low specific activity radioligand was recently described. The purpose of this study was to quantitate benzodiazepine receptor binding in human subjects using PET and high specific activity [(11)C]Iomazenil. Six healthy human subjects underwent PET imaging following a bolus injection of high specific activity (>100 Ci/mmol) [(11)C]iomazenil. Arterial samples were collected at multiple time points after injection for measurement of unmetabolized total and nonprotein-bound parent compound in plasma. Time activity curves of radioligand concentration in brain and plasma were analyzed using two and three compartment model. Kinetic rate constants of transfer of radioligand between plasma, nonspecifically bound brain tissue, and specifically bound brain tissue compartments were fitted to the model. Values for fitted kinetic rate constants were used in the calculation of measures of benzodiazepine receptor binding, including binding potential (the ratio of receptor density to affinity), and product of BP and the fraction of free nonprotein-bound parent compound (V(3)'). Use of the three compartment model improved the goodness of fit in comparison to the two compartment model. Values for kinetic rate constants and measures of benzodiazepine receptor binding, including BP and V(3)', were similar to results obtained with the SPECT radioligand [(123)I]iomazenil, and a prior report with low specific activity [(11)C]Iomazenil. Kinetic modeling using the three compartment model with PET and high specific activity [(11)C]Iomazenil provides a reliable measure of benzodiazepine receptor binding. Synapse 35:68-77, 2000. Published 2000 Wiley-Liss, Inc.

Hap1 and GABA: thinking about food intake.

PubMed

Woods, Stephen C; Seeley, Randy J

2006-06-01

GABA stimulation of hypothalamic GABAA receptors increases food intake and body weight. Huntingtin-associated protein-1 (Hap1), is highly expressed in the hypothalamus and increases activity at GABAA receptors; mice lacking Hap1 are hypophagic. A recent paper (Sheng et al.,2006) further explores the role of Hap1 in the control of food intake.

GABA-benzodiazepine receptor function in alcohol dependence: a combined 11C-flumazenil PET and pharmacodynamic study.

PubMed

Lingford-Hughes, A R; Wilson, S J; Cunningham, V J; Feeney, A; Stevenson, B; Brooks, D J; Nutt, D J

2005-08-01

Gamma-aminobutyric acid (GABA)-benzodiazepine receptor function is hypothesised to be reduced in alcohol dependence. We used positron emission tomography (PET) with [11C]flumazenil, a non-selective tracer for brain GABA-benzodiazepine (GABA-BDZ) receptor binding, to determine in vivo the relationship between BDZ receptor occupancy by an agonist, midazolam, and its functional effects. Abstinent male alcohol dependent subjects underwent [11C]flumazenil PET to measure occupancy of BDZ receptors by midazolam whilst recording its pharmacodynamic effects on behavioural and physiological measures. Rate constants describing the exchange of [11C]flumazenil between the plasma and brain compartments were derived from time activity curves. A 50% reduction in electroencephalography (EEG)-measured sleep time was seen in the alcohol dependent group despite the same degree of occupancy by midazolam as seen in the control group. The effects of midazolam on other measures of benzodiazepine receptor function, increasing EEG beta1 power and slowing of saccadic eye movements, were similar in the two groups. No differences in midazolam or flumazenil metabolism were found between the groups. In summary, our study suggests that alcohol dependence in man is associated with a reduced EEG sleep response to the benzodiazepine agonist, midazolam, which is not explained by reduced BDZ receptor occupancy, and is consistent with reduced sensitivity in this measure of GABA-BDZ receptor function in alcohol dependence. The lack of change in other functional measures may reflect a differential involvement of particular subtypes of the GABA-BDZ receptor.

Chronic administration of aripiprazole activates GSK3β-dependent signalling pathways, and up-regulates GABAA receptor expression and CREB1 activity in rats

PubMed Central

Pan, Bo; Huang, Xu-Feng; Deng, Chao

2016-01-01

Aripiprazole is a D2-like receptor (D2R) partial agonist with a favourable clinical profile. Previous investigations indicated that acute and short-term administration of aripiprazole had effects on PKA activity, GSK3β-dependent pathways, GABAA receptors, NMDA receptor and CREB1 in the brain. Since antipsychotics are used chronically in clinics, the present study investigated the long-term effects of chronic oral aripiprazole treatment on these cellular signalling pathways, in comparison with haloperidol (a D2R antagonist) and bifeprunox (a potent D2R partial agonist). We found that the Akt-GSK3β pathway was activated by aripiprazole and bifeprunox in the prefrontal cortex; NMDA NR2A levels were reduced by aripiprazole and haloperidol. In the nucleus accumbens, all three drugs increased Akt-GSK3β signalling; in addition, both aripiprazole and haloperidol, but not bifeprunox, increased the expression of Dvl-3, β-catenin and GABAA receptors, NMDA receptor subunits, as well as CREB1 phosphorylation levels. The results suggest that chronic oral administration of aripiprazole affects schizophrenia-related cellular signalling pathways and markers (including Akt-GSK3β signalling, Dvl-GSK3β-β-catenin signalling, GABAA receptor, NMDA receptor and CREB1) in a brain-region-dependent manner; the selective effects of aripiprazole on these signalling pathways might be associated with its unique clinical effects. PMID:27435909

Ethanol-induced dopamine elevation in the rat--modulatory effects by subchronic treatment with nicotinic drugs.

PubMed

Löf, Elin; Chau, Pei Pei; Stomberg, Rosita; Söderpalm, Bo

2007-01-26

Chronic nicotine administration is associated with increased ethanol consumption in laboratory animals and in humans. Some smokers report less sedation during acute ethanol intoxication after nicotine administration and the sedative effects from ethanol are mediated by inhibitory GABA(A)-receptors. In a series of in vivo microdialysis experiments we investigated whether subchronic pre-treatment with nicotinic drugs known to enhance ethanol consumption in the rat (nicotine or the peripheral nicotinic antagonist hexamethonium) could modulate the alterations in extracellular dopamine observed in response to administration of ethanol or the sedative GABA(A)-agonist diazepam. In the nucleus accumbens and the dorsal striatum, systemic and/or local ethanol administration resulted in transient increases in extracellular dopamine levels that returned to baseline before the local levels of ethanol started to decline. In hexamethonium pre-treated rats, however, the nucleus accumbens dopamine levels were time-locked to the ethanol levels in the same area after systemic or local ethanol administration. Perfusion of diazepam into the nucleus accumbens produced a significant reduction in nucleus accumbens dopamine in controls. Prior subchronic treatment with nicotine or hexamethonium abolished this effect. The present results suggest that subchronic treatment with the nicotinic acetylcholine receptor antagonist hexamethonium reduces a GABA(A)-R mediated counteraction of the nucleus accumbens dopamine response to ethanol. Additionally, we demonstrate that modulation of nicotinic receptors may reduce the sensitivity of GABA(A) receptors to benzodiazepines. These phenomena may offer a novel explanation to why nicotine and alcohol are often co-abused.

Encephalitis with refractory seizures, status epilepticus, and antibodies to the GABAA receptor: a case series, characterisation of the antigen, and analysis of the effects of antibodies.

PubMed

Petit-Pedrol, Mar; Armangue, Thaís; Peng, Xiaoyu; Bataller, Luis; Cellucci, Tania; Davis, Rebecca; McCracken, Lindsey; Martinez-Hernandez, Eugenia; Mason, Warren P; Kruer, Michael C; Ritacco, David G; Grisold, Wolfgang; Meaney, Brandon F; Alcalá, Carmen; Sillevis-Smitt, Peter; Titulaer, Maarten J; Balice-Gordon, Rita; Graus, Francesc; Dalmau, Josep

2014-03-01

Increasing evidence suggests that seizures and status epilepticus can be immune-mediated. We aimed to describe the clinical features of a new epileptic disorder, and to establish the target antigen and the effects of patients' antibodies on neuronal cultures. In this observational study, we selected serum and CSF samples for antigen characterisation from 140 patients with encephalitis, seizures or status epilepticus, and antibodies to unknown neuropil antigens. The samples were obtained from worldwide referrals of patients with disorders suspected to be autoimmune between April 28, 2006, and April 25, 2013. We used samples from 75 healthy individuals and 416 patients with a range of neurological diseases as controls. We assessed the samples using immunoprecipitation, mass spectrometry, cell-based assay, and analysis of antibody effects in cultured rat hippocampal neurons with confocal microscopy. Neuronal cell-membrane immunoprecipitation with serum of two index patients revealed GABAA receptor sequences. Cell-based assay with HEK293 expressing α1/β3 subunits of the GABAA receptor showed high titre serum antibodies (>1:160) and CSF antibodies in six patients. All six patients (age 3-63 years, median 22 years; five male patients) developed refractory status epilepticus or epilepsia partialis continua along with extensive cortical-subcortical MRI abnormalities; four patients needed pharmacologically induced coma. 12 of 416 control patients with other diseases, but none of the healthy controls, had low-titre GABAA receptor antibodies detectable in only serum samples, five of them also had GAD-65 antibodies. These 12 patients (age 2-74 years, median 26.5 years; seven male patients) developed a broader spectrum of symptoms probably indicative of coexisting autoimmune disorders: six had encephalitis with seizures (one with status epilepticus needing pharmacologically induced coma; one with epilepsia partialis continua), four had stiff-person syndrome (one with seizures and limbic involvement), and two had opsoclonus-myoclonus. Overall, 12 of 15 patients for whom treatment and outcome were assessable had full (three patients) or partial (nine patients) response to immunotherapy or symptomatic treatment, and three died. Patients' antibodies caused a selective reduction of GABAA receptor clusters at synapses, but not along dendrites, without altering NMDA receptors and gephyrin (a protein that anchors the GABAA receptor). High titres of serum and CSF GABAA receptor antibodies are associated with a severe form of encephalitis with seizures, refractory status epilepticus, or both. The antibodies cause a selective reduction of synaptic GABAA receptors. The disorder often occurs with GABAergic and other coexisting autoimmune disorders and is potentially treatable. The National Institutes of Health, the McKnight Neuroscience of Brain Disorders, the Fondo de Investigaciones Sanitarias, Fundació la Marató de TV3, the Netherlands Organisation for Scientific Research (Veni-incentive), the Dutch Epilepsy Foundation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Reduced GABAA Receptor α6 Expression in The Trigeminal Ganglion Enhanced Myofascial Nociceptive Response

PubMed Central

Kramer, P. R.; Bellinger, L. L.

2013-01-01

Activation of the GABAA receptor results in inhibition of neuronal activity. One subunit of this multi-subunit receptor termed alpha 6 (Gabrα6) contributed to inflammatory temporomandibular joint (TMJ) nociception but TMJ disorders often include myofascial pain. To address Gabrα6 role in myofascial pain we hypothesized that Gabrα6 has an inhibitory role in myofascial nociceptive responses similar to inflammatory TMJ arthritis. To test this hypothesis a, myofascial nociceptive response was induced by placing a ligature bilaterally on the tendon attachment of the anterior superficial part of a male rat's masseter muscle. Four days after ligature placement Gabrα6 expression was reduced by infusing the trigeminal ganglia (TG) with small interfering RNA (siRNA) having homology to either the Gabrα6 gene (Gabra6 siRNA) or no known gene (control siRNA). After siRNA infusion nociceptive behavioral responses were measured, i.e., feeding behavior and head withdrawal after pressing upon the region above the ligature with von Frey filaments. Neuronal activity in the TG and trigeminal nucleus caudalis and upper cervical region (Vc–C1) was measured by quantitating the amount of phosphorylated extracellular signalregulated kinase (p-ERK). Total Gabrα6 and GABAA receptor contents in the TG and Vc–C1 were determined. Gabrα6 siRNA infusion reduced Gabrα6 and GABAA receptor expression and significantly increased the nociceptive response in both nociceptive assays. Gabra6 siRNA infusion also significantly increased TG p-ERK expression of the ligated rats. From these results we conclude GABAA receptors consisting of the Gabrα6 subunit inhibit TG nociceptive sensory afferents in the trigeminal pathway and have an important role in the regulation of myofascial nociception. PMID:23602886

Differential Potency of 2,6-Dimethylcyclohexanol Isomers for Positive Modulation of GABAA Receptor Currents.

PubMed

Chowdhury, Luvana; Croft, Celine J; Goel, Shikha; Zaman, Naina; Tai, Angela C-S; Walch, Erin M; Smith, Kelly; Page, Alexandra; Shea, Kevin M; Hall, C Dennis; Jishkariani, D; Pillai, Girinath G; Hall, Adam C

2016-06-01

GABAA receptors meet all of the pharmacological requirements necessary to be considered important targets for the action of general anesthetic agents in the mammalian brain. In the following patch-clamp study, the relative modulatory effects of 2,6-dimethylcyclohexanol diastereomers were investigated on human GABAA (α1β3γ2s) receptor currents stably expressed in human embryonic kidney cells. Cis,cis-, trans,trans-, and cis,trans-isomers were isolated from commercially available 2,6-dimethylcyclohexanol and were tested for positive modulation of submaximal GABA responses. For example, the addition of 30 μM cis,cis-isomer resulted in an approximately 2- to 3-fold enhancement of the EC20 GABA current. Coapplications of 30 μM 2,6-dimethylcyclohexanol isomers produced a range of positive enhancements of control GABA responses with a rank order for positive modulation: cis,cis > trans,trans ≥ mixture of isomers > > cis,trans-isomer. In molecular modeling studies, the three cyclohexanol isomers bound with the highest binding energies to a pocket within transmembrane helices M1 and M2 of the β3 subunit through hydrogen-bonding interactions with a glutamine at the 224 position and a tyrosine at the 220 position. The energies for binding to and hydrogen-bond lengths within this pocket corresponded with the relative potencies of the agents for positive modulation of GABAA receptor currents (cis,cis > trans,trans > cis,trans-2,6-dimethylcyclohexanol). In conclusion, the stereochemical configuration within the dimethylcyclohexanols is an important molecular feature in conferring positive modulation of GABAA receptor activity and for binding to the receptor, a consideration that needs to be taken into account when designing novel anesthetics with enhanced therapeutic indices. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Increased efficiency of the GABAA and GABAB receptor–mediated neurotransmission in the Ts65Dn mouse model of Down syndrome

PubMed Central

Kleschevnikov, Alexander M.; Belichenko, Pavel V.; Gall, Jessica; George, Lizzy; Nosheny, Rachel; Maloney, Michael T.; Salehi, Ahmad; Mobley, William C.

2011-01-01

Cognitive impairment in Down syndrome (DS) involves the hippocampus. In the Ts65Dn mouse model of DS, deficits in hippocampus-dependent learning and synaptic plasticity were linked to enhanced inhibition. However, the mechanistic basis of changes in inhibitory efficiency remains largely unexplored, and efficiency of the GABAergic synaptic neurotransmission has not yet been investigated in direct electrophysiological experiments. To investigate this important feature of neurobiology of DS, we examined synaptic and molecular properties of the GABAergic system in the dentate gyrus (DG) of adult Ts65Dn mice. Both GABAA and GABAB receptor-mediated components of evoked inhibitory postsynaptic currents (IPSCs) were significantly increased in Ts65Dn vs. control (2N) DG granule cells. These changes were unaccompanied by alterations in hippocampal levels of GABAA (α1, α2, α3, α5 and γ2) or GABAB (Gbr1a and Gbr1b) receptor subunits. Immunoreactivity for GAD65, a marker for GABAergic terminals, was also unchanged. In contrast, there was a marked change in functional parameters of GABAergic synapses. Paired stimulations showed reduced paired-pulse ratios of both GABAA and GABAB receptor-mediated IPSC components (IPSC2/IPSC1), suggesting an increase in presynaptic release of GABA. Consistent with increased gene dose, the level of the Kir3.2 subunit of potassium channels, effectors for postsynaptic GABAB receptors, was increased. This change was associated with enhanced postsynaptic GABAB/Kir3.2 signaling following application of the GABAB receptor agonist baclofen. Thus, both GABAA and GABAB receptor-mediated synaptic efficiency is increased in the Ts65Dn DG, thus likely contributing to deficient synaptic plasticity and poor learning in DS. PMID:22062771

Short-term memory impairment after isoflurane in mice is prevented by the α5 γ-aminobutyric acid type A receptor inverse agonist L-655,708.

PubMed

Saab, Bechara J; Maclean, Ashley J B; Kanisek, Marijana; Zurek, Agnieszka A; Martin, Loren J; Roder, John C; Orser, Beverley A

2010-11-01

Memory blockade is an essential component of the anesthetic state. However, postanesthesia memory deficits represent an undesirable and poorly understood adverse effect. Inhibitory α5 subunit-containing γ-aminobutyric acid subtype A receptors (α5GABAA) are known to play a critical role in memory processes and are highly sensitive to positive modulation by anesthetics. We postulated that inhibiting the activity of α5GABAA receptors during isoflurane anesthesia would prevent memory deficits in the early postanesthesia period. Mice were pretreated with L-655,708, an α5GABAA receptor-selective inverse agonist, or vehicle. They were then exposed to isoflurane for 1 h (1.3%, or 1 minimum alveolar concentration, or air-oxygen control). Then, either 1 or 24 h later, mice were conditioned in fear-associated contextual and cued learning paradigms. In addition, the effect of L-655,708 on the immobilizing dose of isoflurane was studied. Motor coordination, sedation, anxiety, and the concentration of isoflurane in the brain at 5 min, 1 h, and 24 h after isoflurane were also examined. Motor and sensory function recovered within minutes after termination of isoflurane administration. In contrast, a robust deficit in contextual fear memory persisted for at least 24 h. The α5GABAA receptor inverse agonist, L-655,708, completely prevented memory deficits without changing the immobilizing dose of isoflurane. Trace concentrations of isoflurane were measured in the brain 24 h after treatment. Memory deficits occurred long after the sedative, analgesic, and anxiolytic effects of isoflurane subsided. L-655,708 prevented memory deficit, suggesting that an isoflurane interaction at α5GABAA receptors contributes to memory impairment during the early postanesthesia period.

Expression of specific ionotropic glutamate and GABA-A receptor subunits is decreased in central amygdala of alcoholics.

PubMed

Jin, Zhe; Bhandage, Amol K; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R; Birnir, Bryndis

2014-01-01

The central amygdala (CeA) has a role for mediating fear and anxiety responses. It is also involved in emotional imbalance caused by alcohol abuse and dependence and in regulating relapse to alcohol abuse. Growing evidences suggest that excitatory glutamatergic and inhibitory γ-aminobutyric acid-ergic (GABAergic) transmissions in the CeA are affected by chronic alcohol exposure. Human post-mortem CeA samples from male alcoholics (n = 9) and matched controls (n = 9) were assayed for the expression level of ionotropic glutamate and GABA-A receptors subunit mRNAs using quantitative real-time reverse transcription-PCR (RT-qPCR). Our data revealed that out of the 16 ionotropic glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA1 and GluA4; one kainate receptor subunit GluK2; one NMDA (N-methyl-D-aspartate) receptor subunit GluN2D and one delta receptor subunit GluD2 were significantly decreased in the CeA of alcoholics. In contrast, of the 19 GABA-A receptor subunits, only the mRNA encoding the α2 subunit was significantly down-regulated in the CeA of the alcoholics as compared with control subjects. Our findings imply that the down-regulation of specific ionotropic glutamate and GABA-A receptor subunits in the CeA of alcoholics may represent one of the molecular substrates underlying the new balance between excitatory and inhibitory neurotransmission in alcohol dependence.

Expression of specific ionotropic glutamate and GABA-A receptor subunits is decreased in central amygdala of alcoholics

PubMed Central

Jin, Zhe; Bhandage, Amol K.; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R.; Birnir, Bryndis

2014-01-01

The central amygdala (CeA) has a role for mediating fear and anxiety responses. It is also involved in emotional imbalance caused by alcohol abuse and dependence and in regulating relapse to alcohol abuse. Growing evidences suggest that excitatory glutamatergic and inhibitory γ-aminobutyric acid-ergic (GABAergic) transmissions in the CeA are affected by chronic alcohol exposure. Human post-mortem CeA samples from male alcoholics (n = 9) and matched controls (n = 9) were assayed for the expression level of ionotropic glutamate and GABA-A receptors subunit mRNAs using quantitative real-time reverse transcription-PCR (RT-qPCR). Our data revealed that out of the 16 ionotropic glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA1 and GluA4; one kainate receptor subunit GluK2; one NMDA (N-methyl-D-aspartate) receptor subunit GluN2D and one delta receptor subunit GluD2 were significantly decreased in the CeA of alcoholics. In contrast, of the 19 GABA-A receptor subunits, only the mRNA encoding the α2 subunit was significantly down-regulated in the CeA of the alcoholics as compared with control subjects. Our findings imply that the down-regulation of specific ionotropic glutamate and GABA-A receptor subunits in the CeA of alcoholics may represent one of the molecular substrates underlying the new balance between excitatory and inhibitory neurotransmission in alcohol dependence. PMID:25278838

Hippocampal GABAA Receptor and Pain Sensitivity during Estrous Cycle in the Rat

PubMed Central

Taherianfard, Mahnaz; Mosavi, Mahnaz

2011-01-01

Background: Estradiol and progesterone as well as hippocampal GABAA receptors are believed to play a role in the modulation of pain. The aim of present study was to investigate the effect of intrahippocampal injections of GABAA receptor agonist (muscimol) and GABAA receptor antagonist (picrotoxin) on pain sensitivity during estrous cycle. Methods: Pain sensitivity was evaluated in rats by formalin test during all stages of estrous cycle. Animals were divided into five groups including; 1- control (intact animal); 2- sham 1 receiving 0.75 µl artificial cerebrospinal fluids (ACSF); 3- sham 2 receiving 0.75 µl alcoholic ACSF; 4- experimental 1 receiving 250 or 500 µg/rat of muscimol in 0.75 µl vehicle, and 5- experimental 2 receiving 20 or 30 µg/rat picrotoxin in 0.75 µl vehicle. Data were analyzed by Kruskal-Wallis followed by Tucky's test for pairwise comparisons using a P value of ≤0.50 for statistical significance. Results: Muscimol significantly (P<0.05) decreased pain sensitivity in all stages of estrous cycle, and the analgesic effect was higher during proestrus and estrus stages of estrous cycle than that during metestrus and diestrus stages. Picrotoxin significantly (P<0.05) increased pain sensitivity in all stages of estrous cycle, and such a hyperalgesic effect was lower during proestrus and estrus stages of estrous cycle than that during metestrus and diestrus stages. Conclusion: The findings of the present study indicate that the role of hippocampal GABAA receptor in the control of the pain sensitivity can be modulated by variation in gonadal steroids during different stages of the estrous cycle. PMID:23115414

Fluoxetine disrupts motivation and GABAergic signaling in adolescent female hamsters.

PubMed

Shannonhouse, John L; DuBois, Dustin W; Fincher, Annette S; Vela, Alejandra M; Henry, Morgan M; Wellman, Paul J; Frye, Gerald D; Morgan, Caurnel

2016-08-01

Initial antidepressant treatment can paradoxically worsen symptoms in depressed adolescents by undetermined mechanisms. Interestingly, antidepressants modulate GABAA receptors, which mediate paradoxical effects of other therapeutic drugs, particularly in females. Although the neuroanatomic site of action for this paradox is unknown, elevated GABAA receptor signaling in the nucleus accumbens can disrupt motivation. We assessed fluoxetine's effects on motivated behaviors in pubescent female hamsters - anhedonia in the reward investigational preference (RIP) test as well as anxiety in the anxiety-related feeding/exploration conflict (AFEC) test. We also assessed accumbal signaling by RT-PCR and electrophysiology. Fluoxetine initially worsened motivated behaviors at puberty, relative to adulthood. It also failed to improve these behaviors as pubescent hamsters transitioned into adulthood. Low accumbal mRNA levels of multiple GABAA receptor subunits and GABA-synthesizing enzyme, GAD67, assessed by RT-PCR, suggested low GABAergic tone at puberty. Nonetheless, rapid fluoxetine-induced reductions of α5GABAA receptor and BDNF mRNA levels at puberty were consistent with age-related differences in GABAergic responses to fluoxetine and disruption of the motivational state. Whole-cell patch clamping of accumbal slices also suggested low GABAergic tone by the low amplitude of miniature inhibitory postsynaptic currents (mIPSCs) at puberty. It also confirmed age-related differences in GABAergic responses to fluoxetine. Specifically, fluoxetine potentiated mIPSC amplitude and frequency at puberty, but attenuated the amplitude during adulthood. These results implicate GABAergic tone and GABAA receptor plasticity in adverse motivational responses and resistance to fluoxetine during adolescence. Copyright © 2016 Elsevier Inc. All rights reserved.

The active analog approach applied to the pharmacophore identification of benzodiazepine receptor ligands

NASA Astrophysics Data System (ADS)

Tebib, Souhail; Bourguignon, Jean-Jacques; Wermuth, Camille-Georges

1987-07-01

Applied to seven potent benzodiazepine-receptor ligands belonging to chemically different classes, the active analog approach allowed the stepwise identification of the pharmacophoric pattern associated with the recognition by the benzodiazepine receptor. A unique pharmacophore model was derived which involves six critical zones: (a) a π-electron rich aromatic (PAR) zone; (b) two electron-rich zones δ1 and δ2 placed at 5.0 and 4.5 Å respectively from the reference centroid in the PAR zone; (c) a freely rotating aromatic ring (FRA) region; (d) an out-of-plane region (OPR), strongly associated with agonist properties; and (e) an additional hydrophobic region (AHR). The model accommodates all presently known ligands of the benzodiazepine receptor, identifies sensitivity to steric hindrance close to the δ1 zone, accounts for R and S differential affinities and distinguishes requirements for agonist versus non-agonist activity profiles.

Fragrances in oolong tea that enhance the response of GABAA receptors.

PubMed

Hossain, Sheikh Julfikar; Aoshima, Hitoshi; Koda, Hirofumi; Kiso, Yoshinobu

2004-09-01

We electrophysiologically investigated the effect of some fragrant compounds in oolong tea on the response of ionotropic gamma-aminobutyric acid (GABA) receptors (GABAA receptors) which were expressed in Xenopus oocytes. Of the tested fragrances in oolong tea, cis-jasmone, jasmine lactone, linalool oxide and methyl jasmonate significantly potentiated the response. Among these, cis-jasmone and methyl jasmonate potently potentiated the response, having a respective dissociation constant of the compound (Kp) and maximum potentiation (Vm) of 0.49 mM and 322% for cis-jasmone, and 0.84 mM and 450% for methyl jasmonate. Inhalation of 0.1% cis-jasmone or methyl jasmonate significantly increased the sleeping time of mice induced by pentobarbital, suggesting that these fragrant compounds were absorbed by the brain and thereby potentiated the GABAA receptor response. Both of these compounds may therefore have a tranquillizing effect on the brain.

Mitochondrial reactive oxygen species regulate the strength of inhibitory GABA-mediated synaptic transmission

NASA Astrophysics Data System (ADS)

Accardi, Michael V.; Daniels, Bryan A.; Brown, Patricia M. G. E.; Fritschy, Jean-Marc; Tyagarajan, Shiva K.; Bowie, Derek

2014-01-01

Neuronal communication imposes a heavy metabolic burden in maintaining ionic gradients essential for action potential firing and synaptic signalling. Although cellular metabolism is known to regulate excitatory neurotransmission, it is still unclear whether the brain’s energy supply affects inhibitory signalling. Here we show that mitochondrial-derived reactive oxygen species (mROS) regulate the strength of postsynaptic GABAA receptors at inhibitory synapses of cerebellar stellate cells. Inhibition is strengthened through a mechanism that selectively recruits α3-containing GABAA receptors into synapses with no discernible effect on resident α1-containing receptors. Since mROS promotes the emergence of postsynaptic events with unique kinetic properties, we conclude that newly recruited α3-containing GABAA receptors are activated by neurotransmitter released onto discrete postsynaptic sites. Although traditionally associated with oxidative stress in neurodegenerative disease, our data identify mROS as a putative homeostatic signalling molecule coupling cellular metabolism to the strength of inhibitory transmission.

Modulation of GABAergic receptor binding by activation of calcium and calmodulin-dependent kinase II membrane phosphorylation.

PubMed

Churn, S B; DeLorenzo, R J

1998-10-26

gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system (CNS). Because of the important role that GABA plays in the CNS, alteration of GABAA receptor function would significantly affect neuronal excitability. Protein phosphorylation is a major mechanism for regulating receptor function in the brain and has been implicated in modulating GABAA receptor function. Therefore, this study was initiated to determine the role of calmodulin-dependent kinase II (CaM kinase II) membrane phosphorylation on GABAA receptor binding. Synaptosomal membrane fractions were tested for CaM kinase II activity towards endogenous substrates. In addition, muscimol binding was evaluated under equilibrium conditions in synaptosomal membrane fractions subjected to either basal (Mg2+ alone) or maximal CaM kinase II-dependent phosphorylation. Activation of endogenous CaM kinase II-dependent phosphorylation resulted in a significant enhancement of the apparent Bmax for muscimol binding without significantly altering the apparent binding affinity. The enhanced muscimol binding could be increased further by the addition of exogenous CaM kinase II to synaptosomal membrane fractions. Co-incubation with inhibitors of kinase activity during the phosphorylation reactions blocked the CaM kinase II-dependent increase in muscimol binding. The data support the hypothesis that activation of CaM kinase II-dependent phosphorylation caused an increased GABAA receptor binding and may play an important role in modulating the function of this inhibitory receptor/chloride ion channel complex. Copyright 1998 Elsevier Science B.V.

Adenovector GAD65 gene delivery into the rat trigeminal ganglion produces orofacial analgesia

PubMed Central

Vit, Jean-Philippe; Ohara, Peter T; Sundberg, Christopher; Rubi, Blanca; Maechler, Pierre; Liu, Chunyan; Puntel, Mariana; Lowenstein, Pedro; Castro, Maria; Jasmin, Luc

2009-01-01

Background Our goal is to use gene therapy to alleviate pain by targeting glial cells. In an animal model of facial pain we tested the effect of transfecting the glutamic acid decarboxylase (GAD) gene into satellite glial cells (SGCs) of the trigeminal ganglion by using a serotype 5 adenovector with high tropisms for glial cells. We postulated that GABA produced from the expression of GAD would reduce pain behavior by acting on GABA receptors on neurons within the ganglion. Results Injection of adenoviral vectors (AdGAD65) directly into the trigeminal ganglion leads to sustained expression of the GAD65 isoform over the 4 weeks observation period. Immunohistochemical analysis showed that adenovirus-mediated GAD65 expression and GABA synthesis were mainly in SGCs. GABAA and GABAB receptors were both seen in sensory neurons, yet only GABAA receptors decorated the neuronal surface. GABA receptors were not found on SGCs. Six days after injection of AdGAD65 into the trigeminal ganglion, there was a statistically significant decrease of pain behavior in the orofacial formalin test, a model of inflammatory pain. Rats injected with control virus (AdGFP or AdLacZ) had no reduction in their pain behavior. AdGAD65-dependent analgesia was blocked by bicuculline, a selective GABAA receptor antagonist, but not by CGP46381, a selective GABAB receptor antagonist. Conclusion Transfection of glial cells in the trigeminal ganglion with the GAD gene blocks pain behavior by acting on GABAA receptors on neuronal perikarya. PMID:19656360

Adenovector GAD65 gene delivery into the rat trigeminal ganglion produces orofacial analgesia.

PubMed

Vit, Jean-Philippe; Ohara, Peter T; Sundberg, Christopher; Rubi, Blanca; Maechler, Pierre; Liu, Chunyan; Puntel, Mariana; Lowenstein, Pedro; Castro, Maria; Jasmin, Luc

2009-08-05

Our goal is to use gene therapy to alleviate pain by targeting glial cells. In an animal model of facial pain we tested the effect of transfecting the glutamic acid decarboxylase (GAD) gene into satellite glial cells (SGCs) of the trigeminal ganglion by using a serotype 5 adenovector with high tropisms for glial cells. We postulated that GABA produced from the expression of GAD would reduce pain behavior by acting on GABA receptors on neurons within the ganglion. Injection of adenoviral vectors (AdGAD65) directly into the trigeminal ganglion leads to sustained expression of the GAD65 isoform over the 4 weeks observation period. Immunohistochemical analysis showed that adenovirus-mediated GAD65 expression and GABA synthesis were mainly in SGCs. GABAA and GABAB receptors were both seen in sensory neurons, yet only GABAA receptors decorated the neuronal surface. GABA receptors were not found on SGCs. Six days after injection of AdGAD65 into the trigeminal ganglion, there was a statistically significant decrease of pain behavior in the orofacial formalin test, a model of inflammatory pain. Rats injected with control virus (AdGFP or AdLacZ) had no reduction in their pain behavior. AdGAD65-dependent analgesia was blocked by bicuculline, a selective GABAA receptor antagonist, but not by CGP46381, a selective GABAB receptor antagonist. Transfection of glial cells in the trigeminal ganglion with the GAD gene blocks pain behavior by acting on GABAA receptors on neuronal perikarya.

A case of succinic semialdehyde dehydrogenase deficiency with status epilepticus and rapid regression.

PubMed

Horino, Asako; Kawawaki, Hisashi; Fukuoka, Masataka; Tsuji, Hitomi; Hattori, Yuka; Inoue, Takeshi; Nukui, Megumi; Kuki, Ichiro; Okazaki, Shin; Tomiwa, Kiyotaka; Hirose, Shinichi

2016-10-01

Clinical phenotypic expression of SSADH deficiency is highly heterogeneous, and some infants may develop refractory secondary generalized seizures. A 9-month-old boy manifested partial seizures, developing severe status epilepticus, and conventional antiepileptic drugs were ineffective. Use of ketamine contributed to the control of status epilepticus, achieving a reduction in frequency of partial seizures, and improving EEG findings without apparent complications. Diffusion-weighted images showed hyperintensities in the bilateral basal ganglia and fornix, and multiple T2 hyperintensity lesions were detected. (123)I-iomazenil (IMZ) SPECT revealed a decrease in binding of (123)I-iomazenil predominantly in the left temporal region by the 18th day of hospitalization. However, repeated IMZ-SPECT on the 46th day of hospitalization demonstrated almost no accumulation across a broad region, sparing the left temporal region. The patient showed rapid regression, refractory myoclonus, and severe progressive brain atrophy. IMZ-SPECT findings demonstrated reduced benzodiazepine receptor binding and its dynamic changes in an SSADH-deficient patient. Considering the down regulation of the GABAA receptor, ketamine should be included in pharmacotherapeutic strategies for treatment of refractory status epilepticus in SSADH-deficient patients. Copyright © 2016 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Rigor, Reproducibility and in vitro CSF assays: The Devil in the Details

PubMed Central

Moody, Olivia A.; Talwar, Sahil; Jenkins, Meagan A.; Freeman, Amanda A.; Trotti, Lynn Marie; García, Paul S.; Bliwise, Donald; Lynch, Joseph W.; Cherson, Brad; Hernandez, Eric M; Feldman, Neil; Saini, Prabhjyot; Rye, David B.; Jenkins, Andrew

2017-01-01

Divergent results and misinterpretation of non-significant findings remain problematic in science – especially in retrospective, hypothesis generating, translational research.1 When such divergence occurs, it is imperative that the cause of the divergence be established. In their recent paper in Annals of Neurology, Dauvilliers et al2 challenged our earlier finding that cerebrospinal fluid (CSF) from some patients with unexplained excessive daytime sleepiness enhances the activation of GABAA receptors (GABAA-R)3. They present data from 15 subjects in which they were unable to find evidence of enhanced activation of GABAA receptors. Here we: 1) establish how flaws in Dauvilliers’ experimental design account for this difference; 2) present new data demonstrating the robustness and reproducibility of our methods and 3) summarize the clinical promise of GABAA-R antagonism in treating IH and related disorders. PMID:28440033

Pharmacology of saccadic eye movements in man. 1. Effects of the benzodiazepine receptor ligands midazolam and flumazenil.

PubMed

Ball, D M; Glue, P; Wilson, S; Nutt, D J

1991-01-01

A paradigm for assessing benzodiazepine receptor sensitivity was developed using intravenous midazolam in normal volunteers. After administration of incremental doses of midazolam, alterations in saccadic eye movement parameters and psychological self ratings were assessed. Significant changes included dose-dependent slowing of peak velocity, peak acceleration, peak deceleration, reduced saccade acceleration/deceleration ratio and saccade accuracy, and increased sedation self-ratings. Changes in saccade variables and sedation ratings were significantly correlated, and also correlated with plasma midazolam concentrations. No significant changes were seen in saccade latency or anxiety self-ratings. Pharmacological specificity of these changes was demonstrated by their reversal with the benzodiazepine antagonist flumazenil. This challenge paradigm appears to be a sensitive means of assessing benzodiazepine receptor function in man.

Developmental changes in expression of GABAA receptor-channels in rat intrinsic cardiac ganglion neurones

PubMed Central

Fischer, Harald; Harper, Alexander A; Anderson, Colin R; Adams, David J

2005-01-01

The effects of γ-aminobutyric acid (GABA) on the electrophysiological properties of intracardiac neurones were investigated in the intracardiac ganglion plexus in situ and in dissociated neurones from neonatal, juvenile and adult rat hearts. Focal application of GABA evoked a depolarizing, excitatory response in both intact and dissociated intracardiac ganglion neurones. Under voltage clamp, both GABA and muscimol elicited inward currents at −60 mV in a concentration-dependent manner. The fast, desensitizing currents were mimicked by the GABAA receptor agonists muscimol and taurine, and inhibited by the GABAA receptor antagonists, bicuculline and picrotoxin. The GABAA0 antagonist (1,2,5,6-tetrahydropyridin-4-yl)methyl phosphonic acid (TPMPA), had no effect on GABA-induced currents, suggesting that GABAA receptor-channels mediate the response. The GABA-evoked current amplitude recorded from dissociated neurones was age dependent whereby the peak current density measured at −100 mV was ∼ 20 times higher for intracardiac neurones obtained from neonatal rats (P2–5) compared with adult rats (P45–49). The decrease in GABA sensitivity occurred during the first two postnatal weeks and coincides with maturation of the sympathetic innervation of the rat heart. Immunohistochemical staining using antibodies against GABA demonstrate the presence of GABA in the intracardiac ganglion plexus of the neonatal rat heart. Taken together, these results suggest that GABA and taurine may act as modulators of neurotransmission and cardiac function in the developing mammalian intrinsic cardiac nervous system. PMID:15731187

Mechanism and Site of Inhibition of AMPA Receptors: Pairing a Thiadiazole with a 2,3-Benzodiazepine Scaffold

PubMed Central

2013-01-01

2,3-Benzodiazepine compounds are synthesized as drug candidates for treatment of various neurological disorders involving excessive activity of AMPA receptors. Here we report that pairing a thiadiazole moiety with a 2,3-benzodiazepine scaffold via the N-3 position yields an inhibitor type with >28-fold better potency and selectivity on AMPA receptors than the 2,3-benzodiazepine scaffold alone. Using whole-cell recording, we characterized two thiadiazolyl compounds, that is, one contains a 1,3,4-thiadiazole moiety and the other contains a 1,2,4-thiadiazole-3-one moiety. These compounds exhibit potent, equal inhibition of both the closed-channel and the open-channel conformations of all four homomeric AMPA receptor channels and two GluA2R-containing complex AMPA receptor channels. Furthermore, these compounds bind to the same receptor site as GYKI 52466 does, a site we previously termed as the “M” site. A thiadiazole moiety is thought to occupy more fully the side pocket of the receptor site or the “M” site, thereby generating a stronger, multivalent interaction between the inhibitor and the receptor binding site. We suggest that, as a heterocycle, a thiadiazole can be further modified chemically to produce a new class of even more potent, noncompetitive inhibitors of AMPA receptors. PMID:24313227

Emamectin is a non-selective allosteric activator of nicotinic acetylcholine receptors and GABAA/C receptors

PubMed Central

Xu, Xiaojun; Sepich, Caraline; Lukas, Ronald J; Zhu, Guonian; Chang, Yongchang

2016-01-01

Avermectins are a group of compounds isolated from a soil-dwelling bacterium. They have been widely used as parasiticides and insecticides, acting by relatively irreversible activation of invertebrate chloride channels. Emamectin is a soluble derivative of an avermectin. It is an insecticide, which persistently activates glutamate-gated chloride channels. However, its effects on mammalian ligand-gated ion channels are unknown. To this end, we tested the effect of emamectin on two cation selective nicotinic receptors and two GABA-gated chloride channels expressed in Xenopus oocytes using two-electrode voltage clamp. Our results demonstrate that emamectin could directly activate α7 nAChR, α4β2 nAChR, α1β2γ2 GABAA receptor and ρ1 GABAC receptor concentration dependently, with similar potencies for each channel. However, the potencies for it to activate these channels were at least two orders of magnitude lower than its potency of activating invertebrate glutamate-gated chloride channel. In contrast, ivermectin only activated the α1β2γ2 GABAA receptor. PMID:27049309

Intra-accumbens baclofen, but not muscimol, mimics the effects of food withdrawal on feeding behaviour.

PubMed

Pulman, K G T; Somerville, E M; Clifton, P G

2010-11-01

Intra-accumbens stimulation of GABA receptors results in a robust increase in food intake. However the differential consequences of stimulating GABA(A) and GABA(B) receptors in the nucleus accumbens have not been extensively explored with respect to feeding behaviour. Here we compare the effects of the GABA(B) receptor agonist baclofen and GABA(A) receptor agonist muscimol, infused into the nucleus accumbens shell, on food intake and related behavior patterns. Baclofen (110-440 ρmol) dose dependently enhanced intake and delayed the onset of satiety within the test period as did the effects of 4-8h food withdrawal. Muscimol (220-660 ρmol) enhanced intake but also disrupted the sequence of associated behaviours at every dose tested. We conclude that GABA(B) receptors in the nucleus accumbens shell may play a role in relation to feeding motivation whereas GABA(A) receptors may, as previously suggested, have a more restricted role in relation to the motor components of approach to food and ingestion. Copyright © 2010 Elsevier Inc. All rights reserved.

Emamectin is a non-selective allosteric activator of nicotinic acetylcholine receptors and GABAA/C receptors.

PubMed

Xu, Xiaojun; Sepich, Caraline; Lukas, Ronald J; Zhu, Guonian; Chang, Yongchang

2016-05-13

Avermectins are a group of compounds isolated from a soil-dwelling bacterium. They have been widely used as parasiticides and insecticides, acting by relatively irreversible activation of invertebrate chloride channels. Emamectin is a soluble derivative of an avermectin. It is an insecticide, which persistently activates glutamate-gated chloride channels. However, its effects on mammalian ligand-gated ion channels are unknown. To this end, we tested the effect of emamectin on two cation selective nicotinic receptors and two GABA-gated chloride channels expressed in Xenopus oocytes using two-electrode voltage clamp. Our results demonstrate that emamectin could directly activate α7 nAChR, α4β2 nAChR, α1β2γ2 GABAA receptor and ρ1 GABAC receptor concentration dependently, with similar potencies for each channel. However, the potencies for it to activate these channels were at least two orders of magnitude lower than its potency of activating invertebrate glutamate-gated chloride channel. In contrast, ivermectin only activated the α1β2γ2 GABAA receptor. Copyright © 2016 Elsevier Inc. All rights reserved.

Neurosteroid modulation of neuronal excitability and synaptic transmission in the rat medial vestibular nuclei.

PubMed

Grassi, Silvarosa; Frondaroli, Adele; Dieni, Cristina; Dutia, Mayank B; Pettorossi, Vito E

2007-07-01

In rat brainstem slices, we investigated the influence of the neurosteroids tetrahydrodeoxycorticosterone (THDOC) and allopregnanolone (ALLO) on the synaptically driven and spontaneous activity of vestibular neurons, by analysing their effects on the amplitude of the field potentials evoked in the medial vestibular nuclei (MVN) by vestibular afferent stimulation and on the spontaneous firing rate of MVN neurons. Furthermore, the interaction with gamma-aminobutyric acid (GABA) and glutamate receptors was analysed by using specific antagonists for GABA(A) (bicuculline), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/ kainate [2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulphonamide disodium salt (NBQX)], N-methyl-D-aspartate (NMDA) [D-(-)-2-amino-5-phosphonopentanoic acid (AP-5)] and group I metabotropic glutamate receptors (mGlu-I) [(R,S)-1-aminoindan-1,5-dicarboxylic acid (AIDA)] receptors. THDOC and ALLO evoked two opposite long-lasting effects, consisting of either a potentiation or a reduction of field potential and firing rate, which showed early and late components, occurring in conjunction or separately after neurosteroid application. The depressions depended on GABA(A) receptors, as they were abolished by bicuculline, while early potentiation involved glutamate AMPA/kainate receptors, as NBQX markedly reduced the incidence of early firing rate enhancement and, in the case of ALLO, even provoked depression. This suggests that THDOC and ALLO enhance the GABA(A) inhibitory influence on the MVN neurons and facilitate the AMPA/kainate facilitatory one. Conversely, a late potentiation effect, which was still induced after glutamate and GABA(A) receptor blockade, might involve a different mechanism. We conclude that the modulation of neuronal activity in the MVN by THDOC and ALLO, through their actions on GABA(A) and AMPA/kainate receptors, may have a physiological role in regulating the vestibular system function under normal conditions and during the stress response that accompanies many forms of vestibular dysfunction.

Affective and cognitive effects of global deletion of alpha3-containing gamma-aminobutyric acid-A receptors.

PubMed

Fiorelli, Roberto; Rudolph, Uwe; Straub, Carolin J; Feldon, Joram; Yee, Benjamin K

2008-09-01

Gamma-aminobutyric acid (GABA)A receptors characterized by the presence of the alpha3 subunit are the major GABAA receptor subtype expressed in brain stem monoaminergic nuclei. These alpha3-GABAA receptors are therefore in a unique position to regulate monoaminergic functions. To characterize the functional properties of alpha3-GABAA receptors, we present a preliminary assessment of the expression of affective and cognitive behaviour in male mice with a targeted deletion of the Gabra3 gene encoding the alpha3 subunit [alpha3 knockout (KO) mice] on a C57BL/6Jx129X1/SvJ F1 hybrid genetic background. The alpha3 KO mice did not exhibit any gross change of anxiety-like behaviour or spontaneous locomotor behaviour. In the Porsolt forced swim test for potential antidepressant activity, alpha3 KO mice exhibited reduced floating and enhanced swimming behaviour relative to wild-type controls. Performance on a two-choice sucrose preference test, however, revealed no evidence for an increase in sucrose preference in the alpha3 KO mice that would have substantiated a potential phenotype for depression-related behaviour. In contrast, a suggestion of an enhanced negative contrast effect was revealed in a one-bottle sucrose consumption test across different sucrose concentrations. These affective phenotypes were accompanied by alterations in the balance between conditioned responding to the discrete conditioned stimulus and to the context, and a suggestion of faster extinction, in the Pavlovian conditioned freezing paradigm. Spatial learning in the water maze reference memory test, however, was largely unchanged in the alpha3 KO mice, except for a trend of preservation during reversal learning. The novel phenotypes following global deletion of the GABAA receptor alpha3 subunit identified here provided relevant insights, in addition to our earlier study, into the potential behavioural relevance of this specific receptor subtypes in the modulation of both affective and cognitive functions.

Nicotine and Nicotine Abstinence Do Not Interfere with GABAA Receptor Neuroadaptations During Alcohol Abstinence.

PubMed

Hillmer, Ansel T; Kloczynski, Tracy; Sandiego, Christine M; Pittman, Brian; Anderson, Jon M; Labaree, David; Gao, Hong; Huang, Yiyun; Deluliis, Giuseppe; O'Malley, Stephanie S; Carson, Richard E; Cosgrove, Kelly P

2016-04-01

Alcohol dependence and tobacco smoking are highly comorbid, and treating both conditions simultaneously is controversial. Previously, we showed that tobacco smoking interferes with GABAA receptor neuroadaptations during alcohol withdrawal in humans, while this effect did not occur with continued nicotine use during alcohol abstinence in nonhuman primates. Here, we extend our previous work by measuring GABAA receptor availability with positron emission tomography (PET) during drug abstinence in nonhuman primates exposed to alcohol alone, nicotine and alcohol together, and alcohol abstinence with continued nicotine exposure. Twenty-four adolescent male rhesus macaques orally self-administered alcohol and nicotine, available separately in water and saccharin, over 20 weeks. The groups included alcohol alone (n = 8); nicotine and alcohol with simultaneous abstinence (n = 8); nicotine and alcohol with alcohol abstinence while nicotine was still available (n = 8); and a pilot group of animals consuming nicotine alone (n = 6). Animals were imaged with [(11)C]flumazenil PET to measure binding potential (BPND), an index of GABAA receptor availability. Imaging occurred at baseline (drug-naíve), and following alcohol and/or nicotine cessation at 1 day, 8 days, and 12 weeks of abstinence. Generalized linear mixed models were used to examine the time course of [(11)C]flumazenil BPND during alcohol abstinence across groups. Animals consumed 3.95 ± 1.22 g/kg/d alcohol and 55.4 ± 35.1 mg/kg/d nicotine. No significant group effects were observed in [(11)C]flumazenil BPND during alcohol abstinence; however, a main effect of time was detected. Post hoc analyses indicated that all groups abstaining from alcohol exhibited significantly increased GABAA receptor availability at 1 day and 8 days (but not 12 weeks) of abstinence relative to baseline, while no changes in [(11)C]flumazenil BPND during nicotine abstinence alone were observed. These data indicate that neither nicotine nor nicotine abstinence interferes with GABAA receptor neuroadaptations during alcohol withdrawal. This conclusion is consistent with our previous study and does not contradict the use of nicotine replacement therapies or non-nicotinic-acting pharmaceuticals to quit smoking during alcohol withdrawal from a GABAergic perspective. Copyright © 2016 by the Research Society on Alcoholism.

Mechanism of the cardiovascular effects of the GABAA receptors of the ventral tegmental area of the rat brain.

PubMed

Yeganeh, Fahimeh; Ranjbar, Afsaneh; Hatam, Masoumeh; Nasimi, Ali

2015-07-23

The ventral tegmental area (VTA) contains GABA terminals involved in the regulation of the cardiovascular system. Previously, we demonstrated that blocking GABAA but not GABAB receptors produced a pressor response accompanied by marked bradycardia. This study was performed to find the possible mechanisms involved in these responses by blocking ganglionic nicotinic receptors, peripheral muscarinic receptors or peripheral V1 vasopressin receptors. Experiments were performed on urethane anesthetized male Wistar rats. Drugs were microinjected unilaterally into the VTA (100 nl). The average changes in mean arterial pressure (MAP) and heart rate (HR) were compared between pre- and post-treatment using paired t-test. Injection of bicuculline methiodide (BMI), a GABAA antagonist, into the VTA caused a significant increase in MAP and a decrease in HR. Administration (i.v.) of the nicotinic receptor blocker, hexamethonium, enhanced the pressor response but abolished the bradycardic response to BMI, which ruled out involvement of the sympathetic nervous system. Blockade of the peripheral muscarinic receptors by homatropine (i.v.) abolished the bradycardic effect of BMI, but had no effect on the pressor response, indicating that bradycardia was produced by the parasympathetic outflow to the heart. Both the pressor and bradycardic responses to BMI were blocked by V1 receptor antagonist (i.v.), indicating that administration of BMI in the VTA disinhibited the release of vasopressin into the circulation. In conclusion, we demonstrated that GABAergic mechanism of the VTA exerts a tonic inhibition on vasopressin release through activation of GABAA receptors. The sympathetic system is not involved in the decrease of blood pressure by GABA of the VTA. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Lack of effect of mossy fiber-released zinc on granule cell GABA(A) receptors in the pilocarpine model of epilepsy.

PubMed

Molnár, P; Nadler, J V

2001-05-01

The recurrent mossy fiber pathway of the dentate gyrus expands dramatically in the epileptic brain and serves as a mechanism for synchronization of granule cell epileptiform activity. It has been suggested that this pathway also promotes epileptiform activity by inhibiting GABA(A) receptor function through release of zinc. Hippocampal slices from pilocarpine-treated rats were used to evaluate this hypothesis. The rats had developed status epilepticus after pilocarpine administration, followed by robust recurrent mossy fiber growth. The ability of exogenously applied zinc to depress GABA(A) receptor function in dentate granule cells depended on removal of polyvalent anions from the superfusion medium. Under these conditions, 200 microM zinc reduced the amplitude of the current evoked by applying muscimol to the proximal portion of the granule cell dendrite (23%). It also reduced the mean amplitude (31%) and frequency (36%) of miniature inhibitory postsynaptic currents. Nevertheless, repetitive mossy fiber stimulation (10 Hz for 1 s, 100 Hz for 1 s, or 10 Hz for 5 min) at maximal intensity did not affect GABA(A) receptor-mediated currents evoked by photorelease of GABA onto the proximal portion of the dendrite, where recurrent mossy fiber synapses were located. These results could not be explained by stimulation-induced depletion of zinc from the recurrent mossy fiber boutons. Negative results were obtained even during exposure to conditions that promoted transmitter release and synchronized granule cell activity (6 mM [K(+)](o), nominally Mg(2+)-free medium, 33 degrees C). These results suggest that zinc released from the recurrent mossy fiber pathway did not reach a concentration at postsynaptic GABA(A) receptors sufficient to inhibit agonist-evoked activation.

Neurodevelopmental disorders among individuals with duplication of 4p13 to 4p12 containing a GABAA receptor subunit gene cluster

PubMed Central

Polan, Michelle B; Pastore, Matthew T; Steingass, Katherine; Hashimoto, Sayaka; Thrush, Devon L; Pyatt, Robert; Reshmi, Shalini; Gastier-Foster, Julie M; Astbury, Caroline; McBride, Kim L

2014-01-01

Recent studies have shown that certain copy number variations (CNV) are associated with a wide range of neurodevelopmental disorders, including autism spectrum disorders (ASD), bipolar disorder and intellectual disabilities. Implicated regions and genes have comprised a variety of post synaptic complex proteins and neurotransmitter receptors, including gamma-amino butyric acid A (GABAA). Clusters of GABAA receptor subunit genes are found on chromosomes 4p12, 5q34, 6q15 and 15q11-13. Maternally inherited 15q11-13 duplications among individuals with neurodevelopmental disorders are well described, but few case reports exist for the other regions. We describe a family with a 2.42 Mb duplication at chromosome 4p13 to 4p12, identified in the index case and other family members by oligonucleotide array comparative genomic hybridization, that contains 13 genes including a cluster of four GABAA receptor subunit genes. Fluorescent in-situ hybridization was used to confirm the duplication. The duplication segregates with a variety of neurodevelopmental disorders in this family, including ASD (index case), developmental delay, dyspraxia and ADHD (brother), global developmental delays (brother), learning disabilities (mother) and bipolar disorder (maternal grandmother). In addition, we identified and describe another individual unrelated to this family, with a similar duplication, who was diagnosed with ASD, ADHD and borderline intellectual disability. The 4p13 to 4p12 duplication appears to confer a susceptibility to a variety of neurodevelopmental disorders in these two families. We hypothesize that the duplication acts through a dosage effect of GABAA receptor subunit genes, adding evidence for alterations in the GABAergic system in the etiology of neurodevelopmental disorders. PMID:23695283

Activation of GABA-A receptors during postnatal brain development increases anxiety- and depression-related behaviors in a time- and dose-dependent manner in adult mice.

PubMed

Salari, Ali-Akbar; Bakhtiari, Amir; Homberg, Judith R

2015-08-01

Disturbances of the gamma-amino butyric acid-ergic (GABAergic) system during postnatal development can have long-lasting consequences for later life behavior, like the individual's response to stress. However, it is unclear which postnatal windows of sensitivity to GABA-ergic modulations are associated with what later-life behavioral outcomes. Therefore, we sought to determine whether neonatal activation of the GABA-A receptor during two postnatal periods, an early window (postnatal day 3-5) and a late window (postnatal day 14-16), can affect anxiety- and depression-related behaviors in male mice in later life. To this end, mice were treated with either saline or muscimol (50, 100, 200, 300 and 500μg/kg) during the early and late postnatal periods. An additional group of mice was treated with the GABA-A receptor antagonist bicuculline+muscimol. When grown to adulthood male mice were exposed to behavioral tests to measure anxiety- and depression-related behaviors. Baseline and stress-induced corticosterone (CORT) levels were also measured. The results indicate that early postnatal and to a lesser extent later postnatal exposure to the GABA-A receptor agonist muscimol increased anxiety-like behavior and stress-induced CORT levels in adults. Moreover, the early postnatal treatment with muscimol increased depression-like behavior with increasing baseline CORT levels. The anxiogenic and depression-like later-life consequences could be antagonized by bicuculline. Our findings suggest that GABA-A receptor signaling during early-life can influence anxiety- and depression-related behaviors in a time- and dose-dependent manner in later life. Our findings help to increase insight in the developmental mechanisms contributing to stress-related disorders. Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.

Neonatal blockade of GABA-A receptors alters behavioral and physiological phenotypes in adult mice.

PubMed

Salari, Ali-Akbar; Amani, Mohammad

2017-04-01

Gamma-aminobutyric acid (GABA) plays an inhibitory role in the mature brain, and has a complex and bidirectional effect in different parts of the immature brain which affects proliferation, migration and differentiation of neurons during development. There is also increasing evidence suggesting that activation or blockade of the GABA-A receptors during early life can induce brain and behavioral abnormalities in adulthood. We investigated whether neonatal blockade of the GABA-A receptors by bicuculline can alter anxiety- and depression-like behaviors, body weight, food intake, corticosterone and testosterone levels in adult mice (postnatal days 80-95). To this end, neonatal mice were treated with either DMSO or bicuculline (70, 150 and 300μg/kg) during postnatal days 7, 9 and 11. When grown to adulthood, mice were exposed to behavioral tests to measure anxiety- (elevated plus-maze and light-dark box) and depression-like behaviors (tail suspension test and forced swim test). Stress-induced serum corticosterone and testosterone levels, body weight and food intake were also evaluated. Neonatal bicuculline exposure at dose of 300μg/kg decreased anxiety-like behavior, stress-induced corticosterone levels and increased testosterone levels, body weight and food intake, without significantly influencing depression-like behavior in adult male mice. However, no significant changes in these parameters were observed in adult females. These findings suggest that neonatal blockade of GABA-A receptors affects anxiety-like behavior, physiological and hormonal parameters in a sex-dependent manner in mice. Taken together, these data corroborate the concept that GABA-A receptors during early life have an important role in programming neurobehavioral phenotypes in adulthood. Copyright © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.

Long-term administration with levonorgestrel decreases allopregnanolone levels and alters GABA(A) receptor subunit expression and anxiety-like behavior.

PubMed

Porcu, Patrizia; Mostallino, Maria Cristina; Sogliano, Cristiana; Santoru, Francesca; Berretti, Roberta; Concas, Alessandra

2012-08-01

Fluctuations in the concentrations of the neuroactive steroid allopregnanolone are thought to influence γ-amino-butyric acid type A (GABA(A)) receptor gene expression and function. Long-term treatment with ethinyl estradiol (EE) plus levonorgestrel (LNG), two of the most widely used steroids in the hormonal contraceptive pill, decreases allopregnanolone levels in rat cerebral cortex and plasma, alters GABA(A) receptor expression and induces anxiety-like behavior. We evaluated which component of the hormonal contraceptive pill is responsible for the aforementioned changes. Female rats were injected subcutaneously (s.c.) with EE (0.030 mg) or LNG (0.125 mg) once a day for 4 weeks. Compared to the respective vehicle-treated control groups, EE decreased cerebral cortical levels of allopregnanolone, progesterone and pregnenolone by 76, 72 and 33%, respectively and hippocampal levels by 52, 56 and 50%, respectively. Likewise, LNG decreased cerebral cortical levels of allopregnanolone, progesterone and pregnenolone by 75, 68 and 33%, respectively, and hippocampal levels by 55, 65 and 60%, respectively. Administration of LNG, but not EE, increased the abundance of the γ2 subunit peptide in cerebral cortex and hippocampus by 38 and 59%, respectively. Further, LNG, but not EE, decreased the time spent and the number of entries into the open arms of the elevated plus maze by 56 and 43%, respectively, an index of anxiety-like behavior. These results suggest that alterations in GABA(A) receptor subunit expression and anxiety-like behavior induced by long-term treatment with combined EE/LNG appear to be caused by LNG. Given that both EE and LNG decrease allopregnanolone levels in a similar manner, these results further suggest that changes in allopregnanolone levels are not associated with GABA(A) receptor expression. Copyright © 2012 Elsevier Inc. All rights reserved.

AZD-3043: A Novel, Metabolically-Labile Sedative/Hypnotic Agent with Rapid and Predictable Emergence from Hypnosis

PubMed Central

Egan, Talmage D.; Obara, Shinju; Jenkins, Thomas E.; Jaw-Tsai, Sarah S.; Amagasu, Shanti; Cook, Daniel R.; Steffensen, Scott C.; Beattie, David T.

2013-01-01

Background Propofol can be associated with delayed awakening after prolonged infusion. The aim of this study was to characterize the preclinical pharmacology of AZD-3043, a positive allosteric modulator of the γ-aminobutyric acidA (GABAA) receptor containing a metabolically-labile ester moiety. We postulated that its metabolic pathway would result in a short acting clinical profile. Methods The effects of AZD-3043, propofol and propanidid were studied on GABAA receptor-mediated chloride currents in embryonic rat cortical neurons. Radioligand binding studies were also performed. The in vitro stability of AZD-3043 in whole blood and liver microsomes was evaluated. The duration of the loss of righting reflex and effects on the electroencephalograph evoked by bolus or infusion intravenous (IV) administration were assessed in rats. A mixed-effects kinetic-dynamic model using minipigs permitted exploration of the clinical pharmacology of AZD-3043. Results AZD-3043 potentiated GABAA receptor-mediated chloride currents and inhibited [35S]tert-butylbicyclophosphorothionate binding to GABAA receptors. AZD-3043 was rapidly hydrolyzed in liver microsomes from humans and animals. AZD-3043 produced hypnosis and electroencephalograph depression in rats. Compared to propofol, AZD-3043 was shorter acting in rats and pigs. Computer simulation using the porcine kinetic-dynamic model demonstrated that AZD-3043 has very short 50 and 80% decrement times independent of infusion duration. Conclusions AZD-3043 is a positive allosteric modulator of the GABAA receptor in vitro and a sedative/hypnotic agent in vivo. The esterase dependent metabolic pathway results in rapid clearance and short duration of action even for long infusions. AZD-3043 may have clinical potential as a sedative/hypnotic agent with rapid and predictable recovery. PMID:22531340

Absence of γ-aminobutyric acid-a receptor potentiation in central hypersomnolence disorders.

PubMed

Dauvilliers, Yves; Evangelista, Elisa; Lopez, Regis; Barateau, Lucie; Jaussent, Isabelle; Cens, Thierry; Rousset, Matthieu; Charnet, Pierre

2016-08-01

The pathophysiology of idiopathic hypersomnia (IH) remains unclear. Recently, cerebrospinal fluid (CSF)-induced enhancement of γ-aminobutyric acid (GABA)-A receptor activity was found in patients with IH compared to controls. Fifteen unrelated patients (2 males and 13 females) affected with typical IH, 12 patients (9 males and 3 females) with narcolepsy type 1, and 15 controls (9 males and 6 females) with unspecified hypersomnolence (n = 7) and miscellaneous neurological conditions (n = 8) were included. A lumbar puncture was performed in all participants to measure CSF hypocretin-1 and GABA-A response. We used a voltage-clamp assay on Xenopus oocytes injected with the RNAs that encode the α1 β2 γ2 or the α2 β2 γ2 subunits of the human GABA-A receptor. A sequence of 6 different applications (GABA, GABA/CSF, and CSF alone) with 2 to 4 oocytes per CSF sample was performed in a whole-cell voltage-clamp assay. Representative current traces from oocytes expressing human α1 β2 γ2 or α2 β2 γ2 GABA-A receptors were recorded in response to 6 successive puffs of GABA diluted in the survival medium (SM), showing stable and reliable response. GABA puffs diluted in SM/CSF solution or SM/CSF solution alone showed no significant differences in the CSF of IH, narcolepsy, or control groups. No associations were found between GABA responses, demographic features, disease duration, or disease severity in the whole population or within groups. Using the Xenopus oocyte assay, we found an absence of GABA-A receptor potentiation with CSF from patients with central hypersomnolence disorders, with no significant differences between hypocretin-deficient and non-hypocretin-deficient patients compared to controls. Ann Neurol 2016;80:259-268. © 2016 American Neurological Association.

Selective GABA(A) α5 positive allosteric modulators improve cognitive function in aged rats with memory impairment.

PubMed

Koh, Ming Teng; Rosenzweig-Lipson, Sharon; Gallagher, Michela

2013-01-01

A condition of excess activity in the hippocampal formation is observed in the aging brain and in conditions that confer additional risk during aging for Alzheimer's disease. Compounds that act as positive allosteric modulators at GABA(A) α5 receptors might be useful in targeting this condition because GABA(A) α5 receptors mediate tonic inhibition of principal neurons in the affected network. While agents to improve cognitive function in the past focused on inverse agonists, which are negative allosteric modulators at GABA(A) α5 receptors, research supporting that approach used only young animals and predated current evidence for excessive hippocampal activity in age-related conditions of cognitive impairment. Here, we used two compounds, Compound 44 [6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one] and Compound 6 [methyl 3,5-diphenylpyridazine-4-carboxylate], with functional activity as potentiators of γ-aminobutyric acid at GABA(A) α5 receptors, to test their ability to improve hippocampal-dependent memory in aged rats with identified cognitive impairment. Improvement was obtained in aged rats across protocols differing in motivational and performance demands and across varying retention intervals. Significant memory improvement occurred after either intracereboventricular infusion with Compound 44 (100 μg) in a water maze task or systemic administration with Compound 6 (3 mg/kg) in a radial arm maze task. Furthermore, systemic administration improved behavioral performance at dosing shown to provide drug exposure in the brain and in vivo receptor occupancy in the hippocampus. These data suggest a novel approach to improve neural network function in clinical conditions of excess hippocampal activity. This article is part of a Special Issue entitled 'Cognitive Enhancers'. Copyright © 2012 Elsevier Ltd. All rights reserved.

Interaction of H+ and Zn2+ on recombinant and native rat neuronal GABAA receptors

PubMed Central

Krishek, Belinda J; Moss, Stephen J; Smart, Trevor G

1998-01-01

The interaction of Zn2+ and H+ ions with GABAA receptors was examined using Xenopus laevis oocytes expressing recombinant GABAA receptors composed of subunits selected from α1, β1, γ2S and δ types, and by using cultured rat cerebellar granule neurones. The potency of Zn2+ as a non-competitive antagonist of GABA-activated responses on α1β1 receptors was reduced by lowering the external pH from 7.4 to 5.4, increasing the Zn2+ IC50 value from 1.2 to 58.3 μm. Zinc-induced inhibition was largely unaffected by alkaline pH up to pH 9.4. For α1β1δ subunits, concentration-response curves for GABA were displaced laterally by Zn2+ in accordance with a novel mixed/competitive-type inhibition. The Zn2+ IC50 at pH 7.4 was 16.3 μm. Acidification of Ringer solution resulted in a reduced antagonism by Zn2+ (IC50, 49.0 μm) without affecting the type of inhibition. At pH 9.4, Zn2+ inhibition remained unaffected. The addition of the γ2S subunit to the α1β1δ construct caused a marked reduction in the potency of Zn2+ (IC50, 615 μm), comparable to that observed with α1β1γ2S receptors (IC50 639 μm). GABA concentration-response curves were depressed in a mixed/non-competitive fashion. In cultured cerebellar granule neurones, Zn2+ inhibited responses to GABA in a concentration-dependent manner. Lowering external pH from 7.4 to 6.4 increased the IC50 from 139 to 253 μm. The type of inhibition exhibited by Zn2+ on cerebellar granule neurones, previously grown in high K+-containing culture media, was complex, with the GABA concentration-response curves shifting laterally with reduced slopes and similar maxima. The Zn2+-induced shift in the GABA EC50 values was reduced by lowering the external pH from 7.4 to 6.4. The interaction of H+ and Zn2+ ions on GABAA receptors suggests that they share either a common regulatory pathway or coincident binding sites on the receptor protein. The apparent competitive mode of block induced by Zn2+ on α1β1δ receptors is shared by GABAA receptors on cerebellar granule neurones, which are known to express δ-subunit-containing receptors. This novel mechanism is masked when a γ2 subunit is incorporated into the receptor complex, revealing further diversity in the response of native GABAA receptors to endogenous cations. PMID:9508826

The insecticide fipronil and its metabolite fipronil sulphone inhibit the rat α1β2γ2L GABAA receptor

PubMed Central

Li, P; Akk, G

2008-01-01

Background and purpose: Fipronil is the active ingredient in a number of widely used insecticides. Human exposure to fipronil leads to symptoms (headache, nausea and seizures) typically associated with the antagonism of GABAA receptors in the brain. In this study, we have examined the modulation of the common brain GABAA receptor subtype by fipronil and its major metabolite, fipronil sulphone. Experimental approach: Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat α1β2γ2L GABAA receptors. Key results: The major effect of fipronil was to increase the rate of current decay in macroscopic recordings. In single-channel recordings, the presence of fipronil resulted in shorter cluster durations without affecting the intracluster open and closed time distributions or the single-channel conductance. The α1V256S mutation, previously shown alleviate channel inhibition by inhibitory steroids and several insecticides, had a relatively small effect on channel block by fipronil. The mode of action of fipronil sulphone was similar to that of its parent compound but the metabolite was less potent at inhibiting the α1β2γ2L receptor. Conclusions and implications: We conclude that exposure to fipronil induces accumulation of receptors in a novel, long-lived blocked state. This process proceeds in parallel with and independently of, channel desensitization. The lower potency of fipronil sulphone indicates that the conversion serves as a detoxifying process in mammalian brain. PMID:18660823

The insecticide fipronil and its metabolite fipronil sulphone inhibit the rat alpha1beta2gamma2L GABA(A) receptor.

PubMed

Li, P; Akk, G

2008-11-01

Fipronil is the active ingredient in a number of widely used insecticides. Human exposure to fipronil leads to symptoms (headache, nausea and seizures) typically associated with the antagonism of GABA(A) receptors in the brain. In this study, we have examined the modulation of the common brain GABA(A) receptor subtype by fipronil and its major metabolite, fipronil sulphone. Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat alpha1beta2gamma2L GABA(A) receptors. The major effect of fipronil was to increase the rate of current decay in macroscopic recordings. In single-channel recordings, the presence of fipronil resulted in shorter cluster durations without affecting the intracluster open and closed time distributions or the single-channel conductance. The alpha1V256S mutation, previously shown alleviate channel inhibition by inhibitory steroids and several insecticides, had a relatively small effect on channel block by fipronil. The mode of action of fipronil sulphone was similar to that of its parent compound but the metabolite was less potent at inhibiting the alpha1beta2gamma2L receptor. We conclude that exposure to fipronil induces accumulation of receptors in a novel, long-lived blocked state. This process proceeds in parallel with and independently of, channel desensitization. The lower potency of fipronil sulphone indicates that the conversion serves as a detoxifying process in mammalian brain.

The effects of agonists of ionotropic GABA(A) and metabotropic GABA(B) receptors on learning.

PubMed

Zyablitseva, Evgeniya A; Kositsyn, Nikolay S; Shul'gina, Galina I

2009-05-01

The research described here investigates the role played by inhibitory processes in the discriminations made by the nervous system of humans and animals between familiar and unfamiliar and significant and nonsignificant events. This research compared the effects of two inhibitory mediators of gamma-aminobutyric acid (GABA): 1) phenibut, a nonselective agonist of ionotropic GABA(A) and metabotropic GABA(B) receptors and 2) gaboxadol a selective agonist of ionotropic GABA(A) receptors on the process of developing active defensive and inhibitory conditioned reflexes in alert non-immobilized rabbits. It was found that phenibut, but not gaboxadol, accelerates the development of defensive reflexes at an early stage of conditioning. Both phenibut and gaboxadol facilitate the development of conditioned inhibition, but the effect of gaboxadol occurs at later stages of conditioning and is less stable than that of phenibut. The earlier and more stable effects of phenibut, as compared to gaboxadol, on storage in memory of the inhibitory significance of a stimulus may occur because GABA(B) receptors play the dominant role in the development of internal inhibition during an early stage of conditioning. On the other hand this may occur because the participation of both GABA(A) and GABA(B) receptors are essential to the process. We discuss the polyfunctionality of GABA receptors as a function of their structure and the positions of the relevant neurons in the brain as this factor can affect regulation of various types of psychological processes.

Neural Modulation of the Immune Response through the Benzodiazepine/GABA Receptor Chloride Ionophore Complex

DTIC Science & Technology

1988-08-30

dose of Alprazolam (a triaobenzodiazepine with high affinty for "central" but not "peripheral" benzodiazepine receptbr). These results suggest that...1987), provide additional support for the hypothesis that the "supramolecular complex" (in the CNS) regulates NK cell activity. 3). Effect of Alprazolam ...this study the effects of alprazolam (a triazolobenzodiazepine with high affinity for "central" but not "peripheral" benzodiazepine receptors) on

GABA, its receptors, and GABAergic inhibition in mouse taste buds.

PubMed

Dvoryanchikov, Gennady; Huang, Yijen A; Barro-Soria, Rene; Chaudhari, Nirupa; Roper, Stephen D

2011-04-13

Taste buds consist of at least three principal cell types that have different functions in processing gustatory signals: glial-like (type I) cells, receptor (type II) cells, and presynaptic (type III) cells. Using a combination of Ca2+ imaging, single-cell reverse transcriptase-PCR and immunostaining, we show that GABA is an inhibitory transmitter in mouse taste buds, acting on GABA(A) and GABA(B) receptors to suppress transmitter (ATP) secretion from receptor cells during taste stimulation. Specifically, receptor cells express GABA(A) receptor subunits β2, δ, and π, as well as GABA(B) receptors. In contrast, presynaptic cells express the GABA(A) β3 subunit and only occasionally GABA(B) receptors. In keeping with the distinct expression pattern of GABA receptors in presynaptic cells, we detected no GABAergic suppression of transmitter release from presynaptic cells. We suggest that GABA may serve function(s) in taste buds in addition to synaptic inhibition. Finally, we also defined the source of GABA in taste buds: GABA is synthesized by GAD65 in type I taste cells as well as by GAD67 in presynaptic (type III) taste cells and is stored in both those two cell types. We conclude that GABA is an inhibitory transmitter released during taste stimulation and possibly also during growth and differentiation of taste buds.

Severe Carisoprodol Withdrawal After a 14-Year Addiction and Acute Overdose.

PubMed

Vo, Kathy T; Horng, Howard; Smollin, Craig G; Benowitz, Neal L

2017-05-01

Carisoprodol, a centrally acting muscle relaxant with a high abuse potential, has barbiturate-like properties at the GABA-A receptor, leading to central nervous system depression and desired effects. Its tolerance and dependence has been previously demonstrated in an animal model, and withdrawal has been described in several recent case reports. Many cases can be effectively managed with a short course of benzodiazepines or antipsychotic agents. However, abrupt cessation in a patient with a history of long-term and high-dose carisoprodol abuse may result in symptoms that are more difficult for providers to treat. We present a case of a 34-year-old man with a long history of carisoprodol abuse who was found unresponsive after having ingested 7.5 grams of carisoprodol. He was intubated and admitted to the intensive care unit. He was given propofol, dexmedetomidine, fentanyl, ketamine, lorazepam, midazolam, quetiapine, and haloperidol, some at high-dose infusions, before his agitation and ventilator asynchrony could be controlled. His improvement coincided with the addition of carisoprodol and phenobarbital to his treatment regimen. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Trends show increasing emergency department presentations for drug-related disorders and treatment. This case highlights an uncommon case of carisoprodol withdrawal that may be encountered by emergency physicians, and demonstrates that benzodiazepines may not be sufficient to suppress severe withdrawal symptoms. Treatment with carisoprodol and phenobarbital provided additional benefit and can be considered in cases of severe carisoprodol withdrawal. Copyright © 2016 Elsevier Inc. All rights reserved.

Gene expression in the rat cerebral cortex: comparison of recovery sleep and hypnotic-induced sleep.

PubMed

Wisor, J P; Morairty, S R; Huynh, N T; Steininger, T L; Kilduff, T S

2006-08-11

Most hypnotic medications currently on the market target some aspect of GABAergic neurotransmission. Although all such compounds increase sleep, these drugs differentially affect the activity of the cerebral cortex as measured by the electroencephalogram. Whereas benzodiazepine medications such as triazolam tend to suppress slow wave activity in the cortex, the GABA(B) ligand gamma-hydroxybutyrate greatly enhances slow wave activity and the non-benzodiazepine, zolpidem, which binds to the omega1 site on the GABA(A) receptor/Cl(-) ionophore complex, is intermediate in this regard. Our previous studies have demonstrated that a small number of genes exhibit increased expression in the cerebral cortex of the mouse and rat during recovery sleep after sleep deprivation: egr-3, fra-2, grp78, grp94, ngfi-b, and nr4a3. Using these genes as a panel of biomarkers associated with sleep, we asked whether hypnotic medications induce similar molecular changes in the rat cerebral cortex to those observed when both sleep continuity and slow wave activity are enhanced during recovery sleep. We find that, although each drug increases the expression of a subset of genes in the panel of biomarkers, no drug fully replicates the molecular changes in the cortex associated with recovery sleep. Furthermore, high levels of slow wave activity in the cortex are correlated with increased expression of fra-2 whereas the expression of grp94 is correlated with body temperature. These results demonstrate that sleep-related changes in gene expression may be affected by physiological covariates of sleep and wakefulness rather than by vigilance state per se.

Extracellular pH modulates GABAergic neurotransmission in rat hypothalamus.

PubMed

Chen, Z L; Huang, R Q

2014-06-20

Changes in extracellular pH have a modulatory effect on GABAA receptor function. It has been reported that pH sensitivity of the GABA receptor is dependent on subunit composition and GABA concentration. Most of previous investigations focused on GABA-evoked currents, which only reflect the postsynaptic receptors. The physiological relevance of pH modulation of GABAergic neurotransmission is not fully elucidated. In the present studies, we examined the influence of extracellular pH on the GABAA receptor-mediated inhibitory neurotransmission in rat hypothalamic neurons. The inhibitory postsynaptic currents (IPSCs), tonic currents, and the GABA-evoked currents were recorded with whole-cell patch techniques on the hypothalamic slices from Sprague-Dawley rats at 15-26 postnatal days. The amplitude and frequency of spontaneous GABA IPSCs were significantly increased while the external pH was changed from 7.3 to 8.4. In the acidic pH (6.4), the spontaneous GABA IPSCs were reduced in amplitude and frequency. The pH induced changes in miniature GABA IPSCs (mIPSCs) similar to that in spontaneous IPSCs. The pH effect on the postsynaptic GABA receptors was assessed with exogenously applied varying concentrations of GABA. The tonic currents and the currents evoked by sub-saturating concentration of GABA ([GABA]) (10 μM) were inhibited by acidic pH and potentiated by alkaline pH. In contrast, the currents evoked by saturating [GABA] (1mM) were not affected by pH changes. We also investigated the influence of pH buffers and buffering capacity on pH sensitivity of GABAA receptors on human recombinant α1β2γ2 GABAA receptors stably expressed in HEK 293 cells. The pH influence on GABAA receptors was similar in HEPES- and MES-buffered media, and not dependent on protonated buffers, suggesting that the observed pH effect on GABA response is a specific consequence of changes in extracellular protons. Our data suggest that the hydrogen ions suppress the GABAergic neurotransmission, which is mediated by both presynaptic and postsynaptic mechanisms. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

mGluR2/3 agonist LY379268 rescues NMDA and GABAA receptor level deficits induced in a two-hit mouse model of schizophrenia.

PubMed

Engel, Martin; Snikeris, Peta; Matosin, Natalie; Newell, Kelly Anne; Huang, Xu-Feng; Frank, Elisabeth

2016-04-01

An imbalance of excitatory and inhibitory neurotransmission underlies the glutamate hypothesis of schizophrenia. Agonists of group II metabotropic glutamate receptors, mGluR2/3, have been proposed as novel therapeutic agents to correct this imbalance. However, the influence of mGluR2/3 activity on excitatory and inhibitory neurotransmitter receptors has not been explored. We aimed to investigate the ability of a novel mGluR2/3 agonist, LY379268, to modulate the availability of the excitatory N-methyl-D-aspartate receptor (NMDA-R) and the inhibitory gamma-aminobutyrate-A receptor (GABAA-R), in a two-hit mouse model of schizophrenia. Wild type (WT) and heterozygous neuregulin 1 transmembrane domain mutant mice (NRG1 HET) were treated daily with phencyclidine (10 mg/kg ip) or saline for 14 days. After a 14-day washout, an acute dose of the mGluR2/3 agonist LY379268 (3 mg/kg), olanzapine (antipsychotic drug comparison, 1.5 mg/kg), or saline was administered. NMDA-R and GABAA-R binding densities were examined by receptor autoradiography in several schizophrenia-relevant brain regions. In both WT and NRG1 HET mice, phencyclidine treatment significantly reduced NMDA-R and GABAA-R binding density in the prefrontal cortex, hippocampus, and nucleus accumbens. Acute treatment with LY379268 restored NMDA-R and GABAA-R levels in the two-hit mouse model comparable to olanzapine. We demonstrate that the mGluR2/3 agonist LY379268 restores excitatory and inhibitory deficits with similar efficiency as olanzapine in our two-hit schizophrenia mouse model. This study significantly contributes to our understanding of the mechanisms underlying the therapeutic effects of LY379268 and supports the use of agents aimed at mGluR2/3.

The tuberal lateral hypothalamus is a major target for GABAA--but not GABAB-mediated control of food intake.

PubMed

Turenius, Christine I; Charles, Jonathan R; Tsai, Donna H; Ebersole, Priscilla L; Htut, Myat H; Ngo, Phuong T; Lara, Raul N; Stanley, B Glenn

2009-08-04

The lateral hypothalamus (LH) is a site of integration for control mechanisms of feeding behavior as it has extensive reciprocal connections with multiple intrahypothalamic and extrahypothalamic brain areas. Evidence suggests that blockade of ionotropric gamma-aminobutyric acid (GABA) receptors in the LH elicits eating in satiated rats. To determine whether this GABA(A) receptor antagonist effect is specific to the LH, the antagonist picrotoxin was injected into one of six nearby sites and food intake was measured. Picrotoxin at 133 pmol elicited eating in the LH, but not in surrounding sites (thalamus, lateral preoptic area, ventral tegmental area, dorsomedial hypothalamus, and entopeduncular nucleus). More specifically, picrotoxin injected into the tuberal LH (tLH) elicited eating, but was ineffective when injected into the anterior or posterior LH. We also investigated whether GABA(B) receptors in the LH participated in the control of food intake and found that neither blockade nor activation of these receptors under multiple conditions changed food intake. Collectively, our findings suggest that GABA(A) but not GABA(B) receptors in the tLH act to suppress feeding behavior.

Effect of peripheral benzodiazepine receptor ligands on lipopolysaccharide-induced tumor necrosis factor activity in thioglycolate-treated mice.

PubMed Central

Matsumoto, T; Ogata, M; Koga, K; Shigematsu, A

1994-01-01

To investigate the effect of peripheral and central benzodiazepine receptor ligands on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) activity in mouse macrophages, three types of ligands, 4'-chlorodiazepam (pure peripheral), midazolam (mixed), and clonazepam (pure central), were compared. Midazolam and 4'-chlorodiazepam significantly suppressed LPS (1-microgram/ml)-induced TNF activity in thioglycolate-elicited mouse macrophages. In every concentration examined (0.001 to 100 microM), 4'-chlorodiazepam was the most effective agent, clonazepam was the least effective agent, and midazolam had an effect intermediate between those of the other two ligands. The peripheral benzodiazepine receptor ligands had a dose-dependent suppressive effect, and the 50% inhibitory concentrations were 0.01 microM for 4'-chlorodiazepam and 5 microM for midazolam. Concomitant use of PK 11195 (10 microM), an antagonist of the peripheral benzodiazepine receptor, reversed this suppressive effect with 4'-chlorodiazepam (10 microM) or midazolam (10 microM). PK 11195 showed this antagonistic effect in a dose-dependent manner. Intravenous 4'-chlorodiazepam (5 mg/kg of body weight) significantly suppressed LPS (100-micrograms)-induced TNF activity of sera (2 h postchallenge with LPS) from thioglycolate-treated mice. The present findings suggest that the peripheral benzodiazepine receptor plays an important role in modulating LPS-induced TNF activity in mouse macrophages. PMID:8031051

Design, Synthesis and Anticonvulsant Activity of 2-(2-Phenoxy) phenyl- 1,3,4-oxadiazole Derivatives.

PubMed

Tabatabai, Sayyed Abbas; Barghi Lashkari, Saoka; Zarrindast, Mohammad Reza; Gholibeikian, Mohammadreza; Shafiee, Abbas

2013-01-01

Benzodiazepines are useful drugs for treatment of sleep disorders, anxiety, seizure cases and skeletal muscle cramps. Some derivatives of 2-(2-Phenoxy) phenyl-1, 3, 4-oxadiazole were synthesized as benzodiazepine receptor agonists. Conformational analysis and superimposition of energy minima conformers of the compounds on estazolam, a known benzodiazepine agonist, reveal that the main proposed benzodiazepine pharmacophores were well matched. Anticonvulsant activity of the synthesized compounds, determined by pentylenetetrazole-induced lethal convulsion test, showed that the introduction of an amino substituent in position 5 of 1,3,4- oxadiazole ring generates compound 9 which has a respectable effect. The results are in agreement with SAR of benzodiazepine receptor ligands since the elimination of electronegative substituent in position 2 of phenoxy ring or position 4 of phenyl ring reduces the anticonvulsant activity.

Design, Synthesis and Anticonvulsant Activity of 2-(2-Phenoxy) phenyl- 1,3,4-oxadiazole Derivatives

PubMed Central

Tabatabai, Sayyed Abbas; Barghi Lashkari, Saoka; Zarrindast, Mohammad Reza; Gholibeikian, Mohammadreza; Shafiee, Abbas

2013-01-01

Benzodiazepines are useful drugs for treatment of sleep disorders, anxiety, seizure cases and skeletal muscle cramps. Some derivatives of 2-(2-Phenoxy) phenyl-1, 3, 4-oxadiazole were synthesized as benzodiazepine receptor agonists. Conformational analysis and superimposition of energy minima conformers of the compounds on estazolam, a known benzodiazepine agonist, reveal that the main proposed benzodiazepine pharmacophores were well matched. Anticonvulsant activity of the synthesized compounds, determined by pentylenetetrazole-induced lethal convulsion test, showed that the introduction of an amino substituent in position 5 of 1,3,4- oxadiazole ring generates compound 9 which has a respectable effect. The results are in agreement with SAR of benzodiazepine receptor ligands since the elimination of electronegative substituent in position 2 of phenoxy ring or position 4 of phenyl ring reduces the anticonvulsant activity. PMID:24250678

GABA-A receptors in mPOAH simultaneously regulate sleep and body temperature in freely moving rats.

PubMed

Jha, S K; Yadav, V; Mallick, B N

2001-09-01

Sleep-wakefulness and body temperature are two circadian rhythmic biological phenomena. The role of GABAergic inputs in the medial preoptico-anterior hypothalamus (mPOAH) on simultaneous regulation of those phenomena was investigated in freely moving normally behaving rats. The GABA-A receptors were blocked by microinjecting picrotoxin, and the effects on electrophysiological parameters signifying sleep-wakefulness, rectal temperature and brain temperature were recorded simultaneously. The results suggest that, normally, GABA in the medial preoptic area acts through GABA-A receptor that induces sleep and prevents an excessive rise in body temperature. However, the results do not allow us to comment on the cause and effect relationship, if any, between changes in sleep-wakefulness and body temperature. The changes in brain and rectal temperatures showed a positive correlation, however, the former varied within a narrower range than that of the latter.

GABA-A Receptors Mediate Tonic Inhibition and Neurosteroid Sensitivity in the Brain.

PubMed

Reddy, Doodipala Samba

2018-01-01

Neurosteroids like allopregnanolone (AP) are positive allosteric modulators of synaptic and extrasynaptic GABA-A receptors. AP and related neurosteroids exhibit a greater potency for δ-containing extrasynaptic receptors. The δGABA-A receptors, which are expressed extrasynaptically in the dentate gyrus and other regions, contribute to tonic inhibition, promoting network shunting as well as reducing seizure susceptibility. Levels of endogenous neurosteroids fluctuate with ovarian cycle. Natural and synthetic neurosteroids maximally potentiate tonic inhibition in the hippocampus and provide robust protection against a variety of limbic seizures and status epilepticus. Recently, a consensus neurosteroid pharmacophore model has been proposed at extrasynaptic δGABA-A receptors based on structure-activity relationship for functional activation of tonic currents and seizure protection. Aside from anticonvulsant actions, neurosteroids have been found to be powerful anxiolytic and anesthetic agents. Neurosteroids and Zn 2+ have preferential affinity for δ-containing receptors. Thus, Zn 2+ can prevent neurosteroid activation of extrasynaptic δGABA-A receptor-mediated tonic inhibition. Recently, we demonstrated that Zn 2+ selectively inhibits extrasynaptic δGABA-A receptors and thereby fully prevents AP activation of tonic inhibition and seizure protection. We confirmed that neurosteroids exhibit greater sensitivity at extrasynaptic δGABA-A receptors. Overall, extrasynaptic GABA-A receptors are primary mediators of tonic inhibition in the brain and play a key role in the pathophysiology of epilepsy and other neurological disorders. © 2018 Elsevier Inc. All rights reserved.

Mechanisms and environmental factors that underlying the intensification of 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy)-induced serotonin syndrome in rats

PubMed Central

Tao, Rui; Shokry, Ibrahim M.; Callanan, John J.; Adams, H. Daniel; Ma, Zhiyuan

2014-01-01

Rationale Illicit use of MDMA (3,4-methylenedioxymethamphetamine; Ecstasy) may cause a mild or severe form of the serotonin syndrome. The syndrome intensity is not just influenced by drug doses but also by environmental factors. Objectives Warm environmental temperatures and physical activity are features of raves. The purpose of this study was to assess how these two factors can potentially intensify the syndrome. Methods Rats were administered MDMA at doses of 0.3, 1 or 3 mg/kg, and examined in the absence or presence of warm temperature and physical activity. The syndrome intensity was estimated by visual scoring for behavioral syndrome and also instrumentally measuring changes in symptoms of the syndrome. Results Our results showed that MDMA at 3 mg/kg, but not 0.3 or 1 mg/kg, caused a mild serotonin syndrome in rats. Each environmental factor alone moderately intensified the syndrome. When the two factors were combined, the intensification became more severe than each factor alone highlighting a synergistic effect. This intensification was blocked by the 5-HT2A receptor antagonist M100907, competitive NMDA receptor antagonist CGS19755, autonomic ganglionic blocker hexamethonium, and the benzodiazepine-GABAA receptor agonist midazolam, but not by the 5-HT1A receptor antagonist WAY100635 or nicotinic receptor antagonist methyllycaconitine. Conclusions Our data suggest that, in the absence of environmental factors, the MDMA-induced syndrome is mainly mediated through the serotonergic transmission (5HT-dependent mechanism), and therefore, is relatively mild. Warm temperature and physical activity facilitate serotonergic and other neural systems such as glutamatergic and autonomic transmissions, resulting in intensification of the syndrome (non-5HT mechanisms). PMID:25300903

Negative modulation of α5 GABAA receptors in rats may partially prevent memory impairment induced by MK-801, but not amphetamine- or MK-801-elicited hyperlocomotion

PubMed Central

Stamenić, Tamara Timić; Joksimović, Srdjan; Biawat, Poonam; Stanković, Tamara; Marković, Bojan; Cook, James M; Savić, Miroslav M

2016-01-01

Reportedly, negative modulation of α5 GABAA receptors may improve cognition in normal and pharmacologically-impaired animals, and such modulation has been proposed as an avenue for treatment of cognitive symptoms in schizophrenia. This study assessed the actions of PWZ-029, administered at doses (2, 5 and 10 mg/kg) at which it reached micromolar concentrations in brain tissue with estimated free concentrations adequate for selective modulation of α5 GABAA receptors, in three cognitive tasks in male Wistar rats acutely treated with the noncompetitive N-methyl-D-aspartate – receptor antagonist, MK-801 (0.1 mg/kg), as well in tests of locomotor activity potentiated by MK-801 (0.2 mg/kg) or amphetamine (0.5 mg/kg). In a hormetic-like manner, only 5 mg/kg PWZ-029 reversed MK-801-induced deficits in novel object recognition test (visual recognition memory), whereas in the Morris water maze, the 2 mg/kg dose of PWZ-029 exerted partial beneficial effects on spatial learning impairment. PWZ-029 did not affect recognition memory deficits in social novelty discrimination procedure. Motor hyperactivity induced with MK-801 or amphetamine was not preventable by PWZ-029. Our results show that certain MK-801-induced memory deficits can be ameliorated by negative modulation of α5 GABAA receptors, and point to the need for further elucidation of their translational relevance to cognitive deterioration in schizophrenia. PMID:26105958

Negative modulation of α₅ GABAA receptors in rats may partially prevent memory impairment induced by MK-801, but not amphetamine- or MK-801-elicited hyperlocomotion.

PubMed

Timić Stamenić, Tamara; Joksimović, Srdjan; Biawat, Poonam; Stanković, Tamara; Marković, Bojan; Cook, James M; Savić, Miroslav M

2015-09-01

Reportedly, negative modulation of α5 GABAA receptors may improve cognition in normal and pharmacologically-impaired animals, and such modulation has been proposed as an avenue for treatment of cognitive symptoms in schizophrenia. This study assessed the actions of PWZ-029, administered at doses (2, 5, and 10 mg/kg) at which it reached micromolar concentrations in brain tissue with estimated free concentrations adequate for selective modulation of α5 GABAA receptors, in three cognitive tasks in male Wistar rats acutely treated with the noncompetitive N-methyl-d-aspartate receptor antagonist, MK-801 (0.1 mg/kg), as well in tests of locomotor activity potentiated by MK-801 (0.2 mg/kg) or amphetamine (0.5 mg/kg). In a hormetic-like manner, only 5 mg/kg PWZ-029 reversed MK-801-induced deficits in novel object recognition test (visual recognition memory), whereas in the Morris water maze, the 2 mg/kg dose of PWZ-029 exerted partial beneficial effects on spatial learning impairment. PWZ-029 did not affect recognition memory deficits in social novelty discrimination procedure. Motor hyperactivity induced with MK-801 or amphetamine was not preventable by PWZ-029. Our results show that certain MK-801-induced memory deficits can be ameliorated by negative modulation of α5 GABAA receptors, and point to the need for further elucidation of their translational relevance to cognitive deterioration in schizophrenia. © The Author(s) 2015.

Noni as an anxiolytic and sedative: a mechanism involving its gamma-aminobutyric acidergic effects.

PubMed

Deng, S; West, B J; Palu, A K; Zhou, B-N; Jensen, C J

2007-08-01

Noni (Morinda citrifolia) is increasing in worldwide popularity as a food or dietary supplement with versatile health benefits. The aim of this study was to investigate the effects of Noni fruit on anxiety symptoms in vitro. To this end, a competitive GABAa receptor-binding assay was developed. Our preliminary study indicates that the methanol crude extract of Noni fruit showed significant affinity to the gamma-aminobutyric acid A (GABAa) inhibitory neurotransmitter receptors, and displayed 75% binding inhibition of the agonist radioligand [3H] muscimol at a concentration of 100 microg/ml. Further experiments demonstrated that the MeOH extract, and its BuOH and H2O partitions, exhibited IC50 values of 22.8, 27.2, and 17.1 microg/ml, respectively, in the GABAa-binding assay. Experimental results with Noni fruit indicate the presence of competitive ligand(s), which may bind to the GABAa receptor as an agonist, and thus induce its anxiolytic and sedative effects. The study provides an in vitro rationale for one of Noni's versatile and traditional uses. In addition, an HPLC fingerprint profile of the methanolic extract of Noni fruit has been established for quality control purpose.

A neuronal mechanism of propofol-induced central respiratory depression in newborn rats.

PubMed

Kashiwagi, Masanori; Okada, Yasumasa; Kuwana, Shun-Ichi; Sakuraba, Shigeki; Ochiai, Ryoichi; Takeda, Junzo

2004-07-01

The neural mechanisms of propofol-induced central respiratory depression remain poorly understood. In the present study, we studied these mechanisms and the involvement of gamma-aminobutyric acid (GABA)A receptors in propofol-induced central respiratory depression. The brainstem and the cervical spinal cord of 1- to 4-day-old rats were isolated, and preparations were maintained in vitro with oxygenated artificial cerebrospinal fluid. Rhythmic inspiratory burst activity was recorded from the C4 spinal ventral root. The activity of respiratory neurons in the ventrolateral medulla was recorded using a perforated patch-clamp technique. We found that bath-applied propofol decreased C4 inspiratory burst rate, which could be reversed by the administration of a GABAA antagonist, bicuculline. Propofol caused resting membrane potentials to hyperpolarize and suppressed the firing of action potentials in preinspiratory and expiratory neurons. In contrast, propofol had little effect on resting membrane potentials and action potential firing in inspiratory neurons. Our findings suggest that the depressive effects of propofol are, at least in part, mediated by the agonistic action of propofol on GABAA receptors. It is likely that the GABAA receptor-mediated hyperpolarization of preinspiratory neurons serves as the neuronal basis of propofol-induced respiratory depression in the newborn rat.

Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors

PubMed Central

Hoffmann, Katrin M.; Herbrechter, Robin; Ziemba, Paul M.; Lepke, Peter; Beltrán, Leopoldo; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

2016-01-01

Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and their constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, a broad screening of Kampo remedies was performed to look for pharmacologically relevant 5-HT3A and GABAA receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness, or insomnia. Therefore, the pharmacological effects of 121 herbal drugs from Kampo medicine were analyzed as ethanol tinctures on heterologously expressed 5-HT3A and GABAA receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix) and Leonurus japonicus (herba) were the most effective inhibitory compounds on the 5-HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5-HT3A receptor antagonist. Several potentiating herbs (e.g., Magnolia officinalis (cortex), Syzygium aromaticum (flos), and Panax ginseng (radix)) were also identified for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects Salvia miltiorrhiza (radix) were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing for a better understanding of their physiological effects and clinical applications. PMID:27524967

Estrous Cycle Regulation of Extrasynaptic δ-Containing GABAA Receptor-Mediated Tonic Inhibition and Limbic Epileptogenesis

PubMed Central

Wu, Xin; Gangisetty, Omkaram; Carver, Chase Matthew

2013-01-01

The ovarian cycle affects susceptibility to behavioral and neurologic conditions. The molecular mechanisms underlying these changes are poorly understood. Deficits in cyclical fluctuations in steroid hormones and receptor plasticity play a central role in physiologic and pathophysiologic menstrual conditions. It has been suggested that synaptic GABAA receptors mediate phasic inhibition in the hippocampus and extrasynaptic receptors mediate tonic inhibition in the dentate gyrus. Here we report a novel role of extrasynaptic δ-containing GABAA receptors as crucial mediators of the estrous cycle–related changes in neuronal excitability in mice, with hippocampus subfield specificity. In molecular and immunofluorescence studies, a significant increase occurred in δ-subunit, but not α4- and γ2-subunits, in the dentate gyrus during diestrus. However, δ-subunit upregulation was not evident in the CA1 region. The δ-subunit expression was undiminished by age and ovariectomy and in mice lacking progesterone receptors, but it was significantly reduced by finasteride, a neurosteroid synthesis inhibitor. Electrophysiologic studies confirmed greater potentiation of GABA currents by progesterone-derived neurosteroid allopregnanolone in dissociated dentate gyrus granule cells in diestrus than in CA1 pyramidal cells. The baseline conductance and allopregnanolone potentiation of tonic currents in dentate granule cells from hippocampal slices were higher than in CA1 pyramidal cells. In behavioral studies, susceptibility to hippocampus kindling epileptogenesis was lower in mice during diestrus. These results demonstrate the estrous cycle–related plasticity of neurosteroid-sensitive, δ-containing GABAA receptors that mediate tonic inhibition and seizure susceptibility. These findings may provide novel insight on molecular cascades of menstrual disorders like catamenial epilepsy, premenstrual syndrome, and migraine. PMID:23667248

Disinhibition of perifornical hypothalamic neurones activates noradrenergic neurones and blocks pontine carbachol-induced REM sleep-like episodes in rats

PubMed Central

Lu, Jackie W; Fenik, Victor B; Branconi, Jennifer L; Mann, Graziella L; Rukhadze, Irma; Kubin, Leszek

2007-01-01

Studies in behaving animals suggest that neurones located in the perifornical (PF) region of the posterior hypothalamus promote wakefulness and suppress sleep. Among such cells are those that synthesize the excitatory peptides, orexins (ORX). Lack of ORX, or their receptors, is associated with narcolepsy/cataplexy, a disorder characterized by an increased pressure for rapid eye movement (REM) sleep. We used anaesthetized rats in which pontine microinjections of a cholinergic agonist, carbachol, can repeatedly elicit REM sleep-like episodes to test whether activation of PF cells induced by antagonism of endogenous, GABAA receptor-mediated, inhibition suppresses the ability of the brainstem to generate REM sleep-like state. Microinjections of the GABAA receptor antagonist, bicuculline (20 nl, 1 mm), into the PF region elicited cortical and hippocampal activation, increased the respiratory rate and hypoglossal nerve activity, induced c-fos expression in ORX and other PF neurones, and increased c-fos expression in pontine A7 and other noradrenergic neurones. The ability of pontine carbachol to elicit any cortical, hippocampal or brainstem component of the REM sleep-like response was abolished during the period of bicuculline-induced activation. The activating and REM sleep-suppressing effect of PF bicuculline was not attenuated by systemic administration of the ORX type 1 receptor antagonist, SB334867. Thus, activation of PF neurones that are endogenously inhibited by GABAA receptors is sufficient to turn off the brainstem REM sleep-generating network; the effect is, at least in part, due to activation of pontine noradrenergic neurones, but is not mediated by ORX type 1 receptors. A malfunction of the pathway that originates in GABAA receptor-expressing PF neurones may cause narcolepsy/cataplexy. PMID:17495048

GABA(B) receptors, schizophrenia and sleep dysfunction: a review of the relationship and its potential clinical and therapeutic implications.

PubMed

Kantrowitz, Joshua; Citrome, Leslie; Javitt, Daniel

2009-08-01

Evidence for an intrinsic relationship between sleep, cognition and the symptomatic manifestations of schizophrenia is accumulating. This review presents evidence for the possible utility of GABA(B) receptor agonists for the treatment of subjective and objective sleep abnormalities related to schizophrenia. At the phenotypic level, sleep disturbance occurs in 16-30% of patients with schizophrenia and is related to reduced quality of life and poor coping skills. On the neurophysiological level, studies suggest that sleep deficits reflect a core component of schizophrenia. Specifically, slow-wave sleep deficits, which are inversely correlated with cognition scores, are seen. Moreover, sleep plays an increasingly well documented role in memory consolidation in schizophrenia. Correlations of slow-wave sleep deficits with impaired reaction time and declarative memory have also been reported. Thus, both behavioural insomnia and sleep architecture are critical therapeutic targets in patients with schizophrenia. However, long-term treatment with antipsychotics often results in residual sleep dysfunction and does not improve slow-wave sleep, and adjunctive GABA(A) receptor modulators, such as benzodiazepines and zolpidem, can impair sleep architecture and cognition in schizophrenia. GABA(B) receptor agonists have therapeutic potential in schizophrenia. These agents have minimal effect on rapid eye movement sleep while increasing slow-wave sleep. Preclinical associations with increased expression of genes related to slow-wave sleep production and circadian rhythm function have also been reported. GABA(B) receptor deficits result in a sustained hyperdopaminergic state and can be reversed by a GABA(B) receptor agonist. Genetic, postmortem and electrophysiological studies also associate GABA(B) receptors with schizophrenia. While studies thus far have not shown significant effects, prior focus on the use of GABA(B) receptor agonists has been on the positive symptoms of schizophrenia, with minimal investigation of GABA(B) receptor agonists such as baclofen or gamma-hydroxybutyric acid and their effects on sleep architecture, cognition and negative symptoms in patients with schizophrenia. Further study is needed.

What can be learned from the effects of benzodiazepines on exploratory behavior?

PubMed

File, S E

1985-01-01

The purpose of this review is to assess the value of using tests of exploratory behavior to study the actions of benzodiazepines. The methods of measuring exploration and the factors influencing it are briefly described. The effects of benzodiazepines on exploratory behavior of rats and mice are reviewed; and the dangers of interpreting the results of such tests in terms of any of the clinical effects of the benzodiazepines is stressed. Finally, the interactions between benzodiazepines and other drugs acting at the GABA-benzodiazepine receptor complex are described. The results of these experiments caution against global classification of compounds as benzodiazepine "antagonists."

CO2-induced ocean acidification increases anxiety in Rockfish via alteration of GABAA receptor functioning

PubMed Central

Hamilton, Trevor James; Holcombe, Adam; Tresguerres, Martin

2014-01-01

The average surface pH of the ocean is dropping at a rapid rate due to the dissolution of anthropogenic CO2, raising concerns for marine life. Additionally, some coastal areas periodically experience upwelling of CO2-enriched water with reduced pH. Previous research has demonstrated ocean acidification (OA)-induced changes in behavioural and sensory systems including olfaction, which is due to altered function of neural gamma-aminobutyric acid type A (GABAA) receptors. Here, we used a camera-based tracking software system to examine whether OA-dependent changes in GABAA receptors affect anxiety in juvenile Californian rockfish (Sebastes diploproa). Anxiety was estimated using behavioural tests that measure light/dark preference (scototaxis) and proximity to an object. After one week in OA conditions projected for the next century in the California shore (1125 ± 100 µatm, pH 7.75), anxiety was significantly increased relative to controls (483 ± 40 µatm CO2, pH 8.1). The GABAA-receptor agonist muscimol, but not the antagonist gabazine, caused a significant increase in anxiety consistent with altered Cl− flux in OA-exposed fish. OA-exposed fish remained more anxious even after 7 days back in control seawater; however, they resumed their normal behaviour by day 12. These results show that OA could severely alter rockfish behaviour; however, this effect is reversible. PMID:24285203

CO2-induced ocean acidification increases anxiety in rockfish via alteration of GABAA receptor functioning.

PubMed

Hamilton, Trevor James; Holcombe, Adam; Tresguerres, Martin

2014-01-22

The average surface pH of the ocean is dropping at a rapid rate due to the dissolution of anthropogenic CO2, raising concerns for marine life. Additionally, some coastal areas periodically experience upwelling of CO2-enriched water with reduced pH. Previous research has demonstrated ocean acidification (OA)-induced changes in behavioural and sensory systems including olfaction, which is due to altered function of neural gamma-aminobutyric acid type A (GABAA) receptors. Here, we used a camera-based tracking software system to examine whether OA-dependent changes in GABAA receptors affect anxiety in juvenile Californian rockfish (Sebastes diploproa). Anxiety was estimated using behavioural tests that measure light/dark preference (scototaxis) and proximity to an object. After one week in OA conditions projected for the next century in the California shore (1125 ± 100 µatm, pH 7.75), anxiety was significantly increased relative to controls (483 ± 40 µatm CO2, pH 8.1). The GABAA-receptor agonist muscimol, but not the antagonist gabazine, caused a significant increase in anxiety consistent with altered Cl(-) flux in OA-exposed fish. OA-exposed fish remained more anxious even after 7 days back in control seawater; however, they resumed their normal behaviour by day 12. These results show that OA could severely alter rockfish behaviour; however, this effect is reversible.

GABA(A) receptor antagonism in the ventrocaudal periaqueductal gray increases anxiety in the anxiety-resistant postpartum rat.

PubMed

Miller, Stephanie M; Piasecki, Christopher C; Peabody, Mitchell F; Lonstein, Joseph S

2010-06-01

Postpartum mammals show suppressed anxiety, which is necessary for their ability to appropriately care for offspring. It is parsimonious to suggest that the neurobiological basis of this reduced anxiety is similar to that of non-parturient animals, involving GABA(A) receptor activity in sites including the midbrain periaqueductal gray (PAG). In Experiment 1, postpartum and diestrous virgin female rats received an intraperitoneal injection of the GABA(A) receptor antagonist (+)-bicuculline (0, 2 and 4 mg/kg) and anxiety-related behavior was assessed with an elevated plus maze. The 4 mg/kg dose of (+)-bicuculline significantly increased anxiety-related behavior, particularly in the postpartum females. Experiment 2 revealed that bicuculline's action was within the central nervous system, because anxiety in neither dams nor virgins was significantly affected by intraperitoneal injection of bicuculline methiodide (0, 2 and 6 mg/kg), which does not readily cross the blood-brain-barrier. In Experiment 3, bicuculline methiodide (2.5 ng/side) was directly infused into the ventrocaudal PAG (cPAGv) and significantly increased dams' anxiety compared to saline-infused controls. These studies expand our knowledge of how GABA(A) receptor modulators affect anxiety behaviors in postpartum rats to the widely-used elevated plus maze, and indicate that the postpartum suppression of anxiety is in part a consequence of elevated GABAergic neurotransmission in the cPAGv. Copyright 2010 Elsevier Inc. All rights reserved.

RNAi screening of subtracted transcriptomes reveals tumor suppression by taurine-activated GABAA receptors involved in volume regulation

PubMed Central

van Nierop, Pim; Vormer, Tinke L.; Foijer, Floris; Verheij, Joanne; Lodder, Johannes C.; Andersen, Jesper B.; Mansvelder, Huibert D.; te Riele, Hein

2018-01-01

To identify coding and non-coding suppressor genes of anchorage-independent proliferation by efficient loss-of-function screening, we have developed a method for enzymatic production of low complexity shRNA libraries from subtracted transcriptomes. We produced and screened two LEGO (Low-complexity by Enrichment for Genes shut Off) shRNA libraries that were enriched for shRNA vectors targeting coding and non-coding polyadenylated transcripts that were reduced in transformed Mouse Embryonic Fibroblasts (MEFs). The LEGO shRNA libraries included ~25 shRNA vectors per transcript which limited off-target artifacts. Our method identified 79 coding and non-coding suppressor transcripts. We found that taurine-responsive GABAA receptor subunits, including GABRA5 and GABRB3, were induced during the arrest of non-transformed anchor-deprived MEFs and prevented anchorless proliferation. We show that taurine activates chloride currents through GABAA receptors on MEFs, causing seclusion of cell volume in large membrane protrusions. Volume seclusion from cells by taurine correlated with reduced proliferation and, conversely, suppression of this pathway allowed anchorage-independent proliferation. In human cholangiocarcinomas, we found that several proteins involved in taurine signaling via GABAA receptors were repressed. Low GABRA5 expression typified hyperproliferative tumors, and loss of taurine signaling correlated with reduced patient survival, suggesting this tumor suppressive mechanism operates in vivo. PMID:29787571

Synthesis and in vivo evaluation of [11C]zolpidem, an imidazopyridine with agonist properties at central benzodiazepine receptors.

PubMed

Dumont, Filip; Waterhouse, Rikki N; Montoya, Julie A; Mattner, Filomena; Katsifis, Andrew; Kegeles, Lawrence S; Laruelle, Marc

2003-05-01

The synthesis and evaluation of [(11)C]zolpidem, an imidazopyridine with agonist properties at central benzodiazepine receptors, is reported herein. The reaction of desmethylzolpidem with [(11)C] methyl iodide afforded the title compound [(11)C]zolpidem in a yield of 19.19 +/- 3.23% in 41 +/- 2 min in specific activities of 0.995-1.19 Ci/micromol (1.115 +/- 0.105 Ci/micromol) (n = 3; decay corrected, EOB). The amount of radioactivity in the brain after tail vein injection in male Wistar rats was low, and the regional distribution was homogeneous and not consistent with the known distribution of the central benzodiazepine receptors. The frontal cortex/cerebellum ratio was not significantly greater than one (1.007 +/- 0.266 at 5 min) and did not increase from 5 to 40 min post-injection. A PET brain imaging study in one baboon confirmed the results obtained in rats. Therefore, it can be concluded that [(11)C]zolpidem is not a suitable tracer for in vivo visualization of central benzodiazepine receptors.

Postsynaptic activity reverses the sign of the acetylcholine-induced long-term plasticity of GABAA inhibition

PubMed Central

Domínguez, Soledad; Fernández de Sevilla, David; Buño, Washington

2014-01-01

Acetylcholine (ACh) regulates forms of plasticity that control cognitive functions but the underlying mechanisms remain largely unknown. ACh controls the intrinsic excitability, as well as the synaptic excitation and inhibition of CA1 hippocampal pyramidal cells (PCs), cells known to participate in circuits involved in cognition and spatial navigation. However, how ACh regulates inhibition in function of postsynaptic activity has not been well studied. Here we show that in rat PCs, a brief pulse of ACh or a brief stimulation of cholinergic septal fibers combined with repeated depolarization induces strong long-term enhancement of GABAA inhibition (GABAA-LTP). Indeed, this enhanced inhibition is due to the increased activation of α5βγ2 subunit-containing GABAA receptors by the GABA released. GABAA-LTP requires the activation of M1-muscarinic receptors and an increase in cytosolic Ca2+. In the absence of PC depolarization ACh triggered a presynaptic depolarization-induced suppression of inhibition (DSI), revealing that postsynaptic activity gates the effects of ACh from presynaptic DSI to postsynaptic LTP. These results provide key insights into mechanisms potentially linked with cognitive functions, spatial navigation, and the homeostatic control of abnormal hyperexcitable states. PMID:24938789

2-Aminoethyl Methylphosphonate, a Potent and Rapidly Acting Antagonist of GABAA-ρ1 Receptors

PubMed Central

Xie, An; Yan, Jun; Yue, Lan; Feng, Feng; Mir, Fozia; Abdel-Halim, Heba; Chebib, Mary; Le Breton, Guy C.; Standaert, Robert F.; Qian, Haohua

2011-01-01

2-Aminoethyl methylphosphonate (2-AEMP), an analog of GABA, has been found to exhibit antagonist activity at GABAA-ρ1 (also known as ρ1 GABAC) receptors. The present study was undertaken to elucidate 2-AEMP's action and to test the activities of 2-AEMP analogs. Whole-cell patch-clamp techniques were used to record membrane currents in neuroblastoma cells stably transfected with human GABAA-ρ1 receptors. The action of 2-AEMP was compared with that of 1,2,5,6-tetrahydropyridin-4-yl methylphosphinic acid (TPMPA), a commonly used GABAA-ρ1 antagonist. With 10 μM GABA, 2-AEMP's IC50 (18 μM) differed by less than 2.5-fold from that of TPMPA (7 μM), and results obtained were consistent with a primarily competitive mode of inhibition by 2-AEMP. Terminating the presentation of 2-AEMP or TPMPA in the presence of GABA produced a release from inhibition. However, the rate of inhibition release upon the termination of 2-AEMP considerably exceeded that determined with termination of TPMPA. Moreover, when presented at concentrations near their respective IC50 values, the preincubation period associated with 2-AEMP's onset of inhibition was much shorter than that for TPMPA. Analogs of 2-AEMP possessing a benzyl or n-butyl rather than a methyl substituent at the phosphorus atom, as well as analogs bearing a C-methyl substituent on the aminoethyl side chain, exhibited reduced potency relative to 2-AEMP. Of these analogs, only (R)-2-aminopropyl methylphosphonate significantly diminished the response to 10 μM GABA. Structure-activity relationships are discussed in the context of molecular modeling of ligand binding to the antagonist binding site of the GABAA-ρ1 receptor. PMID:21810922

The adjustment of γ-aminobutyric acidA tonic subunits in Huntington's disease: from transcription to translation to synaptic levels into the neostriatum

PubMed Central

Rosas-Arellano, Abraham; Estrada-Mondragón, Argel; Mantellero, Carola A.; Tejeda-Guzmán, Carlos; Castro, Maite A.

2018-01-01

γ-Aminobutyric acid (GABA), plays a key role in all stages of life, also is considered the main inhibitory neurotransmitter. GABA activates two kind of membrane receptors known as GABAA and GABAB, the first one is responsible to render tonic inhibition by pentameric receptors containing α4−6, β3, δ, or ρ1−3 subunits, they are located at perisynaptic and/or in extrasynaptic regions. The biophysical properties of GABAA tonic inhibition have been related with cellular protection against excitotoxic injury and cell death in presence of excessive excitation. On this basis, GABAA tonic inhibition has been proposed as a potential target for therapeutic intervention of Huntington's disease. Huntington's disease is a neurodegenerative disorder caused by a genetic mutation of the huntingtin protein. For experimental studies of Huntington's disease mouse models have been developed, such as R6/1, R6/2, HdhQ92, HdhQ150, as well as YAC128. In all of them, some key experimental reports are focused on neostriatum. The neostriatum is considered as the most important connection between cerebral cortex and basal ganglia structures, its cytology display two pathways called direct and indirect constituted by medium sized spiny neurons expressing dopamine D1 and D2 receptors respectively, they display strong expression of many types of GABAA receptors, including tonic subunits. The studies about of GABAA tonic subunits and Huntington's disease into the neostriatum are rising in recent years, suggesting interesting changes in their expression and localization which can be used as a strategy to delay the cellular damage caused by the imbalance between excitation and inhibition, a hallmark of Huntington's disease. PMID:29722299

GABAA overactivation potentiates the effects of NMDA blockade during the brain growth spurt in eliciting locomotor hyperactivity in juvenile mice.

PubMed

Oliveira-Pinto, Juliana; Paes-Branco, Danielle; Cristina-Rodrigues, Fabiana; Krahe, Thomas E; Manhães, Alex C; Abreu-Villaça, Yael; Filgueiras, Cláudio C

2015-01-01

Both NMDA receptor blockade and GABAA receptor overactivation during the brain growth spurt may contribute to the hyperactivity phenotype reminiscent of attention-deficit/hyperactivity disorder. Here, we evaluated the effects of exposure to MK801 (a NMDA antagonist) and/or to muscimol (a GABAA agonist) during the brain growth spurt on locomotor activity of juvenile Swiss mice. This study was carried out in two separate experiments. In the first experiment, pups received a single i.p. injection of either saline solution (SAL), MK801 (MK, 0.1, 0.3 or 0.5 mg/kg) or muscimol (MU, 0.02, 0.1 or 0.5 mg/kg) at the second postnatal day (PND2), and PNDs 4, 6 and 8. In the second experiment, we investigated the effects of a combined injection of MK (0.1 mg/kg) and MU (doses: 0.02, 0.1 or 0.5 mg/kg) following the same injection schedule of the first experiment. In both experiments, locomotor activity was assessed for 15 min at PND25. While MK promoted a dose-dependent increase in locomotor activity, exposure to MU failed to elicit significant effects. The combined exposure to the highest dose of MU and the lowest dose of MK induced marked hyperactivity. Moreover, the combination of the low dose of MK and the high dose of MU resulted in a reduced activity in the center of the open field, suggesting an increased anxiety-like behavior. These findings suggest that, during the brain growth spurt, the blockade of NMDA receptors induces juvenile locomotor hyperactivity whereas hyperactivation of GABAA receptors does not. However, GABAA overactivation during this period potentiates the effects of NMDA blockade in inducing locomotor hyperactivity. Copyright © 2015 Elsevier Inc. All rights reserved.

Quantitative autoradiography of muscarinic and benzodiazepine receptors in the forebrain of the turtle, Pseudemys scripta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schlegel, J.R.; Kriegstein, A.R.

1987-11-22

The distribution of muscarinic and benzodiazepine receptors was investigated in the turtle forebrain by the technique of in vitro receptor autoradiography. Muscarinic binding sites were labeled with 1 nM /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and benzodiazepine sites were demonstrated with the aid of 1 nM /sup 3/H-flunitrazepam (/sup 3/H-FLU). Autoradiograms generated on /sup 3/H-Ultrofilm apposed to tissue slices revealed regionally specific distributions of muscarinic and benzodiazepine binding sites that are comparable with those for mammalian brain. Dense benzodiazepine binding was found in the anterior olfactory nucleus, the lateral and dorsal cortices, and the dorsal ventricular ridge (DVR), a structure withmore » no clear mammalian homologue. Muscarinic binding sites were most dense in the striatum, accumbens, DVR, lateral geniculate, and the anterior olfactory nucleus. Cortical binding sites were studied in greater detail by quantitative analysis of autoradiograms generated by using emulsion-coated coverslips. Laminar gradients of binding were observed that were specific for each radioligand; /sup 3/H-QNB sites were most dense in the inner molecular layer in all cortical regions, whereas /sup 3/H-FLU binding was generally most concentrated in the outer molecular layer and was least dense through all layers in the dorsomedial cortex. Because pyramidal cells are arranged in register in turtle cortex, the laminar patterns of receptor binding may reflect different receptor density gradients along pyramidal cell dendrites.« less

Differential expression of benzodiazepine anticonvulsant cross-tolerance according to time following flurazepam or diazepam treatment.

PubMed

Rosenberg, H C

1995-01-01

In previous studies in which the anti-pentylenetetrazol (PTZ) effect of benzodiazepines was used to measure tolerance, the results depended on the benzodiazepine used for chronic treatment as well as the benzodiazepine given acutely to test for tolerance. In this study, the time course of tolerance reversal was studied in rats given two treatments known to cause anticonvulsant tolerance, 1-week flurazepam (FZP), and 3-week diazepam (DZP). Neither treatment altered convulsive threshold for IV PTZ, but both treatments decreased the convulsive threshold for bicuculline. Withdrawing DZP, but not FZP, treatment resulted in a loss of body weight. Twelve hours after 1-week FZP treatment, all benzodiazepines were significantly less effective, showing tolerance. Forty-eight hours after the 1-week FZP treatment, tolerance was still observed with DZP, FZP, and zolpidem, but was no longer present with clonazepam or bretazenil. After the 3-week DZP treatment, rats were tolerant to all benzodiazepines tested at 12 h of withdrawal, but had lost tolerance to all the drugs except bretazenil by 48 h. The results suggest differences in the way these benzodiazepines interact with their receptors, allowing differential expression of tolerance, and that chronic DZP and FZP treatments affected interactions of the benzodiazepines with their receptors, but not in the same fashion.

Decrement of GABAA receptor-mediated inhibitory postsynaptic currents in dentate granule cells in epileptic hippocampus.

PubMed

Isokawa, M

1996-05-01

1. Inhibitory postsynaptic currents (IPSCs) were studied in hippocampal dentate granule cells (DGCs) in the pilocarpine model and human temporal lobe epilepsy, with the use of the whole cell patch-clamp recording technique in slice preparations. 2. In the pilocarpine model, hippocampal slices were prepared from rats that were allowed to experience spontaneous seizures for 2 mo. Human hippocampal specimens were obtained from epileptic patients who underwent surgical treatment for medically intractable seizures. 3. IPSCs were generated by single perforant path stimulation and recorded at a membrane potential (Vm) of 0 mV near the reversal potential of glutamate excitatory postsynaptic currents in the voltage-clamp recording. IPSCs were pharmacologically identified as gamma-aminobutyric acid-A (GABAA) IPSCs by 10 microM bicuculline methiodide. 4. During low-frequency stimulation, IPSCs were not different in amplitude among non-seizure-experienced rat hippocampi, human nonsclerotic hippocampi, seizure-experienced rat hippocampi, and human sclerotic hippocampi. In the last two groups of DGCs, current-clamp recordings indicated the presence of prolonged excitatory postsynaptic potentials (EPSPs) mediated by the N-methyl-D-aspartate (NMDA) receptor. 5. High-frequency stimulation, administered at Vm = -30 mV to activate NMDA currents, reduced GABAA IPSC amplitude specifically in seizure-experienced rat hippocampi (t = 2.5, P < 0.03) and human sclerotic hippocampi (t = 7.7, P < 0.01). This reduction was blocked by an NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV) (50 microM). The time for GABAA IPSCs to recover to their original amplitude was also shortened by the application of APV. 6. I conclude that, when intensively activated, NMDA receptor-mediated excitatory transmission may interact with GABAergic synaptic inhibition in DGCs in seizure-experienced hippocampus to transiently reduce GABA(A) receptor-channel function. Such interactions may contribute to give rise to epileptic excitation in chronically seizure-prone hippocampus.

STRAIN-SPECIFIC PROGRAMMING OF PRENATAL ETHANOL EXPOSURE ACROSS GENERATIONS

PubMed Central

Popoola, Daniel O.; Nizhnikov, Michael E.; Cameron, Nicole M.

2017-01-01

Behavioral consequences of prenatal alcohol exposure (PAE) can be transmitted from in utero-exposed F1 generation to their F2 offspring. This type of transmission is modulated by genetic and epigenetic mechanism. This study investigated the intergenerational consequences of prenatal exposure to low ethanol dose (1g/kg) during gestational days 17–20, on ethanol-induced hypnosis in adolescent male F1 and F2 generations, in two strains of rats. Adolescent Long Evans and Sprague Dawley male rats were tested for sensitivity to ethanol-induced hypnosis at 3.5g/kg or 4.5g/kg ethanol dose using the loss of righting reflex (LORR) paradigm. We hypothesized that PAE would attenuate sensitivity to ethanol-induced hypnosis in the ethanol-exposed animals in these two strains and in both generations. Interestingly, we only found this effect in Sprague Dawley rats. Lastly, we investigated PAE related changes in expression of GABAA receptor α1, α4, and δ subunits in the cerebral cortex of the PAE sensitive Sprague Dawley strain. We hypothesized a reduction in the cerebral cortex GABAA receptor subunits’ expression in the F1 and F2 PAE groups compared to control animals. GABAA receptor α1, α4, and δ subunits protein expressions were quantified in the cerebral cortex of F1 and F2 male adolescents by western blotting. PAE didn’t alter cerebral cortical GABAA receptor subunit expressions in the F1 generation, but it decreased GABAA receptor α4 and δ subunits’ expressions in the F2 generation, and had a tendency to decrease α1 subunit expression. We also found correlations between some of the subunits in both generations. These strain-dependent vulnerabilities to ethanol sensitivity, and intergenerational PAE-mediated changes in sensitivity to alcohol indicate that genetic and epigenetic factors interact to determine the outcomes of PAE animals and their offspring. PMID:28433421

Intra-accumbens baclofen, but not muscimol, increases second order instrumental responding for food reward in rats.

PubMed

Pulman, Kim G T; Somerville, Elizabeth M; Clifton, Peter G

2012-01-01

Stimulation of either GABA(A) or GABA(B) receptors within the nucleus accumbens shell strongly enhances food intake in rats. However the effects of subtype-selective stimulation of GABA receptors on instrumental responses for food reward are less well characterized. Here we contrast the effects of the GABA(A) receptor agonist muscimol and GABA(B) receptor agonist baclofen on instrumental responding for food using a second order reinforcement schedule. Bilateral intra-accumbens administration of baclofen (220-440 pmol) stimulated responding but a higher dose (660 pmol) induced stereotyped oral behaviour that interfered with responding. Baclofen (220-660 pmol) also stimulated intake of freely available chow. Muscimol (220-660 pmol) was without effect on responding for food on this schedule but did stimulate intake of freely available chow. Unilateral administration of either baclofen or muscimol (220 pmol) induced similar patterns of c-fos immunoreactivity in several hypothalamic sites but differed in its induction in the central nucleus of the amygdala. We conclude that stimulation of GABA(A) or GABA(B) receptors in the nucleus accumbens shell of rats produces clearly distinguishable effects on operant responding for food.

Interactions of L-3,5,3'-Triiodothyronine, Allopregnanolone, and Ivermectin with the GABAA Receptor: Evidence for Overlapping Intersubunit Binding Modes

PubMed Central

Westergard, Thomas; Salari, Reza; Martin, Joseph V.; Brannigan, Grace

2015-01-01

Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3’-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone. PMID:26421724

Interactions of L-3,5,3'-Triiodothyronine [corrected], Allopregnanolone, and Ivermectin with the GABAA Receptor: Evidence for Overlapping Intersubunit Binding Modes.

PubMed

Westergard, Thomas; Salari, Reza; Martin, Joseph V; Brannigan, Grace

2015-01-01

Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3'-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone.

Tyrosine Phosphorylation of GABAA Receptor γ2-Subunit Regulates Tonic and Phasic Inhibition in the Thalamus

PubMed Central

Nani, Francesca; Bright, Damian P.; Revilla-Sanchez, Raquel; Tretter, Verena; Moss, Stephen J.

2013-01-01

GABA-mediated tonic and phasic inhibition of thalamic relay neurons of the dorsal lateral geniculate nucleus (dLGN) was studied after ablating tyrosine (Y) phosphorylation of receptor γ2-subunits. As phosphorylation of γ2 Y365 and Y367 reduces receptor internalization, to understand their importance for inhibition we created a knock-in mouse in which these residues are replaced by phenylalanines. On comparing wild-type (WT) and γ2Y365/367F+/− (HT) animals (homozygotes are not viable in utero), the expression levels of GABAA receptor α4-subunits were increased in the thalamus of female, but not male mice. Raised δ-subunit expression levels were also observed in female γ2Y365/367F +/− thalamus. Electrophysiological analyses revealed no difference in the level of inhibition in male WT and HT dLGN, while both the spontaneous inhibitory postsynaptic activity and the tonic current were significantly augmented in female HT relay cells. The sensitivity of tonic currents to the δ-subunit superagonist THIP, and the blocker Zn2+, were higher in female HT relay cells. This is consistent with upregulation of extrasynaptic GABAA receptors containing α4- and δ-subunits to enhance tonic inhibition. In contrast, the sensitivity of GABAA receptors mediating inhibition in the female γ2Y356/367F +/− to neurosteroids was markedly reduced compared with WT. We conclude that disrupting tyrosine phosphorylation of the γ2-subunit activates a sex-specific increase in tonic inhibition, and this most likely reflects a genomic-based compensation mechanism for the reduced neurosteroid sensitivity of inhibition measured in female HT relay neurons. PMID:23904608

Missense Gamma-Aminobutyric Acid Receptor Polymorphisms Are Associated with Reaction Time, Motor Time, and Ethanol Effects in Vivo.

PubMed

García-Martín, Elena; Ramos, María I; Cornejo-García, José A; Galván, Segismundo; Perkins, James R; Rodríguez-Santos, Laura; Alonso-Navarro, Hortensia; Jiménez-Jiménez, Félix J; Agúndez, José A G

2018-01-01

Background: The Gamma-aminobutyric acid type A receptor (GABA-A receptor) is affected by ethanol concentrations equivalent to those reached during social drinking. At these concentrations, ethanol usually causes impairment in reaction and motor times in most, but not all, individuals. Objectives: To study the effect of GABA-A receptor variability in motor and reaction times, and the effect of low ethanol doses. Methods: Two hundred and fifty healthy subjects received one single dose of 0.5 g/Kg ethanol per os . Reaction and motor times were determined before ethanol challenge (basal), and when participants reached peak ethanol concentrations. We analyzed all common missense polymorphisms described in the 19 genes coding for the GABA-A receptor subunits by using TaqMan probes. Results: The GABRA6 rs4454083 T/C polymorphisms were related to motor times, with individuals carrying the C/C genotype having faster motor times, both, at basal and at peak ethanol concentrations. The GABRA4 rs2229940 T/T genotype was associated to faster reaction times and with lower ethanol effects, determined as the difference between basal reaction time and reaction time at peak concentrations. All these associations remained significant after correction for multiple comparisons. No significant associations were observed for the common missense SNPs GABRB3 rs12910925, GABRG2 rs211035, GABRE rs1139916, GABRP rs1063310, GABRQ rs3810651, GABRR1 rs12200969 or rs1186902, GABRR2 rs282129, and GABRR3 rs832032. Conclusions: This study provides novel information supporting a role of missense GABA-A receptor polymorphisms in reaction time, motor time and effects of low ethanol doses in vivo .

An Investigation of the Differential Effects of Ursane Triterpenoids from Centella asiatica, and Their Semisynthetic Analogues, on GABAA Receptors.

PubMed

Hamid, Kaiser; Ng, Irene; Tallapragada, Vikram J; Váradi, Linda; Hibbs, David E; Hanrahan, Jane; Groundwater, Paul W

2016-09-01

The ursane triterpenoids, asiatic acid 1 and madecassic acid 2, are the major pharmacological constituents of Centella asiatica, commonly known as Gotu Kola, which is used traditionally for the treatment of anxiety and for the improvement of cognition and memory. Using the two-electrode voltage-clamp technique, these triterpenes, and some semisynthetic derivatives, were found to exhibit selective negative modulation of different subtypes of the GABAA receptor expressed in Xenopus laevis oocytes. Despite differing by only one hydroxyl group, asiatic acid 1 was found to be a negative modulator of the GABA-induced current at α1 β2 γ2L, α2 β2 γ2L and α5 β3 γ2L GABAA receptors, while madecassic acid 2 was not. Asiatic acid 1 exhibited the greatest effect at α1 β2 γ2L (IC50 37.05 μm), followed by α5 β3 γ2L (IC50 64.05 μm) then α2 β2 γ2L (IC50 427.2 μm) receptors. Conversion of the carboxylic acid group of asiatic acid 1 to a carboxamide group (2α,3β,23-trihydroxy-urs-12-en-28-amide 5) resulted in enhanced inhibition at both the α1 β2 γ2L (IC50 14.07 μm) and α2 β2 γ2L receptor subtypes (IC50 28.41 μm). The results of this study, and the involvement of α5 -containing GABAA receptors in cognition and memory, suggest that asiatic acid 1 may be a lead compound for the enhancement of cognition and memory. © 2016 John Wiley & Sons A/S.

Comparison of Steroid Modulation of Spontaneous Inhibitory Postsynaptic Currents in Cultured Hippocampal Neurons and Steady-State Single-Channel Currents from Heterologously Expressed α1β2γ2L GABAA Receptors

PubMed Central

Chakrabarti, Sampurna; Qian, Mingxing; Krishnan, Kathiresan; Covey, Douglas F.; Mennerick, Steven

2016-01-01

Neuroactive steroids are efficacious modulators of γ-aminobutyric acid type A receptor (GABAA) receptor function. The effects of steroids on the GABAA receptor are typically determined by comparing steady-state single-channel open probability or macroscopic peak responses elicited by GABA in the absence and presence of a steroid. Due to differences in activation conditions (exposure duration, concentration of agonist), it is not obvious whether modulation measured using typical experimental protocols can be used to accurately predict the effect of a modulator on native receptors under physiologic conditions. In the present study, we examined the effects of 14 neuroactive steroids and analogs on the properties of spontaneous inhibitory postsynaptic currents (sIPSCs) in cultured rat hippocampal neurons. The goal was to determine whether the magnitude of modulation of the decay time course of sIPSCs correlates with the extent of modulation and kinetic properties of potentiation as determined in previous single-channel studies. The steroids were selected to cover a wide range of efficacy on heterologously expressed rat α1β2γ2L GABAA receptors, ranging from essentially inert to highly efficacious (strong potentiators of single-channel and macroscopic peak responses). The data indicate a strong correlation between prolongation of the decay time course of sIPSCs and potentiation of single-channel open probability. Furthermore, changes in intracluster closed time distributions were the single best predictor of prolongation of sIPSCs. We infer that the information obtained in steady-state single-channel recordings can be used to forecast modulation of synaptic currents. PMID:26769414

A Residue in Loop 9 of the β2-Subunit Stabilizes the Closed State of the GABAA Receptor*

PubMed Central

Williams, Carrie A.; Bell, Shannon V.; Jenkins, Andrew

2010-01-01

In γ-aminobutyric acid type A (GABAA) receptors, the structural elements that couple ligand binding to channel opening remain poorly defined. Here, site-directed mutagenesis was used to determine if Loop 9 on the non-GABA binding site interface of the β2-subunit may be involved in GABAA receptor activation. Specifically, residues Gly170-Gln185 of the β2-subunit were mutated to alanine, co-expressed with wild-type α1- and γ2S-subunits in human embryonic kidney (HEK) 293 cells and assayed for their activation by GABA, the intravenous anesthetic propofol and the endogenous neurosteroid pregnanolone using whole cell macroscopic recordings. Three mutants, G170A, V175A, and G177A, produced 2.5-, 6.7-, and 5.6-fold increases in GABA EC50 whereas one mutant, Q185A, produced a 5.2-fold decrease in GABA EC50. None of the mutations affected the ability of propofol or pregnanolone to potentiate a submaximal GABA response, but the Q185A mutant exhibited 8.3- and 3.5-fold increases in the percent direct activation by propofol and pregnanolone, respectively. Mutant Q185A receptors also had an increased leak current that was sensitive to picrotoxin, indicating an increased gating efficiency. Further Q185E, Q185L, and Q185W substitutions revealed a strong correlation between the hydropathy of the amino acid at this position and the GABA EC50. Taken together, these results indicate that β2 Loop 9 is involved in receptor activation by GABA, propofol, and pregnanolone and that β2(Q185) participates in hydrophilic interactions that are important for stabilizing the closed state of the GABAA receptor. PMID:20007704

Multiple Non-Equivalent Interfaces Mediate Direct Activation of GABAA Receptors by Propofol.

PubMed

Eaton, Megan M; Germann, Allison L; Arora, Ruby; Cao, Lily Q; Gao, Xiaoyi; Shin, Daniel J; Wu, Albert; Chiara, David C; Cohen, Jonathan B; Steinbach, Joe Henry; Evers, Alex S; Akk, Gustav

2016-01-01

Propofol is a sedative agent that at clinical concentrations acts by allosterically activating or potentiating the γ-aminobutyric acid type A (GABAA) receptor. Mutational, modeling, and photolabeling studies with propofol and its analogues have identified potential interaction sites in the transmembrane domain of the receptor. At the &quot;+&quot; of the β subunit, in the β-α interface, meta-azipropofol labels the M286 residue in the third transmembrane domain. Substitution of this residue with tryptophan results in loss of potentiation by propofol. At the &quot;-&quot; side of the β subunit, in the α-β interface (or β-β interface, in the case of homomeric β receptors), ortho-propofol diazirine labels the H267 residue in the second transmembrane domain. Structural modeling indicates that the β(H267) residue lines a cavity that docks propofol with favorable interaction energy. We used two-electrode voltage clamp to determine the functional effects of mutations to the "+" and "-" sides of the β subunit on activation of the α1β3 GABAA receptor by propofol. We found that while the individual mutations had a small effect, the combination of the M286W mutation with tryptophan mutations of selected residues at the α-β interface leads to strong reduction in gating efficacy for propofol. We conclude that α1β3 GABAA receptors can be activated by propofol interactions with the β-β, α-β, and β-α interfaces, where distinct, non-equivalent regions control channel gating. Any interface can mediate activation, hence substitutions at all interfaces are required for loss of activation by propofol.

Multiple Non-Equivalent Interfaces Mediate Direct Activation of GABAA Receptors by Propofol

PubMed Central

Eaton, Megan M.; Germann, Allison L.; Arora, Ruby; Cao, Lily Q.; Gao, Xiaoyi; Shin, Daniel J.; Wu, Albert; Chiara, David C.; Cohen, Jonathan B.; Steinbach, Joe Henry; Evers, Alex S.; Akk, Gustav

2016-01-01

Abstract: Background Propofol is a sedative agent that at clinical concentrations acts by allosterically activating or potentiating the γ-aminobutyric acid type A (GABAA) receptor. Mutational, modeling, and photolabeling studies with propofol and its analogues have identified potential interaction sites in the transmembrane domain of the receptor. At the “+” of the β subunit, in the β-α interface, meta-azipropofol labels the M286 residue in the third transmembrane domain. Substitution of this residue with tryptophan results in loss of potentiation by propofol. At the “-” side of the β subunit, in the α-β interface (or β-β interface, in the case of homomeric β receptors), ortho-propofol diazirine labels the H267 residue in the second transmembrane domain. Structural modeling indicates that the β(H267) residue lines a cavity that docks propofol with favorable interaction energy. Method We used two-electrode voltage clamp to determine the functional effects of mutations to the 
“+” and “-” sides of the β subunit on activation of the α1β3 GABAA receptor by propofol. Results We found that while the individual mutations had a small effect, the combination of the M286W mutation with tryptophan mutations of selected residues at the α-β interface leads to strong reduction in gating efficacy for propofol. Conclusion We conclude that α1β3 GABAA receptors can be activated by propofol interactions with the β-β, α-β, and β-α interfaces, where distinct, non-equivalent regions control channel gating. Any interface can mediate activation, hence substitutions at all interfaces are required for loss of activation by propofol. PMID:26830963

Discovery of an imidazopyridine-containing 1,4-benzodiazepine nonpeptide vitronectin receptor (alpha v beta 3) antagonist with efficacy in a restenosis model.

PubMed

Keenan, R M; Lago, M A; Miller, W H; Ali, F E; Cousins, R D; Hall, L B; Hwang, S M; Jakas, D R; Kwon, C; Louden, C; Nguyen, T T; Ohlstein, E H; Rieman, D J; Ross, S T; Samanen, J M; Smith, B R; Stadel, J; Takata, D T; Vickery, L; Yuan, C C; Yue, T L

1998-11-17

In the 3-oxo-1,4-benzodiazepine-2-acetic acid series of vitronectin receptor (alpha v beta 3) antagonists, a compound containing an imidazopyridine arginine mimetic was discovered which had sufficient potency and i.v. pharmacokinetics for demonstration of efficacy in a rat restenosis model.

Protein kinase and phosphatase modulation of quail brain GABA(A) and non-NMDA receptors co-expressed in Xenopus oocytes.

PubMed

Moon, C; Fraser, S P; Djamgoz, M B

2000-02-01

The GABA(A) receptor and the non-NMDA subtype of the ionotropic glutamate receptor were co-expressed in Xenopus oocytes by injection of quail brain mRNA. The oocytes were treated with various protein kinase (PK) and protein phosphatase (PP) activators and inhibitors and the effects on receptor functioning were monitored. Two phorbol esters, 4-beta-phorbol 12-myristate-13-acetate (PMA) and 4-beta-phorbol 12,13-dibutyrate (PDBu); the cGMP-dependent PK activators sodium nitroprusside (SNP) and S-nitrosoglutathione (SNOG); and the PP inhibitor okadaic acid (OA) reduced the amplitude of the GABA-induced currents, whilst the PK inhibitor staurosporine potentiated it. In addition, PMA, PDBu, SNP, and OA reduced the desensitization of the GABA-induced response. Identical treatments generally had similar but less pronounced effects on responses generated by kainate (KA) but the desensitization characteristic of the non-NMDA receptor was not affected. None of the treatments had any effect on the reversal potentials of the induced currents. Immunoblots revealed that the oocytes express endogenous PKG and guanylate cyclase. The results are discussed in terms of the molecular structures of GABA(A) and non-NMDA receptors and the potential functional consequences of phosphorylation/dephosphorylation.

Insulin-Independent GABAA Receptor-Mediated Response in the Barrel Cortex of Mice with Impaired Met Activity

PubMed Central

Lo, Fu-Sun; Erzurumlu, Reha S.

2016-01-01

Autism spectrum disorder (ASD) is a neurodevelopmental disorder caused by genetic variants, susceptibility alleles, and environmental perturbations. The autism associated gene MET tyrosine kinase has been implicated in many behavioral domains and endophenotypes of autism, including abnormal neural signaling in human sensory cortex. We investigated somatosensory thalamocortical synaptic communication in mice deficient in Met activity in cortical excitatory neurons to gain insights into aberrant somatosensation characteristic of ASD. The ratio of excitation to inhibition is dramatically increased due to decreased postsynaptic GABAA receptor-mediated inhibition in the trigeminal thalamocortical pathway of mice lacking active Met in the cerebral cortex. Furthermore, in contrast to wild-type mice, insulin failed to increase GABAA receptor-mediated response in the barrel cortex of mice with compromised Met signaling. Thus, lacking insulin effects may be a risk factor in ASD pathogenesis. SIGNIFICANCE STATEMENT A proposed common cause of neurodevelopmental disorders is an imbalance in excitatory neural transmission, provided by the glutamatergic neurons, and the inhibitory signals from the GABAergic interneurons. Many genes associated with autism spectrum disorders impair synaptic transmission in the expected cell type. Previously, inactivation of the autism-associated Met tyrosine kinase receptor in GABAergic interneurons led to decreased inhibition. In thus report, decreased Met signaling in glutamatergic neurons had no effect on excitation, but decimated inhibition. Further experiments indicate that loss of Met activity downregulates GABAA receptors on glutamatergic neurons in an insulin independent manner. These data provide a new mechanism for the loss of inhibition and subsequent abnormal excitation/inhibition balance and potential molecular candidates for treatment or prevention. PMID:27030755

Dose-dependent EEG effects of zolpidem provide evidence for GABA(A) receptor subtype selectivity in vivo.

PubMed

Visser, S A G; Wolters, F L C; van der Graaf, P H; Peletier, L A; Danhof, M

2003-03-01

Zolpidem is a nonbenzodiazepine GABA(A) receptor modulator that binds in vitro with high affinity to GABA(A) receptors expressing alpha(1) subunits but with relatively low affinity to receptors expressing alpha(2), alpha(3), and alpha(5) subunits. In the present study, it was investigated whether this subtype selectivity could be detected and quantified in vivo. Three doses (1.25, 5, and 25 mg) of zolpidem were administered to rats in an intravenous infusion over 5 min. The time course of the plasma concentrations was determined in conjunction with the change in the beta-frequency range of the EEG as pharmacodynamic endpoint. The concentration-effect relationship of the three doses showed a dose-dependent maximum effect and a dose-dependent potency. The data were analyzed for one- or two-site binding using two pharmacodynamic models based on 1) the descriptive model and 2) a novel mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model for GABA(A) receptor modulators that aims to separates drug- and system-specific properties, thereby allowing the estimation of in vivo affinity and efficacy. The application of two-site models significantly improved the fits compared with one-site models. Furthermore, in contrast to the descriptive model, the mechanism-based PK/PD model yielded dose-independent estimates for affinity (97 +/- 40 and 33,100 +/- 14,800 ng x ml(-1)). In conclusion, the mechanism-based PK/PD model is able to describe and explain the observed dose-dependent EEG effects of zolpidem and suggests the subtype selectivity of zolpidem in vivo.

Effect of ganaxolone and THIP on operant and limited-access ethanol self-administration

PubMed Central

Ramaker, Marcia J.; Strong, Moriah N.; Ford, Matthew M.; Finn, Deborah A.

2013-01-01

Recent evidence suggests that GABAA receptor ligands may regulate ethanol intake via effects at both synaptic and extrasynaptic receptors. For example, the endogenous neurosteroid, allopregnanolone (ALLO) has a similar pharmacological profile as ethanol, and it alters ethanol intake in rodent models. Additionally, recent evidence suggests that δ-subunit containing extrasynaptic GABAA receptors may confer high sensitivity to both ethanol and neurosteroids. The purpose of the present study was to determine the effects of ganaxolone (GAN; an ALLO analogue) and gaboxadol (THIP; a GABAA receptor agonist with selectivity for the extrasynaptic δ-subunit) on ethanol intake, drinking patterns, and bout characteristics in operant and limited access self-administration procedures. In separate studies, the effects of GAN (0 – 10 mg/kg) and THIP (2 – 16 mg/kg) were tested in C57BL/6J male mice provided with two-hour access to a two-bottle choice of water or 10% ethanol or trained to respond for 30 minutes of access to 10% ethanol. GAN had no overall significant effect on operant ethanol self-administration, but tended to decrease the latency to consume the first bout. In the limited-access procedure, GAN dose-dependently decreased ethanol intake. THIP dose-dependently decreased ethanol intake in both paradigms, altering both the consummatory and appetitive processes of operant self-administration as well as shifting the drinking patterns in both procedures. These results add to literature suggesting time-dependent effects of neurosteroids to promote the onset, and to subsequently decrease, ethanol drinking behavior, and they support a role for extrasynaptic GABAA receptor activation in ethanol reinforcement. PMID:22613838

Glycosylation of β2 Subunits Regulates GABAA Receptor Biogenesis and Channel Gating*

PubMed Central

Lo, Wen-yi; Lagrange, Andre H.; Hernandez, Ciria C.; Harrison, Rebecca; Dell, Anne; Haslam, Stuart M.; Sheehan, Jonathan H.; Macdonald, Robert L.

2010-01-01

γ-Aminobutyric acid type A (GABAA) receptors are heteropentameric glycoproteins. Based on consensus sequences, the GABAA receptor β2 subunit contains three potential N-linked glycosylation sites, Asn-32, Asn-104, and Asn-173. Homology modeling indicates that Asn-32 and Asn-104 are located before the α1 helix and in loop L3, respectively, near the top of the subunit-subunit interface on the minus side, and that Asn-173 is located in the Cys-loop near the bottom of the subunit N-terminal domain. Using site-directed mutagenesis, we demonstrated that all predicted β2 subunit glycosylation sites were glycosylated in transfected HEK293T cells. Glycosylation of each site, however, produced specific changes in α1β2 receptor surface expression and function. Although glycosylation of Asn-173 in the Cys-loop was important for stability of β2 subunits when expressed alone, results obtained with flow cytometry, brefeldin A treatment, and endo-β-N-acetylglucosaminidase H digestion suggested that glycosylation of Asn-104 was required for efficient α1β2 receptor assembly and/or stability in the endoplasmic reticulum. Patch clamp recording revealed that mutation of each site to prevent glycosylation decreased peak α1β2 receptor current amplitudes and altered the gating properties of α1β2 receptor channels by reducing mean open time due to a reduction in the proportion of long open states. In addition to functional heterogeneity, endo-β-N-acetylglucosaminidase H digestion and glycomic profiling revealed that surface β2 subunit N-glycans at Asn-173 were high mannose forms that were different from those of Asn-32 and N104. Using a homology model of the pentameric extracellular domain of α1β2 channel, we propose mechanisms for regulation of GABAA receptors by glycosylation. PMID:20639197

RIC-3 phosphorylation enables dual regulation of excitation and inhibition of Caenorhabditis elegans muscle

PubMed Central

Safdie, Gracia; Liewald, Jana F.; Kagan, Sarah; Battat, Emil; Gottschalk, Alexander; Treinin, Millet

2016-01-01

Brain function depends on a delicate balance between excitation and inhibition. Similarly, Caenorhabditis elegans motor system function depends on a precise balance between excitation and inhibition, as C. elegans muscles receive both inhibitory, GABAergic and excitatory, cholinergic inputs from motor neurons. Here we show that phosphorylation of the ER-resident chaperone of nicotinic acetylcholine receptors, RIC-3, leads to increased muscle excitability. RIC-3 phosphorylation at Ser-164 depends on opposing functions of the phosphatase calcineurin (TAX-6), and of the casein kinase II homologue KIN-10. Effects of calcineurin down-regulation and of phosphorylated RIC-3 on muscle excitability are mediated by GABAA receptor inhibition. Thus RIC-3 phosphorylation enables effects of this chaperone on GABAA receptors in addition to nAChRs. This dual effect provides coordinated regulation of excitation and inhibition and enables fine-tuning of the excitation–inhibition balance. Moreover, regulation of inhibitory GABAA signaling by calcineurin, a calcium- and calmodulin-dependent phosphatase, enables homeostatic balancing of excitation and inhibition. PMID:27489343

Bumetanide is not capable of terminating status epilepticus but enhances phenobarbital efficacy in different rat models.

PubMed

Töllner, Kathrin; Brandt, Claudia; Erker, Thomas; Löscher, Wolfgang

2015-01-05

In about 20-40% of patients, status epilepticus (SE) is refractory to standard treatment with benzodiazepines, necessitating second- and third-line treatments that are not always successful, resulting in increased mortality. Rat models of refractory SE are instrumental in studying the changes underlying refractoriness and to develop more effective treatments for this severe medical emergency. Failure of GABAergic inhibition is a likely cause of the development of benzodiazepine resistance during SE. In addition to changes in GABAA receptor expression, trafficking, and function, alterations in Cl(-) homeostasis with increased intraneuronal Cl(-) levels may be involved. Bumetanide, which reduces intraneuronal Cl(-) by inhibiting the Cl(-) intruding Na(+), K(+), Cl(-) cotransporter NKCC1, has been reported to interrupt SE induced by kainate in urethane-anesthetized rats, indicating that this diuretic drug may be an interesting candidate for treatment of refractory SE. In this study, we evaluated the effects of bumetanide in the kainate and lithium-pilocarpine models of SE as well as a model in which SE is induced by sustained electrical stimulation of the basolateral amygdala. Unexpectedly, bumetanide alone was ineffective to terminate SE in both conscious and anesthetized adult rats. However, it potentiated the anticonvulsant effect of low doses of phenobarbital, although this was only seen in part of the animals; higher doses of phenobarbital, particularly in combination with diazepam, were more effective to terminate SE than bumetanide/phenobarbital combinations. These data do not suggest that bumetanide, alone or in combination with phenobarbital, is a valuable option in the treatment of refractory SE in adult patients. Copyright © 2014 Elsevier B.V. All rights reserved.

In Vivo Measurement of GABA Transmission in Healthy Subjects and Schizophrenia Patients

PubMed Central

Frankle, W. Gordon; Cho, Raymond Y.; Prasad, Konasale M.; Mason, N. Scott; Paris, Jennifer; Himes, Michael L.; Walker, Christopher; Lewis, David A.; Narendran, Rajesh

2016-01-01

Objective Postmortem studies in schizophrenia reveal alterations in gene products that regulate the release and extracellular persistence of GABA. However, results of in vivo studies of schizophrenia measuring total tissue GABA with magnetic resonance spectroscopy (MRS) have been inconsistent. Neither the postmortem nor the MRS studies directly address the physiological properties of GABA neurotransmission. The present study addresses this question through an innovative positron emission tomography (PET) paradigm. Method The binding of [11C]flumazenil, a benzodiazepine-specific PET radiotracer, was measured before and after administration of tiagabine (0.2 mg/kg of body weight), a GABA membrane transporter (GAT1) blocker, in 17 off-medication patients with schizophrenia and 22 healthy comparison subjects. Increased extracellular GABA, through GAT1 blockade, enhances the affinity of GABAA receptors for benzodiazepine ligands, detected as an increase in [11C]flumazenil tissue distribution volume (VT). Results [11C]Flumazenil VT was significantly increased across all cortical brain regions in the healthy comparison group but not in the schizophrenia group. This lack of effect was most prominent in the antipsychotic-naive schizophrenia group. In this subgroup, [11C]flumazenil ΔVT in the medial temporal lobe was correlated with positive symptoms, and baseline [11C] flumazenil VT in the medial temporal lobe was negatively correlated with visual learning. In the healthy comparison group but not the schizophrenia group, [11C]flumazenil ΔVT was positively associated with gamma-band oscillation power. Conclusions This study demonstrates, for the first time, an in vivo impairment in GABA transmission in schizophrenia, most prominent in antipsychotic-naive individuals. The impairment in GABA transmission appears to be linked to clinical symptoms, disturbances in cortical oscillations, and cognition. PMID:26133962

In vivo measurement of GABA transmission in healthy subjects and schizophrenia patients.

PubMed

Frankle, W Gordon; Cho, Raymond Y; Prasad, Konasale M; Mason, N Scott; Paris, Jennifer; Himes, Michael L; Walker, Christopher; Lewis, David A; Narendran, Rajesh

2015-11-01

Postmortem studies in schizophrenia reveal alterations in gene products that regulate the release and extracellular persistence of GABA. However, results of in vivo studies of schizophrenia measuring total tissue GABA with magnetic resonance spectroscopy (MRS) have been inconsistent. Neither the postmortem nor the MRS studies directly address the physiological properties of GABA neurotransmission. The present study addresses this question through an innovative positron emission tomography (PET) paradigm. The binding of [(11)C]flumazenil, a benzodiazepine-specific PET radiotracer, was measured before and after administration of tiagabine (0.2 mg/kg of body weight), a GABA membrane transporter (GAT1) blocker, in 17 off-medication patients with schizophrenia and 22 healthy comparison subjects. Increased extracellular GABA, through GAT1 blockade, enhances the affinity of GABAA receptors for benzodiazepine ligands, detected as an increase in [(11)C]flumazenil tissue distribution volume (VT). [(11)C]Flumazenil VT was significantly increased across all cortical brain regions in the healthy comparison group but not in the schizophrenia group. This lack of effect was most prominent in the antipsychotic-naive schizophrenia group. In this subgroup, [(11)C]flumazenil ΔVT in the medial temporal lobe was correlated with positive symptoms, and baseline [(11)C]flumazenil VT in the medial temporal lobe was negatively correlated with visual learning. In the healthy comparison group but not the schizophrenia group, [(11)C]flumazenil ΔVT was positively associated with gamma-band oscillation power. This study demonstrates, for the first time, an in vivo impairment in GABA transmission in schizophrenia, most prominent in antipsychotic-naive individuals. The impairment in GABA transmission appears to be linked to clinical symptoms, disturbances in cortical oscillations, and cognition.

Sensitivity of thalamic GABAergic currents to clonazepam does not differ between control and genetic absence epilepsy rats.

PubMed

Badiu, Carmen-Ionela

2004-11-12

Mutations in GABA-A receptor subunits have been reported in a number of idiopathic generalized epilepsies including childhood absence epilepsy. One of these mutations is located within a high-affinity benzodiazepine-binding domain, and clonazepam is clinically used as an anti-absence drug. The intrathalamic loop consisting of the GABAergic neurons of the nucleus reticularis thalami (NRT) and the thalamocortical (TC) neurons of sensory thalamic nuclei plays an essential role in spike and wave discharges. In a well-established genetic model of absence epilepsy (Genetic Absence Epilepsy rat from Strasbourg, GAERS), systemic injections of benzodiazepines have been shown to suppress spike-and-waves discharges. The aim of this study, therefore, was to determine whether the sensitivity of GABAergic synaptic currents to clonazepam in NRT and TC neurons was different in GAERS and non-epileptic control (NEC) rats. In both pre-seizure GAERS and NEC clonazepam (100 nM) had no effect on the mIPSCs recorded from TC neurons while it increased the decay time constant of the mIPSCs recorded in NRT neurons by a similar amount in GAERS (54.5+/-5%) and NEC (50.7+/-5%). Similar results have been obtained in the presence of 100 microM Cd2+, showing that the effect of clonazepam did not occur via modulation of voltage-activated Ca2+ currents. These results are relevant to understand that in GAERS, the clonazepam anti-absence actions cannot be fully explained by the enhancement of the intra-NRT inhibition and the modulation of the GABAergic synaptic currents in other brain areas, in particular the cortex, must be taken into consideration.

Opposing roles for GABAA and GABAC receptors in short-term memory formation in young chicks.

PubMed

Gibbs, M E; Johnston, G A R

2005-01-01

The inhibitory neurotransmitter GABA has both inhibitory and enhancing effects on short-term memory for a bead discrimination task in the young chick. Low doses of GABA (1-3 pmol/hemisphere) injected into the multimodal association area of the chick forebrain, inhibit strongly reinforced memory, whereas higher doses (30-100 pmol/hemisphere) enhance weakly reinforced memory. The effect of both high and low doses of GABA is clearly on short-term memory in terms of both the time of injection and in the time that the memory loss occurs. We argue on the basis of relative sensitivities to GABA and to selective GABA receptor antagonists that low doses of GABA act at GABAC receptors (EC50 approximately 1 microM) and the higher doses of GABA act via GABAA receptors (EC50 approximately 10 microM). The selective GABAA receptor antagonist bicuculline inhibited strongly reinforced memory in a dose and time dependent manner, whereas the selective GABAC receptor antagonists TPMPA and P4MPA enhanced weakly reinforced in a dose and time dependent manner. Confirmation that different levels of GABA affect different receptor subtypes was demonstrated by the shift in the GABA dose-response curves to the selective antagonists. It is clear that GABA is involved in the control of short-term memory formation and its action, enhancing or inhibiting, depends on the level of GABA released at the time of learning.

Hypocretin and GABA interact in the pontine reticular formation to increase wakefulness.

PubMed

Brevig, Holly N; Watson, Christopher J; Lydic, Ralph; Baghdoyan, Helen A

2010-10-01

Hypocretin-1/orexin A administered directly into the oral part of rat pontine reticular formation (PnO) causes an increase in wakefulness and extracellular gamma-aminobutyric acid (GABA) levels. The receptors in the PnO that mediate these effects have not been identified. Therefore, this study tested the hypothesis that the increase in wakefulness caused by administration of hypocretin-1 into the PnO occurs via activation of GABAA receptors and hypocretin receptors. Within/between subjects. University of Michigan. Twenty-three adult male Crl:CD*(SD) (Sprague Dawley) rats. Microinjection of hypocretin-1, bicuculline (GABAA receptor antagonist), SB-334867 (hypocretin receptor-1 antagonist), and Ringer solution (vehicle control) into the PnO. Hypocretin-1 caused a significant concentration-dependent increase in wakefulness and decrease in rapid eye movement (REM) sleep and non-REM (NREM) sleep. Coadministration of SB-334867 and hypocretin-1 blocked the hypocretin-1-induced increase in wakefulness and decrease in both the NREM and REM phases of sleep. Coadministration of bicuculline and hypocretin-1 blocked the hypocretin-1-induced increase in wakefulness and decrease in NREM sleep caused by hypocretin-1. The increase in wakefulness caused by administering hypocretin-1 to the PnO is mediated by hypocretin receptors and GABAA receptors in the PnO. These results show for the first time that hypocretinergic and GABAergic transmission in the PnO can interact to promote wakefulness.

Ventral tegmental area GABA neurons and opiate motivation

PubMed Central

Vargas-Perez, Hector; Mabey, Jennifer K.; Shin, Samuel I.; Steffensen, Scott C.; van der Kooy, Derek

2013-01-01

Rational Past research has demonstrated that when an animal changes from a previously drug-naive to an opiate-dependent and withdrawn state, morphine’s motivational effects are switched from a tegmental pedunculopontine nucleus (TPP)-dependent to a dopamine-dependent pathway. Interestingly, a corresponding change is observed in ventral tegmental area (VTA) GABAA receptors, which change from mediating hyperpolarization of VTA GABA neurons to mediating depolarization. Objectives The present study investigated whether pharmacological manipulation of VTA GABAA receptor activity could directly influence the mechanisms underlying opiate motivation. Results Using an unbiased place conditioning procedure, we demonstrated that in Wistar rats, intra-VTA administration of furosemide, a Cl− cotransporter inhibitor, was able to promote a switch in the mechanisms underlying morphine’s motivational properties, one which is normally observed only after chronic opiate exposure. This behavioral switch was prevented by intra-VTA administration of acetazolamide, an inhibitor of the bicarbonate ion-producing carbonic anhydrase enzyme. Electrophysiological recordings of mouse VTA showed that furosemide reduced the sensitivity of VTA GABA neurons to inhibition by the GABAA receptor agonist muscimol, instead increasing the firing rate of a significant subset of these GABA neurons. Conclusion Our results suggest that the carbonic anhydrase enzyme may constitute part of a common VTA GABA neuron-based biological pathway responsible for controlling the mechanisms underlying opiate motivation, supporting the hypothesis that VTA GABAA receptor hyperpolarization or depolarization is responsible for selecting TPP- or dopamine-dependent motivational outputs, respectively. PMID:23392354

Removal of GABAA Receptor γ2 Subunits from Parvalbumin Neurons Causes Wide-Ranging Behavioral Alterations

PubMed Central

Leppä, Elli; Linden, Anni-Maija; Vekovischeva, Olga Y.; Swinny, Jerome D.; Rantanen, Ville; Toppila, Esko; Höger, Harald; Sieghart, Werner; Wulff, Peer; Wisden, William; Korpi, Esa R.

2011-01-01

We investigated the behavioral significance of fast synaptic inhibition by αβγ2-type GABAA receptors on parvalbumin (Pv) cells. The GABAA receptor γ2 subunit gene was selectively inactivated in Pv-positive neurons by Cre/loxP recombination. The resulting Pv-Δγ2 mice were relatively healthy in the first postnatal weeks; but then as Cre started to be expressed, the mice progressively developed wide-ranging phenotypic alterations including low body weight, motor deficits and tremor, decreased anxiety levels, decreased pain sensitivity and deficient prepulse inhibition of the acoustic startle reflex and impaired spatial learning. Nevertheless, the deletion was not lethal, and mice did not show increased mortality even after one year. Autoradiography with t-butylbicyclophosphoro[35S]thionate suggested an increased amount of GABAA receptors with only α and β subunits in central nervous system regions that contained high levels of parvalbumin neurons. Using BAC-transgenesis, we reduced some of the Pv-Δγ2 phenotype by selectively re-expressing the wild-type γ2 subunit back into some Pv cells (reticular thalamic neurons and cerebellar Pv-positive neurons). This produced less severe impairments of motor skills and spatial learning compared with Pv-Δγ2 mice, but all other deficits remained. Our results reveal the widespread significance of fast GABAergic inhibition onto Pv-positive neurons for diverse behavioral modalities, such as motor coordination, sensorimotor integration, emotional behavior and nociception. PMID:21912668

Biased signaling of the proton-sensing receptor OGR1 by benzodiazepines.

PubMed

Pera, Tonio; Deshpande, Deepak A; Ippolito, Michael; Wang, Bin; Gavrila, Adelina; Michael, James V; Nayak, Ajay P; Tompkins, Eric; Farrell, Eleni; Kroeze, Wesley K; Roth, Bryan L; Panettieri, Reynold A; Benovic, Jeffrey L; An, Steven S; Dulin, Nickolai O; Penn, Raymond B

2018-02-01

GPCRs have diverse signaling capabilities, based on their ability to assume various conformations. Moreover, it is now appreciated that certain ligands can promote distinct receptor conformations and thereby bias signaling toward a specific pathway to differentially affect cell function. The recently deorphanized G protein-coupled receptor OGR1 [ovarian cancer G protein-coupled receptor 1 ( GPR68)] exhibits diverse signaling events when stimulated by reductions in extracellular pH. We recently demonstrated airway smooth muscle cells transduce multiple signaling events, reflecting a diverse capacity to couple to multiple G proteins. Moreover, we recently discovered that the benzodiazepine lorazepam, more commonly recognized as an agonist of the γ-aminobutyric acid A (GABA A ) receptor, can function as an allosteric modulator of OGR1 and, similarly, can promote multiple signaling events. In this study, we demonstrated that different benzodiazepines exhibit a range of biases for OGR1, with sulazepam selectively activating the canonical Gs of the G protein signaling pathway, in heterologous expression systems, as well as in several primary cell types. These findings highlight the potential power of biased ligand pharmacology for manipulating receptor signaling qualitatively, to preferentially activate pathways that are therapeutically beneficial.-Pera, T., Deshpande, D. A., Ippolito, M., Wang, B., Gavrila, A., Michael, J. V., Nayak, A. P., Tompkins, E., Farrell, E., Kroeze, W. K., Roth, B. L., Panettieri, R. A. Jr Benovic, J. L., An, S. S., Dulin, N. O., Penn, R. B. Biased signaling of the proton-sensing receptor OGR1 by benzodiazepines.

A new meaning for “Gin & Tonic”

PubMed Central

Mody, Istvan; Glykys, Joseph; Wei, Weizheng

2007-01-01

Gamma-aminobutyric acid (GABA) is the main chemical inhibitory neurotransmitter in the brain. In the central nervous system (CNS) it acts on two distinct types of receptor: an ion channel, i.e., an “ionotropic” receptor permeable to Cl- and HCO3- (GABAA receptors) and a G-protein coupled “metabotropic” receptor that is linked to various effector mechanisms (GABAB receptors). This review will summarize novel developments in the physiology and pharmacology of GABAA receptors (GABAARs), specifically those found outside synapses. The focus will be on a particular combination of GABAAR subunits responsible for mediating tonic inhibition and sensitive to concentrations of ethanol legally considered to be sobriety impairing. Since the same receptors are also a preferred target for the metabolites of steroid hormones synthesized in the brain (neurosteroids), the ethanol-sensitive tonic inhibition may be a common pathway for interactions between the effects of alcohol and those of ovarian and stress-related neurosteroids. PMID:17521846

The imidazobenzodiazepine Ro 15-4513 antagonizes methoxyflurane anesthesia.

PubMed

Moody, E J; Skolnick, P

1988-01-01

Parenteral administration of the imidazobenzodiazepine Ro 15-4513 (a high affinity ligand of the benzodiazepine receptor with partial inverse agonist qualities) produced a dose dependent reduction in sleep time of mice exposed to the inhalation anesthetic, methoxyflurane. The reductions in methoxyflurane sleep time ranged from approximately 20% at 4 mg/kg to approximately 38% at 32 mg/kg of Ro 15-4513. Co-administration of the benzodiazepine receptor antagonist Ro 15-1788 (16 mg/kg) or the inverse agonists DMCM (5-20 mg/kg) and FG 7142 (22.5 mg/kg) blocks this effect which suggests that the reductions in methoxyflurane sleep time produced by Ro 15-4513 are mediated via occupation of benzodiazepine receptors. Moreover, neither DMCM (5-20 mg/kg) nor FG 7142 (22.5 mg/kg) reduced methoxyflurane sleep time which suggests this effect of Ro 15-4513 cannot be attributed solely to its partial inverse agonist properties. These observations support recent findings that inhalation anesthetics may produce their depressant effects via perturbation of the benzodiazepine/GABA receptor chloride channel complex, and suggest that Ro 15-4513 may serve as a prototype of agents capable of antagonizing the depressant effects of inhalation anesthetics such as methoxyflurane.

The Use of Benzodiazepine Receptor Agonists and Risk of Respiratory Failure in Patients with Chronic Obstructive Pulmonary Disease: A Nationwide Population-Based Case-Control Study

PubMed Central

Chen, Su-Jung; Yeh, Chiu-Mei; Chao, Tze-Fan; Liu, Chia-Jen; Wang, Kang-Ling; Chen, Tzeng-Ji; Chou, Pesus; Wang, Fu-Der

2015-01-01

Study Objectives: Insomnia is prevalent in patients with chronic obstructive pulmonary disease (COPD), and benzodiazepine receptor agonists (BZRAs) are the most commonly used drugs despite their adverse effects on respiratory function. The aim of this study was to investigate whether the use of BZRAs was associated with an increased risk of respiratory failure (RF) in COPD patients. Design: Matched case-control study. Setting: National Health Insurance Research Database (NHIRD) in Taiwan. Participants: The case group consisted of 2,434 COPD patients with RF, and the control group consisted of 2,434 COPD patients without RF, matched for age, sex, and date of enrollment. Measurements and Results: Exposure to BZRAs during the 180-day period preceding the index date was analyzed and compared in the case and control groups. Conditional logistic regression was performed, and the use of BZRAs was associated with an increased risk of RF (adjusted odds ratio [aOR] 1.56, 95% confidence interval [CI] 1.14–2.13). In subgroup analysis, we found that the benzodiazepine (BZD) users had a higher risk of RF (aOR 1.58, 95% CI 1.14–2.20), whereas the risk in non-benzodiazepine (non-BZD) users was insignificant (aOR 0.85, 95% CI 0.51–1.44). A greater than 2-fold increase in risk was found in those who received two or more kinds of BZRAs and those using a combination of BZD and non-BZD medications. Conclusions: The use of benzodiazepine receptor agonists was a significant risk factor for respiratory failure in patients with chronic obstructive pulmonary disease (COPD). Compared to benzodiazepine, the prescription of non-benzodiazepine may be safer for the management of insomnia in COPD patients. Citation: Chen SJ, Yeh CM, Chao TF, Liu CJ, Wang KL, Chen TJ, Chou P, Wang FD. The use of benzodiazepine receptor agonists and risk of respiratory failure in patients with chronic obstructive pulmonary disease: a nationwide population-based case-control study. SLEEP 2015;38(7):1045–1050. PMID:25669186

The pharmacokinetic and pharmacodynamic effects of SL65.1498, a GABA-A alpha2,3 selective agonist, in comparison with lorazepam in healthy volunteers.

PubMed

de Haas, S L; Franson, K L; Schmitt, J A J; Cohen, A F; Fau, J B; Dubruc, C; van Gerven, J M A

2009-08-01

Benzodiazepines are effective short-term treatments for anxiety disorders, but their use is limited by undesirable side effects related to Central Nervous System impairment and tolerance development. SL65.1498 is a new compound that acts in vitro as a full agonist at the gamma-aminobutyric acid(A) 2 and 3 receptor and as a partial agonist at the 1 and 5 receptor subtypes. It is thought that the compound could be anxiolytic by its activation at the alpha2 and alpha3 receptor subtypes, without causing unfavourable side effects, which are believed to be mediated by the alpha1 and alpha5 subtypes. This study was a double-blind, five-way cross-over study to investigate the effects of three doses of SL65.1498 in comparison with placebo and lorazepam 2 mg in healthy volunteers. The objective was to select a dose level (expected to be therapeutically active), free of any significant deleterious effect. Psychomotor and cognitive effects were measured using a validated battery of measurements, including eye movements, body sway, memory tests, reaction-time assessments, and visual analogue scales. The highest dose of SL65.1498 showed slight effects on saccadic peak velocity and smooth pursuit performance, although to a much lesser extent than lorazepam. In contrast to lorazepam, none of the SL65.1498 doses affected body sway, visual analogue scale alertness, attention, or memory tests. This study showed that the three doses of SL65.1498 were well tolerated and induced no impairments on memory, sedation, psychomotor, and cognitive functions.

Estrogen increases latencies to seizures and levels of 5α-pregnan-3α-ol-20-one in hippocampus of wild-type, but not 5α-reductase knockout, mice

PubMed Central

Osborne, Danielle M.; Frye, Cheryl A.

2013-01-01

Sex steroids can influence seizures. Estrogen (E2), progesterone (P4), and its metabolite, 5α-pregnan-3α-ol-20-one (3α,5α-THP), in particular, have received much attention for exerting these effects. Typically, it is thought that E2 precipitates seizures, and progestogens, such as P4 and 3α,5α-THP, attenuate seizures. However, E2 may also have antiseizure effects, perhaps in part through its enhancement of the formation of 3α,5α-THP, which has GABAA/benzodiazepine receptor agonist-like actions. To test this hypothesis, male and female, castrated or ovariectomized, wild-type and 5α-reductase knockout mice were implanted with Silastic capsules of E2 or vehicle and then administered pentylenetetrazol (85 mg/kg, ip). Wild-type, but not 5α-reductase knockout, mice administered E2 had significantly longer latencies to myoclonus and increased levels of 3α,5α-THP in the hippocampus. Thus, some of the anticonvulsive effects of E2 may involve formation of 3α,5α-THP in the hippocampus. PMID:19782646

Reducing excessive GABAergic tonic inhibition promotes post-stroke functional recovery

PubMed Central

Clarkson, Andrew N.; Huang, Ben S.; MacIsaac, Sarah E.; Mody, Istvan; Carmichael, S. Thomas

2010-01-01

Stroke is a leading cause of disability; but no pharmacological therapy is currently available for promoting recovery. The brain region adjacent to stroke damage, the peri-infarct zone, is critical for rehabilitation, as it exhibits heightened neuroplasticity, allowing sensorimotor functions to re-map from damaged areas1–3. Thus, understanding the neuronal properties constraining this plasticity is important to developing new treatments. Here we show that after a stroke in mice, tonic neuronal inhibition is increased in the peri-infarct zone. This increased tonic inhibition is mediated by extrasynaptic GABAA receptors (GABAARs) and is caused by an impairment in GABA transporter (GAT-3/4) function. To counteract the heightened inhibition, we administered in vivo a benzodiazepine inverse agonist specific for the α5-subunit-containing extrasynaptic GABAARs at a delay after stroke. This treatment produced an early and sustained recovery of motor function. Genetically lowering the number of α5 or δ-subunit-containing GABAARs responsible for tonic inhibition also proved beneficial for post-stroke recovery, consistent with the therapeutic potential of diminishing extrasynaptic GABAAR function. Together, our results identify new pharmacological targets and provide the rationale for a novel strategy to promote recovery after stroke and possibly other brain injuries. PMID:21048709

Zolpidem generalization and antagonism in male and female cynomolgus monkeys trained to discriminate 1.0 or 2.0 g/kg ethanol.

PubMed

Helms, Christa M; Rogers, Laura S M; Waters, Courtney A; Grant, Kathleen A

2008-07-01

The subtypes of gamma-aminobutyric acid (GABA)(A) receptors mediating the discriminative stimulus effects of ethanol in nonhuman primates are not completely identified. The GABA(A) receptor positive modulator zolpidem has high, intermediate, and low activity at receptors containing alpha(1), alpha(2/3), and alpha(5) subunits, respectively, and partially generalizes from ethanol in several species. The partial inverse agonist Ro15-4513 has the greatest affinity for alpha(4/6)-containing receptors, higher affinity for alpha(5)- and lower, but equal, affinity for alpha(1)- and alpha(2/3)-, containing GABA(A) receptors, and antagonizes the discriminative stimulus effects of ethanol. This study assessed Ro15-4513 antagonism of the generalization of zolpidem from ethanol in male (n = 9) and female (n = 8) cynomolgus monkeys (Macaca fascicularis) trained to discriminate 1.0 g/kg (n = 10) or 2.0 g/kg (n = 7) ethanol (i.g.) from water with a 30-minute pretreatment interval. Zolpidem (0.017 to 5.6 mg/kg, i.m.) completely generalized from ethanol (>or=80% of total session responses on the ethanol-appropriate lever) for 6/7 monkeys trained to discriminate 2.0 g/kg and 4/10 monkeys trained to discriminate 1.0 g/kg ethanol. Zolpidem partially generalized from 1.0 or 2.0 g/kg ethanol in 6/7 remaining monkeys. Ro15-4513 (0.003 to 0.30 mg/kg, i.m., 5-minute pretreatment) shifted the zolpidem dose-response curve to the right in all monkeys showing generalization. Analysis of apparent pK(B) from antagonism tests suggested that the discriminative stimulus effects of ethanol common with zolpidem are mediated by low-affinity Ro15-4513 binding sites. Main effects of sex and training dose indicated greater potency of Ro15-4513 in males and in monkeys trained to discriminate 1.0 g/kg ethanol. Ethanol and zolpidem share similar discriminative stimulus effects most likely through GABA(A) receptors that contain alpha(1) subunits, however, antagonism by Ro15-4513 of zolpidem generalization from the lower training dose of ethanol (1.0 g/kg) may involve additional zolpidem-sensitive GABA(A) receptor subtypes (e.g., alpha(2/3) and alpha(5)).

Alcohol-induced tolerance and physical dependence in mice with ethanol insensitive α1 GABAA receptors

PubMed Central

Werner, David F.; Swihart, Andrew R.; Ferguson, Carolyn; Lariviere, William R.; Harrison, Neil L.; Homanics, Gregg E.

2009-01-01

Background Although many people consume alcohol (ethanol), it remains unknown why some become addicted. Elucidating the molecular mechanisms of tolerance and physical dependence (withdrawal) may provide insight into alcohol addiction. While the exact molecular mechanisms of ethanol action are unclear, γ-aminobutyric acid type A receptors (GABAA-Rs) have been extensively implicated in ethanol action. The α1 GABAA-R subunit is associated with tolerance and physical dependence, but its exact role remains unknown. In this report, we tested the hypothesis that α1-GABAA-Rs mediate in part these effects of ethanol. Methods Ethanol-induced behavioral responses related to tolerance and physical dependence were investigated in knockin mice that have ethanol-insensitive α1 GABAA-Rs and wildtype controls. Acute functional tolerance (AFT) was assessed using the stationary dowel and loss of righting reflex assays. Chronic tolerance was assessed on the loss of righting reflex, fixed speed rotarod, hypothermia, and radiant tail flick assays following ten consecutive days of ethanol exposure. Withdrawal-related hyperexcitability was assessed by handling-induced convulsions following 3 cycles of ethanol vapor exposure/withdrawal. Immunoblots were used to assess α1 protein levels. Results Compared to controls, knockin mice displayed decreased AFT and chronic tolerance to ethanol-induced motor ataxia, and also displayed heightened ethanol-withdrawal hyperexcitability. No differences between wildtype and knockin mice were seen in other ethanol-induced behavioral measures. Following chronic exposure to ethanol, control mice displayed reductions in α1 protein levels, but knockins did not. Conclusions We conclude that α1-GABAA-Rs play a role in tolerance to ethanol-induced motor ataxia and withdrawal-related hyperexcitability. However, other aspects of behavioral tolerance and physical dependence do not rely on α1-containing GABAA-Rs. PMID:19032579

Effect of GABA receptor agonists or antagonists injected spinally on the blood glucose level in mice.

PubMed

Sim, Yun-Beom; Park, Soo-Hyun; Kang, Yu-Jung; Kim, Sung-Su; Kim, Chea-Ha; Kim, Su-Jin; Jung, Jun-Sub; Ryu, Ohk-Hyun; Choi, Moon-Gi; Suh, Hong-Won

2013-05-01

The possible roles of gamma-amino butyric acid (GABA) receptors located in the spinal cord for the regulation of the blood glucose level were studied in ICR mice. We found in the present study that intrathecal (i.t.) injection with baclofen (a GABAB receptor agonist; 1-10 μg/5 μl) or bicuculline (a GABAA receptor antagonist; 1-10 μg/5 μl) caused an elevation of the blood glucose level in a dose-dependent manner. The hyperglycemic effect induced by baclofen was more pronounced than that induced by bicuculline. However, muscimol (a GABAA receptor agonist; 1-5 μg/5 μl) or phaclofen (a GABAB receptor antagonist; 5-10 μg/5 μl) administered i.t. did not affect the blood glucose level. Baclofen-induced elevation of the blood glucose was dose-dependently attenuated by phaclofen. Furthermore, i.t. pretreatment with pertussis toxin (PTX; 0.05 or 0.1 μg/5 μl) for 6 days dose-dependently reduced the hyperglycemic effect induced by baclofen. Our results suggest that GABAB receptors located in the spinal cord play important roles for the elevation of the blood glucose level. Spinally located PTX-sensitive G-proteins appear to be involved in hyperglycemic effect induced by baclofen. Furthermore, inactivation of GABAA receptors located in the spinal cord appears to be responsible for tonic up-regulation of the blood glucose level.

Activation of single heteromeric GABAA receptor ion channels by full and partial agonists

PubMed Central

Mortensen, Martin; Kristiansen, Uffe; Ebert, Bjarke; Frølund, Bente; Krogsgaard-Larsen, Povl; Smart, Trevor G

2004-01-01

The linkage between agonist binding and the activation of a GABAA receptor ion channel is yet to be resolved. This aspect was examined on human recombinant α1β2γ2S GABAA receptors expressed in human embryonic kidney cells using the following series of receptor agonists: GABA, isoguvacine, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), isonipecotic acid, piperidine-4-sulphonic acid (P4S), imidazole-4-acetic acid (IAA), 5-(4-piperidyl)-3-isothiazolol (thio-4-PIOL) and 5-(4-piperidyl)-3-isoxazolol (4-PIOL). Whole-cell concentration–response curves enabled the agonists to be categorized into four classes based upon their maximum responses. Single channel analyses revealed that the channel conductance of 25–27 pS was unaffected by the agonists. However, two open states were resolved from the open period distributions with mean open times reduced 5-fold by the weakest partial agonists. Using saturating agonist concentrations, estimates of the channel shutting rate, α, ranged from 200 to 600 s−1. The shut period distributions were described by three or four components and for the weakest partial agonists, the interburst shut periods increased whilst the mean burst durations and longest burst lengths were reduced relative to the full agonists. From the burst analyses, the opening rates for channel activation, β, and the total dissociation rates, k−1, for the agonists leaving the receptor were estimated. The agonist efficacies were larger for the full agonists (E ∼7−9) compared to the weak partial agonists (∼0.4–0.6). Overall, changes in agonist efficacy largely determined the different agonist profiles with contributions from the agonist affinities and the degree of receptor desensitization. From this we conclude that GABAA receptor activation does not occur in a switch-like manner since the agonist recognition sites are flexible, accommodating diverse agonist structures which differentially influence the opening and shutting rates of the ion channel. PMID:14990676

Methods for recording and measuring tonic GABAA receptor-mediated inhibition

PubMed Central

Bright, Damian P.; Smart, Trevor G.

2013-01-01

Tonic inhibitory conductances mediated by GABAA receptors have now been identified and characterized in many different brain regions. Most experimental studies of tonic GABAergic inhibition have been carried out using acute brain slice preparations but tonic currents have been recorded under a variety of different conditions. This diversity of recording conditions is likely to impact upon many of the factors responsible for controlling tonic inhibition and can make comparison between different studies difficult. In this review, we will firstly consider how various experimental conditions, including age of animal, recording temperature and solution composition, are likely to influence tonic GABAA conductances. We will then consider some technical considerations related to how the tonic conductance is measured and subsequently analyzed, including how the use of current noise may provide a complementary and reliable method for quantifying changes in tonic current. PMID:24367296

The effect of propofol on CA1 pyramidal cell excitability and GABAA-mediated inhibition in the rat hippocampal slice.

PubMed

Albertson, T E; Walby, W F; Stark, L G; Joy, R M

1996-05-24

An in vitro paired-pulse orthodromic stimulation technique was used to examine the effects of propofol on excitatory afferent terminals, CA1 pyramidal cells and recurrent collateral evoked inhibition in the rat hippocampal slice. Hippocampal slices 400 microns thick were perfused with oxygenated artificial cerebrospinal fluid, and electrodes were placed in the CA1 region to record extracellular field population spike (PS) or excitatory postsynaptic potential (EPSP) responses to stimulation of Schaffer collateral/commissural fibers. Gamma-aminobutyric acid (GABA)-mediated recurrent inhibition was measured using a paired-pulse technique. The major effect of propofol (7-28 microM) was a dose and time dependent increase in the intensity and duration of GABA-mediated inhibition. This propofol effect could be rapidly and completely reversed by exposure to known GABAA antagonists, including picrotoxin, bicuculline and pentylenetetrazol. It was also reversed by the chloride channel antagonist, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). It was not antagonized by central (flumazenil) or peripheral (PK11195) benzodiazepine antagonists. Reversal of endogenous inhibition was also noted with the antagonists picrotoxin and pentylenetetrazol. Input/output curves constructed using stimulus propofol caused only a small enhancement of EPSPs at higher stimulus intensities but had no effect on PS amplitudes. These studies are consistent with propofol having a GABAA-chloride channel mechanism causing its effect on recurrent collateral evoked inhibition in the rat hippocampal slice.

Radixin regulates synaptic GABAA receptor density and is essential for reversal learning and short-term memory

PubMed Central

Hausrat, Torben J.; Muhia, Mary; Gerrow, Kimberly; Thomas, Philip; Hirdes, Wiebke; Tsukita, Sachiko; Heisler, Frank F.; Herich, Lena; Dubroqua, Sylvain; Breiden, Petra; Feldon, Joram; Schwarz, Jürgen R; Yee, Benjamin K.; Smart, Trevor G.; Triller, Antoine; Kneussel, Matthias

2015-01-01

Neurotransmitter receptor density is a major variable in regulating synaptic strength. Receptors rapidly exchange between synapses and intracellular storage pools through endocytic recycling. In addition, lateral diffusion and confinement exchanges surface membrane receptors between synaptic and extrasynaptic sites. However, the signals that regulate this transition are currently unknown. GABAA receptors containing α5-subunits (GABAAR-α5) concentrate extrasynaptically through radixin (Rdx)-mediated anchorage at the actin cytoskeleton. Here we report a novel mechanism that regulates adjustable plasma membrane receptor pools in the control of synaptic receptor density. RhoA/ROCK signalling regulates an activity-dependent Rdx phosphorylation switch that uncouples GABAAR-α5 from its extrasynaptic anchor, thereby enriching synaptic receptor numbers. Thus, the unphosphorylated form of Rdx alters mIPSCs. Rdx gene knockout impairs reversal learning and short-term memory, and Rdx phosphorylation in wild-type mice exhibits experience-dependent changes when exposed to novel environments. Our data suggest an additional mode of synaptic plasticity, in which extrasynaptic receptor reservoirs supply synaptic GABAARs. PMID:25891999

Effects of gaboxadol on the expression of cocaine sensitization in rats.

PubMed

Silverman, Nora Siegal; Popp, Susanna; Vialou, Vincent; Astafurov, Konstantin; Nestler, Eric J; Dow-Edwards, Diana

2016-04-01

Behavioral sensitization to psychostimulants is associated with changes in dopamine (DA), glutamate, and GABA within the mesocorticolimbic and nigrostriatal DA systems. Because GABAA receptors are highly expressed within these systems, we examined the role of these receptors containing a δ subunit in cocaine behavioral sensitization. Experiment 1 examined the effects of Gaboxadol (GBX, also known as THIP [4,5,6,7-tetrahydro-isoxazolo[5,4-c]pyridin-3-ol]), a selective δ-GABAA receptor agonist, on the locomotor responses to acute cocaine. GBX at 1.25 mg/kg produced locomotor depression in female rats alone. We then examined the effects of GBX on the expression of cocaine-induced locomotion and stereotypy in female and male rats treated with 5 days of cocaine (15 mg/kg) followed by cocaine challenge 7 days later. We administered systemic (Experiment 2) or intranucleus accumbens (intra-NAC; Experiment 3) injections of GBX (0, 1.25, 2.5, 5, or 10 mg/kg subcutaneously, or 1 μmol/L or 1 mM intra-NAC, respectively) prior to cocaine challenge (10 mg/kg). In our experiments females were robustly sensitized to cocaine at low dose whereas males did not show such sensitization-limiting comparisons between the 2 sexes. Sensitized females showed a biphasic response to low (1.25 mg/kg and 1 μmol/L) and high (10 mg/kg and 1 mM) dose GBX whereas nonsensitized males showed this pattern only following intra-NAC injection. Immunohistochemical analysis of the NAC revealed that females have more δ-containing GABAA receptors than do males and that following chronic cocaine injections this difference persisted (Experiment 4). Together, our results support the notion of the key role of extrasynaptic GABAA δ-subunit containing receptors in cocaine sensitization. (c) 2016 APA, all rights reserved).

Perimenstrual-Like Hormonal Regulation of Extrasynaptic δ-Containing GABAA Receptors Mediating Tonic Inhibition and Neurosteroid Sensitivity

PubMed Central

Carver, Chase Matthew; Wu, Xin; Gangisetty, Omkaram

2014-01-01

Neurosteroids are endogenous regulators of neuronal excitability and seizure susceptibility. Neurosteroids, such as allopregnanolone (AP; 3α-hydroxy-5α-pregnan-20-one), exhibit enhanced anticonvulsant activity in perimenstrual catamenial epilepsy, a neuroendocrine condition in which seizures are clustered around the menstrual period associated with neurosteroid withdrawal (NSW). However, the molecular mechanisms underlying such enhanced neurosteroid sensitivity remain unclear. Neurosteroids are allosteric modulators of both synaptic (αβγ2-containing) and extrasynaptic (αβδ-containing) GABAA receptors, but they display greater sensitivity toward δ-subunit receptors in dentate gyrus granule cells (DGGCs). Here we report a novel plasticity of extrasynaptic δ-containing GABAA receptors in the dentate gyrus in a mouse perimenstrual-like model of NSW. In molecular and immunofluorescence studies, a significant increase occurred in δ subunits, but not α1, α2, β2, and γ2 subunits, in the dentate gyrus of NSW mice. Electrophysiological studies confirmed enhanced sensitivity to AP potentiation of GABA-gated currents in DGGCs, but not in CA1 pyramidal cells, in NSW animals. AP produced a greater potentiation of tonic currents in DGGCs of NSW animals, and such enhanced AP sensitivity was not evident in δ-subunit knock-out mice subjected to a similar withdrawal paradigm. In behavioral studies, mice undergoing NSW exhibited enhanced seizure susceptibility to hippocampus kindling. AP has enhanced anticonvulsant effects in fully kindled wild-type mice, but not δ-subunit knock-out mice, undergoing NSW-induced seizures, confirming δ-linked neurosteroid sensitivity. These results indicate that perimenstrual NSW is associated with striking upregulation of extrasynaptic, δ-containing GABAA receptors that mediate tonic inhibition and neurosteroid sensitivity in the dentate gyrus. These findings may represent a molecular rationale for neurosteroid therapy of catamenial epilepsy. PMID:25339733

Furosemide suppresses ileal and colonic contractility via interactions with GABA-A receptor in mice.

PubMed

Kaewsaro, Kannaree; Nualplub, Suparp; Bumrungsri, Sara; Khuituan, Pissared

2017-11-01

The loop diuretic furosemide has an action to inhibit Na + -K + -2Cl - co-transporter at the thick ascending limb of Henle's loop resulting in diuresis. Furosemide also has the non-diuretic effects by binding to GABA-A receptor which may involve the gastrointestinal tract. The aim of this study was to investigate the effects of furosemide on smooth muscle contractions in mice ileum and proximal colon. Each intestinal segment suspended in an organ bath was connected to a force transducer. Signal output of mechanical activity was amplified and recorded for analysis using PowerLab System. After equilibration, the intestine was directly exposed to furosemide, GABA, GABA-A receptor agonist (muscimol), or muscarinic receptor antagonist (atropine). Furosemide (50, 100 and 500 μmol L -1 ) acutely reduced the amplitude of ileal and colonic contraction. In the ileum, 1 mmol L -1 GABA and 10-60 μmol L -1 muscimol significantly increased the amplitude, whereas in the colon, 50-100 mmol L -1 GABA and 60 μmol L -1 muscimol decreased the contractions. The contractions were also significantly suppressed by atropine. To investigate the mechanisms underlying the inhibiting effect of furosemide, furosemide was added to the organ bath prior to the addition of muscimol or atropine. A comparison of furosemide combined with muscimol or atropine group and furosemide group showed no significant difference of the ileal contraction, but the amplitude of colonic contraction significantly decreased when compared to adding furosemide alone. These results suggest that furosemide can reduce the ileal and proximal colonic contraction mediated by blocking and supporting of GABA-A receptor, respectively, resulting in decreased acetylcholine release. © 2017 John Wiley & Sons Australia, Ltd.

Role of the amygdala GABA-A receptors in ACPA-induced deficits during conditioned fear learning.

PubMed

Nasehi, Mohammad; Roghani, Farnaz; Ebrahimi-Ghiri, Mohaddeseh; Zarrindast, Mohammad-Reza

2017-05-01

The basolateral amygdala (BLA) is a key structure for the emotional processing and storage of memories associated with emotional events, especially fear. On the other hand, endocannabinoids and CB1 receptors play a key role in learning and memory partly through long-term synaptic depression of GABAergic synapses in the BLA. The aim of this study was to explore the effects of GABA-A receptor agonist and antagonist in the fear-related memory acquisition deficits induced by ACPA (a selective CB1 cannabinoid receptor agonist). This study used context and tone fear conditioning paradigms to assess fear-related memory in male NMRI mice. Our results showed that the pre-training intraperitoneal administration of ACPA (0.5mg/kg) or (0.1 and 0.5mg/kg) decreased the percentage of freezing time in the contextual and tone fear conditioning, respectively. This indicated an impaired context- or tone-dependent fear memory acquisition. Moreover, the pre-training intra-BLA microinjection of GABA-A receptor agonist, muscimol, at 0.05 and 0.5μg/mouse impaired context-dependent fear memory, while the same doses of GABA-A antagonist, bicuculline, impaired tone-dependent fear memory. However, a subthreshold dose of muscimol or bicuculline increased the effect of ACPA at 0.1 and 0.5 or 0.05mg/kg on context- or tone-dependent fear memory, respectively. In addition, bicuculline at the lower dose increased the ACPA response on locomotor activity compared to its respective group. Such findings highlighted an interaction between BLA GABAergic and cannabinoidergic systems during the acquisition phase of conditioned fear memories. Copyright © 2017 Elsevier Inc. All rights reserved.

The Use of Benzodiazepine Receptor Agonists and Risk of Respiratory Failure in Patients with Chronic Obstructive Pulmonary Disease: A Nationwide Population-Based Case-Control Study.

PubMed

Chen, Su-Jung; Yeh, Chiu-Mei; Chao, Tze-Fan; Liu, Chia-Jen; Wang, Kang-Ling; Chen, Tzeng-Ji; Chou, Pesus; Wang, Fu-Der

2015-07-01

Insomnia is prevalent in patients with chronic obstructive pulmonary disease (COPD), and benzodiazepine receptor agonists (BZRAs) are the most commonly used drugs despite their adverse effects on respiratory function. The aim of this study was to investigate whether the use of BZRAs was associated with an increased risk of respiratory failure (RF) in COPD patients. Matched case-control study. National Health Insurance Research Database (NHIRD) in Taiwan. The case group consisted of 2,434 COPD patients with RF, and the control group consisted of 2,434 COPD patients without RF, matched for age, sex, and date of enrollment. Exposure to BZRAs during the 180-day period preceding the index date was analyzed and compared in the case and control groups. Conditional logistic regression was performed, and the use of BZRAs was associated with an increased risk of RF (adjusted odds ratio [aOR] 1.56, 95% confidence interval [CI] 1.14-2.13). In subgroup analysis, we found that the benzodiazepine (BZD) users had a higher risk of RF (aOR 1.58, 95% CI 1.14-2.20), whereas the risk in non-benzodiazepine (non-BZD) users was insignificant (aOR 0.85, 95% CI 0.51-1.44). A greater than 2-fold increase in risk was found in those who received two or more kinds of BZRAs and those using a combination of BZD and non-BZD medications. The use of benzodiazepine receptor agonists was a significant risk factor for respiratory failure in patients with chronic obstructive pulmonary disease (COPD). Compared to benzodiazepine, the prescription of non-benzodiazepine may be safer for the management of insomnia in COPD patients. © 2015 Associated Professional Sleep Societies, LLC.

Influence of age, body temperature, GABAA receptor inhibition and caffeine on the Hering-Breuer inflation reflex in unanesthetized rat pups

PubMed Central

Arnal, Ashley V.; Gore, Julie L.; Rudkin, Alison; Bartlett, Donald; Leiter, J.C.

2013-01-01

We measured the duration of apnea induced by sustained end-inspiratory lung inflation (the Hering Breuer Reflex; HBR) in unanesthetized infant rat pups aged 4 days (P4) to P20 at body temperatures of 32°C and 36°C. The expiratory prolongation elicited by the HBR lasted longer in the younger pups and lasted longer at the higher body temperature. Blockade of adenosine receptors by caffeine following injection into the cisterna magna (ICM) significantly blunted the thermal prolongation of the HBR. Blockade of gama-amino-butyric acid A (GABAA) receptors by pre-treatment with ICM bicuculline had no effect on the HBR duration at either body temperature. To test the hypothesis that developmental maturation of GABAergic inhibition of breathing was modifying the response to bicuculline, we pretreated rat pups with systemically administered bumetanide to lower the intracellular chloride concentration, and repeated the bicuculline studies. Bicuculline still did not alter the HBR at either temperature after bumetanide treatment. We administered PSB-36, a selective adenosine A1 receptor antagonist, and this drug treatment did not modify the HBR. We conclude that caffeine blunts the thermal prolongation of the HBR, probably by blocking adenosine A2a receptors. The thermally-sensitive adenosinergic prolongation of the HBR in these intact animals does not seem to depend on GABAA receptors PMID:23318703

Astrocytes Modulate a Postsynaptic NMDA–GABAA-Receptor Crosstalk in Hypothalamic Neurosecretory Neurons

PubMed Central

Potapenko, Evgeniy S.; Biancardi, Vinicia C.; Zhou, Yiqiang

2013-01-01

A dynamic balance between the excitatory and inhibitory neurotransmitters glutamate and GABA is critical for maintaining proper neuronal activity in the brain. This balance is partly achieved via presynaptic interactions between glutamatergic and GABAAergic synapses converging into the same targets. Here, we show that in hypothalamic magnocellular neurosecretory neurons (MNCs), a direct crosstalk between postsynaptic NMDA receptors (NMDARs) and GABAA receptors (GABAARs) contributes to the excitatory/inhibitory balance in this system. We found that activation of NMDARs by endogenous glutamate levels controlled by astrocyte glutamate transporters, evokes a transient and reversible potentiation of postsynaptic GABAARs. This inter-receptor crosstalk is calcium-dependent and involves a kinase-dependent phosphorylation mechanism, but does not require nitric oxide as an intermediary signal. Finally, we found the NMDAR–GABAAR crosstalk to be blunted in rats with heart failure, a pathological condition in which the hypothalamic glutamate–GABA balance is tipped toward an excitatory predominance. Together, our findings support a novel form of glutamate–GABA interactions in MNCs, which involves crosstalk between NMDA and GABAA postsynaptic receptors, whose strength is controlled by the activity of local astrocytes. We propose this inter-receptor crosstalk to act as a compensatory, counterbalancing mechanism to dampen glutamate-mediated overexcitation. Finally, we propose that an uncoupling between NMDARs and GABAARs may contribute to exacerbated neuronal activity and, consequently, sympathohumoral activation in such disease conditions as heart failure. PMID:23303942

Mechanism of action of the insecticides, lindane and fipronil, on glycine receptor chloride channels

PubMed Central

Islam, Robiul; Lynch, Joseph W

2012-01-01

BACKGROUND AND PURPOSE Docking studies predict that the insecticides, lindane and fipronil, block GABAA receptors by binding to 6′ pore-lining residues. However, this has never been tested at any Cys-loop receptor. The neurotoxic effects of these insecticides are also thought to be mediated by GABAA receptors, although a recent morphological study suggested glycine receptors mediated fipronil toxicity in zebrafish. Here we investigated whether human α1, α1β, α2 and α3 glycine receptors were sufficiently sensitive to block by either compound as to represent possible neurotoxicity targets. We also investigated the mechanisms by which lindane and fipronil inhibit α1 glycine receptors. EXPERIMENTAL APPROACH Glycine receptors were recombinantly expressed in HEK293 cells and insecticide effects were studied using patch-clamp electrophysiology. KEY RESULTS Both compounds completely inhibited all tested glycine receptor subtypes with IC50 values ranging from 0.2–2 µM, similar to their potencies at vertebrate GABAA receptors. Consistent with molecular docking predictions, both lindane and fipronil interacted with 6′ threonine residues via hydrophobic interactions and hydrogen bonds. In contrast with predictions, we found no evidence for lindane interacting at the 2′ level. We present evidence for fipronil binding in a non-blocking mode in the anaesthetic binding pocket, and for lindane as an excellent pharmacological tool for identifying the presence of β subunits in αβ heteromeric glycine receptors. CONCLUSIONS AND IMPLICATIONS This study implicates glycine receptors as novel vertebrate toxicity targets for fipronil and lindane. Furthermore, lindane interacted with pore-lining 6′ threonine residues, whereas fipronil may have both pore and non-pore binding sites. PMID:22035056

Mechanism of action of the insecticides, lindane and fipronil, on glycine receptor chloride channels.

PubMed

Islam, Robiul; Lynch, Joseph W

2012-04-01

Docking studies predict that the insecticides, lindane and fipronil, block GABA(A) receptors by binding to 6' pore-lining residues. However, this has never been tested at any Cys-loop receptor. The neurotoxic effects of these insecticides are also thought to be mediated by GABA(A) receptors, although a recent morphological study suggested glycine receptors mediated fipronil toxicity in zebrafish. Here we investigated whether human α1, α1β, α2 and α3 glycine receptors were sufficiently sensitive to block by either compound as to represent possible neurotoxicity targets. We also investigated the mechanisms by which lindane and fipronil inhibit α1 glycine receptors. Glycine receptors were recombinantly expressed in HEK293 cells and insecticide effects were studied using patch-clamp electrophysiology. Both compounds completely inhibited all tested glycine receptor subtypes with IC(50) values ranging from 0.2-2 µM, similar to their potencies at vertebrate GABA(A) receptors. Consistent with molecular docking predictions, both lindane and fipronil interacted with 6' threonine residues via hydrophobic interactions and hydrogen bonds. In contrast with predictions, we found no evidence for lindane interacting at the 2' level. We present evidence for fipronil binding in a non-blocking mode in the anaesthetic binding pocket, and for lindane as an excellent pharmacological tool for identifying the presence of β subunits in αβ heteromeric glycine receptors. This study implicates glycine receptors as novel vertebrate toxicity targets for fipronil and lindane. Furthermore, lindane interacted with pore-lining 6' threonine residues, whereas fipronil may have both pore and non-pore binding sites. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

GABA/benzodiazepine receptor complex in long-sleep and short-sleep mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marley, R.J.

LS mice are more sensitive to benzodiazepine-induced anesthesia; however, the two lines do not differ in their hypothermic response to flurazepam. SS mice are more resistant to 3-mercaptopropionic acid-induced seizures and more sensitive to the anticonvulsant effects of benzodiazepines. The various correlates of GABA and benzodiazepine actions probably are the results of different mechanisms of action and/or differential regional control. Bicuculline competition for /sup 3/H-GABA binding sites is greater in SS cerebellar tissue and /sup 3/H-flunitrazepam binding is greater in the mid-brain region of LS mice. GABA enhancement of /sup 3/H-flunitrazepma binding is greater in SS mice. Ethanol also enhancesmore » /sup 3/H-flunitrazepam binding and increases the levels of /sup 3/H-flunitrazepam binding above those observed for GABA. Using correlational techniques on data from LS and SS mice and several inbred mouse strains, it was demonstrated that a positive relationship exists between the degree of receptor coupling within the GABA receptor complex and the degree of resistance to seizures.« less

Human α1β3γ2L gamma-aminobutyric acid type A receptors: High-level production and purification in a functional state.

PubMed

Dostalova, Zuzana; Zhou, Xiaojuan; Liu, Aiping; Zhang, Xi; Zhang, Yinghui; Desai, Rooma; Forman, Stuart A; Miller, Keith W

2014-02-01

Gamma-aminobutyric acid type A receptors (GABA(A)Rs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA(A)Rs determine their function and pharmacological profile. GABAA Rs are heteropentamers of subunits, and (α1)2 (β3)2 (γ2L)1 is a common subtype. Biochemical and biophysical studies of GABA(A)Rs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high-level production of active human α1β3 GABA(A)R using tetracycline-inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline-inducible HEK293-TetR cell line expressing human (N)-FLAG-α1β3γ2L-(C)-(GGS)3 GK-1D4 GABA(A)R. These cells achieved expression levels of 70-90 pmol [(3)H]muscimol binding sites/15-cm plate at a specific activity of 15-30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [(3)H]flunitrazepam to [(3)H]muscimol binding sites and sensitivity of GABA-induced currents to benzodiazepines and zinc. The α1β3γ2L GABA(A)Rs were solubilized in dodecyl-D-maltoside, purified by anti-FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ∼ 30%. Typical purifications yielded 1.0-1.5 nmoles of [(3)H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [(3)H]muscimol binding were maintained in the purified state. © 2013 The Protein Society.

GABAB receptor modulation of the release of substance P from capsaicin-sensitive neurones in the rat trachea in vitro.

PubMed Central

Ray, N. J.; Jones, A. J.; Keen, P.

1991-01-01

1. The role of gamma-aminobutyric acid (GABA) as an inhibitory transmitter in the central nervous system is well documented. Recently, GABAA and GABAB receptors have been identified in the peripheral nervous system, notably on primary afferent neurones (PAN). We have utilised a multi-superfusion system to investigate the effect of selective GABA receptor agonists and antagonists on the release of substance P (SP) from the rat trachea in vitro. 2. GABA (1-100 microM) did not affect spontaneous release of SP-like immunoreactivity (LI) but caused dose-related inhibition of calcium-dependent potassium (60 mM)-stimulated SP-LI release. The greatest inhibition of 77.7 +/- 18.8% was observed at 100 microM. 3. The inhibitory effect of GABA was mimicked by the GABAB receptor agonist, (+/-)-baclofen (1-100 microM), but not the GABAA receptor agonist, 3-amino-1-propane-sulphonic acid (3-APS, 1-100 microM). Baclofen (100 microM) had no effect on SP-LI release stimulated by capsaicin (1 microM). 4. The inhibitory effect of baclofen (30 microM) was significantly reduced by prior and concomitant exposure to the GABAB receptor antagonist, phacolofen (100 microM) but not the GABAA receptor antagonist, bicuculline (10 microM). Neither antagonist, alone, affected spontaneous or potassium-stimulated SP-LI release. 5. We conclude that activation of pre-synaptic GABAB receptors on the peripheral termini of PANs in the rat trachea inhibits SP-LI release and suggest that GABAB receptor agonists may be of value in the therapeutic treatment of asthma. PMID:1713105

Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lummis, S.C.R.; Johnston, G.A.R.; Nicoletti, G.

1991-01-01

Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E. coli. The bacterial ({sup 3}H)diazepam binding sites are modulated by GABA; the modulation is dose dependent and is reduced at high concentrations. The most potent competitors of E.Coli ({sup 3}H)diazepam binding are those that are active in displacing ({sup 3}H)benzodiazepines from vertebrate peripheral benzodiazepine binding sites. These vertebrate sites are not modulated by GABA, in contrast to vertebrate neuronal benzodiazepine binding sites. The E.coli benzodiazepine binding sites therefore differ from both classes of vertebrate benzodiazepine binding sites; however the ligandmore » spectrum and GABA-modulatory properties of the E.coli sites are similar to those found in insects. This intermediate type of receptor in lower species suggests a precursor for at least one class of vertebrate benzodiazepine binding sites may have existed.« less

Chronic intermittent ethanol exposure produces persistent anxiety in adolescent and adult rats.

PubMed

Van Skike, Candice E; Diaz-Granados, Jaime L; Matthews, Douglas B

2015-02-01

Ethanol (EtOH) dependence and tolerance in the adult are marked by increased function of NMDA receptors and decreased function of GABAA receptors, which coincide with altered receptor subunit expression in specific brain regions. Adolescents often use EtOH at levels greater than adults, yet the receptor subunit expression profiles following chronic intermittent EtOH (CIE) exposure in adolescents are not known. Persistent age-dependent changes in receptor subunit alterations coupled with withdrawal-related anxiety may help explain the increase in alcohol abuse following adolescent experimentation with the drug. Adolescent and adult rats received 10 intraperitoneal administrations of 4.0 g/kg EtOH or saline every 48 hours. At either 24 hours or 12 days after the final exposure, anxiety-like behavior was assessed on the elevated plus maze and tissue was collected. Western blotting was used to assess changes in selected NMDA and GABAA receptor subunits in whole cortex and bilateral hippocampus. CIE exposure yields a persistent increase in anxiety-like behavior in both age groups. However, selected NMDA and GABAA receptor subunits were not differentially altered by this CIE exposure paradigm in adolescents or adults. CIE exposure produced persistent anxiety-like behavior, which has important implications for alcohol cessation. Given the reported behavioral and neuropeptide expression changes in response to this dose of EtOH, it is important for future work to consider the circumstances under which these measures are altered by EtOH exposure. Copyright © 2015 by the Research Society on Alcoholism.

Context-Dependent Modulation of αβγ and αβγ GABAA Receptors by Penicillin: Implications for Phasic and Tonic Inhibition

PubMed Central

Feng, Hua-Jun; Botzolakis, Emmanuel J.; Macdonald, Robert L.

2009-01-01

Summary Penicillin, an open-channel blocker of GABAA receptors, was recently reported to inhibit phasic, but not tonic, currents in hippocampal neurons. To distinguish between isoform-specific and context-dependent modulation as possible explanations for this selectivity, the effects of penicillin were evaluated on recombinant GABAA receptors expressed in HEK293T cells. When co-applied with saturating GABA, penicillin decreased peak amplitude, induced rebound, and prolonged deactivation of currents evoked from both synaptic and extrasynaptic receptor isoforms. However, penicillin had isoform-specific effects on the extent of desensitization, reflecting its ability to differentially modulate peak (non-equilibrium) and residual (near-equilibrium) currents. This suggested that the context of activation could determine the apparent sensitivity of a given receptor isoform to penicillin. To test this hypothesis, we explored the ability of penicillin to modulate synaptic and extrasynaptic isoforms that were activated under more physiologically relevant conditions. Interestingly, while currents evoked from synaptic isoforms under phasic conditions (transient activation by a saturating concentration of GABA) were substantially inhibited by penicillin, currents evoked from extrasynaptic isoforms under tonic conditions (prolonged application by a sub-saturating concentration of GABA) were minimally affected. We therefore concluded that the reported inability of penicillin to modulate tonic currents could not simply be attributed to insensitivity of extrasynaptic receptors, but rather, reflected an inability to modulate these receptors in their native context of activation. PMID:18775733

Inhibition of Orexin Signaling Promotes Sleep Yet Preserves Salient Arousability in Monkeys.

PubMed

Tannenbaum, Pamela L; Tye, Spencer J; Stevens, Joanne; Gotter, Anthony L; Fox, Steven V; Savitz, Alan T; Coleman, Paul J; Uslaner, Jason M; Kuduk, Scott D; Hargreaves, Richard; Winrow, Christopher J; Renger, John J

2016-03-01

In addition to enhancing sleep onset and maintenance, a desirable insomnia therapeutic agent would preserve healthy sleep's ability to wake and respond to salient situations while maintaining sleep during irrelevant noise. Dual orexin receptor antagonists (DORAs) promote sleep by selectively inhibiting wake-promoting neuropeptide signaling, unlike global inhibition of central nervous system excitation by gamma-aminobutyric acid (GABA)-A receptor (GABAaR) modulators. We evaluated the effect of DORA versus GABAaR modulators on underlying sleep architecture, ability to waken to emotionally relevant stimuli versus neutral auditory cues, and performance on a sleepiness-sensitive cognitive task upon awakening. DORA-22 and GABAaR modulators (eszopiclone, diazepam) were evaluated in adult male rhesus monkeys (n = 34) with continuous polysomnography recordings in crossover studies of sleep architecture, arousability to a classically conditioned salient versus neutral acoustical stimulus, and psychomotor vigilance task (PVT) performance if awakened. All compounds decreased wakefulness, but only DORA-22 sleep resembled unmedicated sleep in terms of underlying sleep architecture, preserved ability to awaken to salient-conditioned acoustic stimuli while maintaining sleep during neutral acoustic stimuli, and no congnitive impairment in PVT performance. Although GABAaR modulators induced lighter sleep, monkeys rarely woke to salient stimuli and PVT performance was impaired if monkeys were awakened. In nonhuman primates, DORAs' targeted mechanism for promoting sleep protects the ability to selectively arouse to salient stimuli and perform attentional tasks unimpaired, suggesting meaningful differentiation between a hypnotic agent that works through antagonizing orexin wake signaling versus the sedative hypnotic effects of the GABAaR modulator mechanism of action. © 2016 Associated Professional Sleep Societies, LLC.

Peripheral benzodiazepine receptors are decreased during cocaine withdrawal in humans.

PubMed

Javaid, J I; Notorangelo, M P; Pandey, S C; Reddy, P L; Pandey, G N; Davis, J M

1994-07-01

In the present study, homovanillic acid in plasma (pHVA) and benzodiazepine receptors (3H-PK11195 binding) in neutrophil membranes were determined in blood obtained from cocaine-dependent (DSM-III-R) adult male inpatients at baseline-(within 72 hr of last cocaine use) and after 3 weeks of cocaine abstinence, and normal controls. The mean (+/- SEM) pHVA at baseline (10.3 ng/ml +/- 1.1) was similar to normals and did not change after 3 weeks of cocaine abstinence. Similarly, the binding indices of benzodiazepine receptors in cocaine-dependent subjects as a group were not significantly different than in normal controls. In 10 cocaine-dependent subjects, however, where both blood samples were available, the number of 3H-PK11195 binding sites was significantly (p < 0.05) decreased after 3 weeks of cocaine abstinence (mean +/- sem: Bmax = 6371 +/- 657 fmol/mg protein) compared with baseline (Bmax = 7553 +/- 925 fmol/mg protein), although there were no differences in the binding affinity (mean +/- sem: KD = 8.6 +/- 1.2 nmol/L after 3 weeks of abstinence compared with 8.1 +/- 1.0 nmol/L at baseline). These preliminary results suggest that peripheral benzodiazepine receptors may play an important role in the pathophysiology of cocaine withdrawal in cocaine-dependent human subjects.

Neurosteroids for the potential protection of humans against organophosphate toxicity.

PubMed

Reddy, Doodipala Samba

2016-08-01

This article describes the therapeutic potential of neurosteroids as anticonvulsant antidotes for chemical intoxication caused by organophosphate pesticides and nerve agents or gases like sarin and soman. Toxic manifestations following nerve agent exposure, as evident in chemical attacks in Japan and Syria, include hypersecretion, respiratory distress, tremors, convulsions leading to status epilepticus (SE), and death. Benzodiazepines, such as diazepam, are the current anticonvulsants of choice for controlling nerve agent-induced life-threatening seizures, SE, and brain injury. Benzodiazepines can control acute seizures when given early, but they are less effective for delayed treatment of SE, which is characterized by rapid desensitization of synaptic GABA A receptors, benzodiazepine resistance, and brain injury. Neurosteroid-sensitive extrasynaptic GABA A receptors, however, remain unaffected by such events. Thus, anticonvulsant neurosteroids may produce more effective protection than benzodiazepines against a broad spectrum of chemical agents, even when given late after nerve agent exposure. © 2016 New York Academy of Sciences.

Benzodiazepine sensitivity in normal human subjects.

PubMed

Hommer, D W; Matsuo, V; Wolkowitz, O; Chrousos, G; Greenblatt, D J; Weingartner, H; Paul, S M

1986-06-01

Increasing intravenous doses of diazepam or placebo were administered to ten healthy normal volunteers, and the changes in saccadic eye velocity, self-rated sedation and anxiety, and plasma cortisol and growth hormone concentrations were measured. Diazepam administration (4.4 to 140 micrograms/kg, cumulative dose) resulted in a dose-dependent decrease in saccadic eye velocity and plasma cortisol level as well as a dose-dependent increase in self-rated sedation and plasma growth hormone level. Self-rated anxiety was unaffected in these relatively nonanxious subjects. The diazepam-induced changes in saccadic eye velocity, sedation, and growth hormone and cortisol levels were highly correlated with each other and with increasing plasma diazepam concentration. These results are consistent with a benzodiazepine receptor-mediated action of diazepam. The highly quantifiable and dose-dependent decrease in saccadic eye velocity by benzodiazepines should make this a useful measure of benzodiazepine receptor sensitivity in humans.

GABA-independent GABAA Receptor Openings Maintain Tonic Currents

PubMed Central

Wlodarczyk, Agnieszka I.; Sylantyev, Sergiy; Herd, Murray B.; Kersanté, Flavie; Lambert, Jeremy J.; Rusakov, Dmitri A.; Linthorst, Astrid C.E.; Semyanov, Alexey; Belelli, Delia; Pavlov, Ivan; Walker, Matthew C.

2013-01-01

Activation of GABAA receptors (GABAARs) produces two forms of inhibition: ‘phasic’ inhibition generated by the rapid, transient activation of synaptic GABAARs by presynaptic GABA release, and tonic inhibition generated by the persistent activation of peri- or extrasynaptic GABAARs which can detect extracellular GABA. Such tonic GABAAR-mediated currents are particularly evident in dentate granule cells in which they play a major role in regulating cell excitability. Here we show that in rat dentate granule cells in ex-vivo hippocampal slices, tonic currents are predominantly generated by GABA-independent GABAA receptor openings. This tonic GABAAR conductance is resistant to the competitive GABAAR antagonist SR95531, which at high concentrations acts as a partial agonist, but can be blocked by an open channel blocker picrotoxin. When slices are perfused with 200 nM GABA, a concentration that is comparable to cerebrospinal fluid concentrations but is twice that measured by us in the hippocampus in vivo using zero-net-flux microdialysis, negligible GABA is detected by dentate granule cells. Spontaneously opening GABAARs, therefore, maintain dentate granule cell tonic currents in the face of low extracellular GABA concentrations. PMID:23447601

Insomnia medication use and the probability of an accidental event in an older adult population

PubMed Central

Avidan, Alon Y; Palmer, Liisa A; Doan, Justin F; Baran, Robert W

2010-01-01

Objective: This study examined the risk of accidental events in older adults prescribed a sedating antidepressant, long-acting benzodiazepine, short-acting benzodiazepine, and nonbenzodiazepine, relative to a reference group (selective melatonin receptor agonist). Methods: This was a retrospective cohort analysis of older adults (≥65 years) with newly initiated pharmacological treatment of insomnia. Data were collected from the Thomson MarketScan® Medicare Supplemental and Coordination of Benefits databases (January 1, 2000, through June 30, 2006). Probit models were used to evaluate the probability of an accidental event. Results: Data were analyzed for 445,329 patients. Patients taking a long-acting benzodiazepine (1.21 odds ratio [OR]), short-acting benzodiazepine (1.16 OR), or nonbenzodiazepine (1.12 OR) had a significantly higher probability of experiencing an accidental event during the first month following treatment initiation compared with patients taking the reference medication (P < 0.05 for all). A significantly higher probability of experiencing an accidental event was also observed during the 3-month period following the initiation of treatment (1.62 long-acting benzodiazepine, 1.60 short-acting benzodiazepine, 1.48 nonbenzodiazepine, and 1.56 sedating antidepressant; P < 0.05). Conclusions: Older adults taking an SAD or any of the benzodiazepine receptor agonists appear to have a greater risk of an accidental event compared with a reference group taking an MR. PMID:21701634

Etomidate blocks LTP and impairs learning but does not enhance tonic inhibition in mice carrying the N265M point mutation in the beta3 subunit of the GABAA receptor

PubMed Central

Oh, I; Rau, V; Lor, C; Laha, KT; Jurd, R; Rudolph, U; Eger, EI; Pearce, RA

2015-01-01

Enhancement of tonic inhibition mediated by extrasynaptic α5-subunit containing GABAA receptors (GABAARs) has been proposed as the mechanism by which a variety of anesthetics, including the general anesthetic etomidate, impair learning and memory. Since α5 subunits preferentially partner with β3 subunits, we tested the hypothesis that etomidate acts through β3-subunit containing GABAARs to enhance tonic inhibition, block LTP, and impair memory. We measured the effects of etomidate in wild type mice and in mice carrying a point mutation in the GABAAR β3-subunit (β3-N265M) that renders these receptors insensitive to etomidate. Etomidate enhanced tonic inhibition in CA1 pyramidal cells of the hippocampus in wild type but not in mutant mice, demonstrating that tonic inhibition is mediated by β3-subunit containing GABAARs. However, despite its inability to enhance tonic inhibition, etomidate did block LTP in brain slices from mutant mice as well as in those from wild type mice. Etomidate also impaired fear conditioning to context, with no differences between genotypes. In studies of recombinant receptors expressed in HEK293 cells, α5β1γ2L GABAARs were insensitive to amnestic concentrations of etomidate (1 [.proportional]M and below), whereas α5β2γ2L and α5β3γ2L GABAARs were enhanced. We conclude that etomidate enhances tonic inhibition in pyramidal cells through its action on α5β3-containing GABAA receptors, but blocks LTP and impairs learning by other means - most likely by modulating α5β2-containing GABAA receptors. The critical anesthetic targets underlying amnesia might include other forms of inhibition imposed on pyramidal neurons (e.g. slow phasic inhibition), or inhibitory processes on non-pyramidal cells (e.g. interneurons). PMID:25680234

THE ROLE OF DELTA OPIOID RECEPTORS IN THE ANXIOLYTIC ACTIONS OF BENZODIAZEPINES

PubMed Central

Primeaux, Stefany D.; Wilson, Steven P.; McDonald, Alexander J.; Mascagni, Franco; Wilson, Marlene A.

2007-01-01

The anxiolytic effects of benzodiazepines appear to involve opioid processes in the amygdala. In previous experiments, overexpression of enkephalin in the amygdala enhanced the anxiolytic actions of the benzodiazepine agonist diazepam in the elevated plus maze. The effects of systemically administered diazepam are also blocked by injections of naltrexone into the central nucleus of the amygdala. The current studies investigated the role of delta opioid receptors in the anxiety-related effects of diazepam. Three days following bilateral stereotaxic injections of viral vectors containing cDNA encoding proenkephalin or β-galactosidase (control vector), the delta opioid receptor antagonist naltrindole (10 mg/kg, s.c.) attenuated the enhanced anxiolytic effects of 1–2 mg/kg diazepam in rats overexpressing preproenkephalin in the amygdala. Despite this effect, naltrindole failed to attenuate the anxiolytic action of higher diazepam doses (3 mg/kg) in animals with normal amygdalar enkephalin expression. Similarly, the mu opioid receptor antagonist, β-funaltrexamine (20mg/kg, sc), had no effect on the anxiolytic effect of diazepam alone. These data support a role for delta opioid receptors in the opioid-enhanced anxiolytic effects of diazepam. PMID:17109943

Evidence that GABA ρ subunits contribute to functional ionotropic GABA receptors in mouse cerebellar Purkinje cells

PubMed Central

Harvey, Victoria L; Duguid, Ian C; Krasel, Cornelius; Stephens, Gary J

2006-01-01

Ionotropic γ-amino butyric acid (GABA) receptors composed of heterogeneous molecular subunits are major mediators of inhibitory responses in the adult CNS. Here, we describe a novel ionotropic GABA receptor in mouse cerebellar Purkinje cells (PCs) using agents reported to have increased affinity for ρ subunit-containing GABAC over other GABA receptors. Exogenous application of the GABAC-preferring agonist cis-4-aminocrotonic acid (CACA) evoked whole-cell currents in PCs, whilst equimolar concentrations of GABA evoked larger currents. CACA-evoked currents had a greater sensitivity to the selective GABAC antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) than GABA-evoked currents. Focal application of agonists produced a differential response profile; CACA-evoked currents displayed a much more pronounced attenuation with increasing distance from the PC soma, displayed a slower time-to-peak and exhibited less desensitization than GABA-evoked currents. However, CACA-evoked currents were also completely blocked by bicuculline, a selective agent for GABAA receptors. Thus, we describe a population of ionotropic GABA receptors with a mixed GABAA/GABAC pharmacology. TPMPA reduced inhibitory synaptic transmission at interneurone–Purkinje cell (IN–PC) synapses, causing clear reductions in miniature inhibitory postsynaptic current (mIPSC) amplitude and frequency. Combined application of NO-711 (a selective GABA transporter subtype 1 (GAT-1) antagonist) and SNAP-5114 (a GAT-(2)/3/4 antagonist) induced a tonic GABA conductance in PCs; however, TPMPA had no effect on this current. Immunohistochemical studies suggest that ρ subunits are expressed predominantly in PC soma and proximal dendritic compartments with a lower level of expression in more distal dendrites; this selective immunoreactivity contrasted with a more uniform distribution of GABAA α1 subunits in PCs. Finally, co-immunoprecipitation studies suggest that ρ subunits can form complexes with GABAA receptor α1 subunits in the cerebellar cortex. Overall, these data suggest that ρ subunits contribute to functional ionotropic receptors that mediate a component of phasic inhibitory GABAergic transmission at IN–PC synapses in the cerebellum. PMID:16945976

Modifications of Antiepileptic Drugs for Improved Tolerability and Efficacy

PubMed Central

Landmark, Cecilie Johannessen; Johannessen, Svein I.

2008-01-01

Introduction A large number of antiepileptic drugs (AEDs) are available today, but they may not be satisfactory regarding clinical efficacy, tolerance, toxicity or pharmacokinetic properties. The purpose of this review is to focus upon the rationale behind the chemical modifications of several recently marketed AEDs or drugs in development and to categorize them according to the main purposes for the improvements: better efficacy or tolerability accompanied by improved pharmacokinetic properties. Material and Method AEDs that have been chemically modified to new derivatives during the last years are reviewed based on recent publications and PubMed-searches. Results and Discussion Improvement in pharmacokinetic parameters may affect both tolerability and efficacy. Modifications to improve tolerability include various valproate analogues, divided into aliphatic amides, cyclic derivatives or amino acid conjugates. Furthermore, there are the carbamazepine analogues oxcarbazepine and eslicarbazepine, the felbamate analogues fluorofelbamate and carisbamate (RWJ 33369), and the lamotrigine analogue JZP-4. The levetiracetam analogues brivaracetam and seletracetam and the derivatives of gabapentin, pregabalin and XP13512, have improved selectivity compared to their parent compounds. Other new drugs have new mechanisms of action related to GABA and glutamate receptors; the glutamate antagonists like topiramate (talampanel and NS-1209), and GABAA receptor agonists, benzodiazepine or progesterone analogues (ELB-139 and ganaxolone). Conclusion Further challenges for development of new AEDs include investigations of target molecules affected by pathophysiological processes and detailed structure-activity relationships with focus on stereoselectivity. These potential drugs may become of importance in future drug therapy in epilepsy and other CNS disorders. PMID:19787095

Identification and cloning of a gamma 3 subunit splice variant of the human GABA(A) receptor.

PubMed

Poulsen, C F; Christjansen, K N; Hastrup, S; Hartvig, L

2000-05-31

cDNA sequences encoding two forms of the GABA(A) gamma 3 receptor subunit were cloned from human hippocampus. The nucleotide sequences differ by the absence (gamma 3S) or presence (gamma 3L) of 18 bp located in the presumed intracellular loop between transmembrane region (TM) III and IV. The extra 18 bp in the gamma 3L subunit generates a consensus site for phosphorylation by protein kinase C (PKC). Analysis of human genomic DNA encoding the gamma 3 subunit reveals that the 18 bp insert is contiguous with the upstream proximal exon.

Characteristics of concatemeric GABAA receptors containing α4/δ subunits expressed in Xenopus oocytes

PubMed Central

Shu, Hong-Jin; Bracamontes, John; Taylor, Amanda; Wu, Kyle; Eaton, Megan M; Akk, Gustav; Manion, Brad; Evers, Alex S; Krishnan, Kathiresan; Covey, Douglas F; Zorumski, Charles F; Steinbach, Joe Henry; Mennerick, Steven

2012-01-01

BACKGROUND AND PURPOSE GABAA receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABAA receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems. EXPERIMENTAL APPROACH We engineered concatemeric (fused) subunits to ensure δ and α4 subunit expression. We tested the pharmacology of the concatemeric receptors, compared with a common synaptic-like receptor subunit combination (α1 +β2 +γ2L), and with free-subunit α4/δ receptors, expressed in Xenopus oocytes. KEY RESULTS δ-β2 −α4 +β2-α4 cRNA co-injected into Xenopus oocytes resulted in GABA-gated currents with the expected pharmacological properties of α4/δ-containing receptors. Criteria included sensitivity to agonists of different efficacy, sensitivity to the allosteric activator pentobarbital, and modulation of agonist responses by DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide; a δ-selective positive modulator), furosemide, and Zn2+. We used the concatemers to examine neurosteroid sensitivity of extrasynaptic-like, δ-containing receptors. We found no qualitative differences between extrasynaptic-like receptors and synaptic-like receptors in the actions of either negative or positive neurosteroid modulators of receptor function. Quantitative differences were explained by the partial agonist effects of the natural agonist GABA and by a mildly increased sensitivity to low steroid concentrations. CONCLUSIONS AND IMPLICATIONS The neurosteroid structure-activity profile for α4/δ-containing extrasynaptic receptors is unlikely to differ from that of synaptic-like receptors such as α1/β2/γ2-containing receptors. PMID:21950777

Chronic stress in Lizards: Studies on the Behavior and Benzodiazepine Receptors in Liolaemus koslowskyi and Cnemidophorus tergolaevigatus.

PubMed

Soloaga, Alejandra; Pueta, Mariana; Cruz, Félix Benjamín; Kembro, Jackelyn Melissa; Marin, Raul Hector

2016-12-01

Behavioral and physiological adaptive responses of animals facing chronic exposure to a single stressor may allow them to overcome its negative effects for future exposures to similar stressful situations. At chemical level, the GABA A /benzodiazepine complex is considered one of the main receptor systems involved in the modulation of stress-induced responses. Here, we describe the behavioral responses of two different lizard species, Liolaemus koslowskyi and Cnemidophorus tergolaevigatus exposed to three potential chronic stressful treatments: (a) high temperature, (b) forced swimming, and (c) simulated predator. Additionally, we aimed to determine in those lizards whether the central-type benzodiazepine receptor (CBR; an allosteric modulator site of the GABA A receptor) is related to adaptive responses to those stressful stimulations. Our results revealed that the simulated predator was the stress condition that showed the largest difference in behavioral responses between the two species, resembling previously described strategies in nature. The basal affinity of CBRs (obtained from undisturbed animals) showed differences between both species, and the simulated predator was the only stressor that altered the affinity of CBRs. L. koslowskyi CBRs showed a decreased receptor affinity, whereas C. tergolaevigatus showed an increased receptor affinity in comparison to their respective control groups. We show for the first time the effects of different types of stressors upon behavioral responses and CBR biochemical parameters in two lizard species. Our findings suggest a potential GABA/benzodiazepine role in the ability of lizards to cope with a repeated exposure to a stressful (e.g., predator) condition. © 2017 Wiley Periodicals, Inc.

Influence of age, body temperature, GABAA receptor inhibition and caffeine on the Hering-Breuer inflation reflex in unanesthetized rat pups.

PubMed

Arnal, Ashley V; Gore, Julie L; Rudkin, Alison; Bartlett, Donald; Leiter, J C

2013-03-01

We measured the duration of apnea induced by sustained end-inspiratory lung inflation (the Hering Breuer Reflex, HBR) in unanesthetized infant rat pups aged 4 days (P4) to P20 at body temperatures of 32°C and 36°C. The expiratory prolongation elicited by the HBR lasted longer in the younger pups and lasted longer at the higher body temperature. Blockade of adenosine receptors by caffeine following injection into the cisterna magna (ICM) significantly blunted the thermal prolongation of the HBR. Blockade of gama-amino-butyric acid A (GABAA) receptors by pre-treatment with ICM bicuculline had no effect on the HBR duration at either body temperature. To test the hypothesis that developmental maturation of GABAergic inhibition of breathing was modifying the response to bicuculline, we pretreated rat pups with systemically administered bumetanide to lower the intracellular chloride concentration, and repeated the bicuculline studies. Bicuculline still did not alter the HBR at either temperature after bumetanide treatment. We administered PSB-36, a selective adenosine A1 receptor antagonist, and this drug treatment did not modify the HBR. We conclude that caffeine blunts the thermal prolongation of the HBR, probably by blocking adenosine A2a receptors. The thermally sensitive adenosinergic prolongation of the HBR in these intact animals does not seem to depend on GABAA receptors. Copyright © 2013 Elsevier B.V. All rights reserved.

Experiment K-6-18. Study of muscarinic and gaba (benzodiazepine) receptors in the sensory-motor cortex, hippcampus and spinal code

NASA Technical Reports Server (NTRS)

Daunton, N.; Damelio, F.; Krasnov, I.

1990-01-01

Frontal lobe samples of rat brains flown aboard Cosmos 1887 were processed for the study of muscarinic (cholinergic) and GABA (benzodiazepine) receptors and for immunocytochemical localization of the neurotransmitter gamma-aminobutyric acid (GABA) and glial fibrillary acidic protein (GFAP). Although radioactive labeling of both muscarinic cholinergic and GABA (benzodiazepine) receptors proved to be successful with the techniques employed, distinct receptor localization of individual laminae of the frontal neocortex was not possible since the sampling of the area was different in the various groups of animals. In spite of efforts made for proper orientation and regional identification of laminae, it was found that a densitometric (quantitation of autoradiograms) analysis of the tissue did not contribute to the final interpretation of the effects of weightlessness on these receptors. As to the immunocytochemical studies the use of both markers, GFAP and GABA antiserum, confirmed the suitability of the techniques for use in frozen material. However, similar problems to those encountered in the receptor studies prevented an adequate interpretation of the effects of micro-G exposure on the localization and distribution of GABA and GFAP. This study did, however, confirm the feasibility of investigating neurotransmitters and their receptors in future space flight experiments.

Separate and combined effects of the GABAA positive allosteric modulator diazepam and Δ⁹-THC in humans discriminating Δ⁹-THC.

PubMed

Lile, Joshua A; Kelly, Thomas H; Hays, Lon R

2014-10-01

Our previous research suggested the involvement of γ-aminobutyric acid (GABA), in particular the GABAB receptor subtype, in the interoceptive effects of Δ(9)-tetrahydrocannabinol (Δ(9)-THC). The aim of the present study was to determine the potential involvement of the GABAA receptor subtype by assessing the separate and combined effects of the GABAA positive allosteric modulator diazepam and Δ(9)-THC using pharmacologically selective drug-discrimination procedures. Ten cannabis users learned to discriminate 30 mg oral Δ(9)-THC from placebo and then received diazepam (5 and 10mg), Δ(9)-THC (5, 15 and 30 mg) and placebo, alone and in combination. Self-report, task performance and physiological measures were also collected. Δ(9)-THC functioned as a discriminative stimulus, produced subjective effects typically associated with cannabinoids (e.g., High, Stoned, Like Drug) and elevated heart rate. Diazepam alone impaired performance on psychomotor performance tasks and increased ratings on a limited number of self-report questionnaire items (e.g., Any Effect, Sedated), but did not substitute for the Δ(9)-THC discriminative stimulus or alter the Δ(9)-THC discrimination dose-response function. Similarly, diazepam had limited impact on the other behavioral effects of Δ(9)-THC. These results suggest that the GABAA receptor subtype has minimal involvement in the interoceptive effects of Δ(9)-THC, and by extension cannabis, in humans. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Ethanol increases GABAergic transmission at both pre- and postsynaptic sites in rat central amygdala neurons

PubMed Central

Roberto, Marisa; Madamba, Samuel G.; Moore, Scott D.; Tallent, Melanie K.; Siggins, George R.

2003-01-01

We examined the interaction of ethanol with the γ-aminobutyric acid (GABA)ergic system in neurons of slices of the rat central amygdala nucleus (CeA), a brain region thought to be critical for the reinforcing effects of ethanol. Brief superfusion of 11–66 mM ethanol significantly increased GABA type A (GABAA) receptor-mediated inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) in most CeA neurons, with a low apparent EC50 of 20 mM. Acute superfusion of 44 mM ethanol increased the amplitude of evoked GABAA IPSPs and IPSCs in 70% of CeA neurons. The ethanol enhancement of IPSPs and IPSCs occurred to a similar extent in the presence of the GABA type B (GABAB) receptor antagonist CGP 55845A, suggesting that this receptor is not involved in the ethanol effect on CeA neurons. Ethanol superfusion also decreased paired-pulse facilitation of evoked GABAA IPSPs and IPSCs and always increased the frequency and sometimes the amplitude of spontaneous miniature GABAA IPSCs as well as responses to local GABA application, indicating both presynaptic and postsynaptic sites of action for ethanol. Thus, the CeA is the first brain region to reveal, without conditional treatments such as GABAB antagonists, consistent, low-dose ethanol enhancement of GABAergic transmission at both pre- and postsynaptic sites. These findings add further support to the contention that the ethanol–GABA interaction in CeA plays an important role in the reinforcing effects of ethanol. PMID:12566570

GABA(A) receptor blockade in dorsomedial and ventromedial nuclei of the hypothalamus evokes panic-like elaborated defensive behaviour followed by innate fear-induced antinociception.

PubMed

Freitas, Renato Leonardo; Uribe-Mariño, Andrés; Castiblanco-Urbina, Maria Angélica; Elias-Filho, Daoud Hibraim; Coimbra, Norberto Cysne

2009-12-11

Dysfunction in the hypothalamic GABAergic system has been implicated in panic syndrome in humans. Furthermore, several studies have implicated the hypothalamus in the elaboration of pain modulation. Panic-prone states are able to be experimentally induced in laboratory animals to study this phenomenon. The aim of the present work was to investigate the involvement of medial hypothalamic nuclei in the organization of panic-like behaviour and the innate fear-induced oscillations of nociceptive thresholds. The blockade of GABA(A) receptors in the neuronal substrates of the ventromedial or dorsomedial hypothalamus was followed by elaborated defensive panic-like reactions. Moreover, innate fear-induced antinociception was consistently elicited after the escape behaviour. The escape responses organized by the dorsomedial and ventromedial hypothalamic nuclei were characteristically more elaborated, and a remarkable exploratory behaviour was recorded during GABA(A) receptor blockade in the medial hypothalamus. The motor characteristic of the elaborated defensive escape behaviour and the patterns of defensive alertness and defensive immobility induced by microinjection of the bicuculline either into the dorsomedial or into the ventromedial hypothalamus were very similar. This was followed by the same pattern of innate fear-induced antinociceptive response that lasted approximately 40 min after the elaborated defensive escape reaction in both cases. These findings suggest that dysfunction of the GABA-mediated neuronal system in the medial hypothalamus causes panic-like responses in laboratory animals, and that the elaborated escape behaviour organized in both dorsomedial and ventromedial hypothalamic nuclei are followed by significant innate-fear-induced antinociception. Our findings indicate that the GABA(A) receptor of dorsomedial and ventromedial hypothalamic nuclei are critically involved in the modulation of panic-like behaviour.

Responses to GABA(A) receptor activation are altered in NTS neurons isolated from chronic hypoxic rats.

PubMed

Tolstykh, Gleb; Belugin, Sergei; Mifflin, Steve

2004-04-23

The inhibitory amino acid GABA is released within the nucleus of the solitary tract (NTS) during hypoxia and modulates the respiratory response to hypoxia. To determine if responses of NTS neurons to activation of GABA(A) receptors are altered following exposure to chronic hypoxia, GABA(A) receptor-evoked whole cell currents were measured in enzymatically dispersed NTS neurons from normoxic and chronic hypoxic rats. Chronic hypoxic rats were exposed to 10% O(2) for 9-12 days. Membrane capacitance was the same in neurons from normoxic (6.9+/-0.5 pF, n=16) and hypoxic (6.3+/-0.5 pF, n=15) rats. The EC(50) for peak GABA-evoked current density was significantly greater in neurons from hypoxic (21.7+/-2.2 microM) compared to normoxic rats (12.2+/-0.9 microM) (p<0.001). Peak and 5-s adapted GABA currents evoked by 1, 3 and 10 microM were greater in neurons from normoxic compared to hypoxic rats (p<0.05) whereas peak and 5-s adapted responses to 30 and 100 microM GABA were not different comparing normoxic to hypoxic rats. Desensitization of GABA(A)-evoked currents was observed at concentrations greater than 3 microM and, measured as the ratio of the current 5 s after the onset of 100 microM GABA application to the peak GABA current, was the same in neurons from normoxic (0.37+/-0.03) and hypoxic rats (0.33+/-0.04). Reduced sensitivity to GABA(A) receptor-evoked inhibition in chronic hypoxia could influence chemoreceptor afferent integration by NTS neurons.

Ionic plasticity and pain: The loss of descending serotonergic fibers after spinal cord injury transforms how GABA affects pain.

PubMed

Huang, Yung-Jen; Grau, James W

2018-05-02

Activation of pain (nociceptive) fibers can sensitize neural circuits within the spinal cord, inducing an increase in excitability (central sensitization) that can foster chronic pain. The development of spinally-mediated central sensitization is regulated by descending fibers and GABAergic interneurons. In adult animals, the co-transporter KCC2 maintains a low intracellular concentration of the anion Cl - . As a result, when the GABA-A receptor is engaged, Cl - flows in the neuron which has a hyperpolarizing (inhibitory) effect. Spinal cord injury (SCI) can down-regulate KCC2 and reverse the flow of Cl - . Under these conditions, engaging the GABA-A receptor can have a depolarizing (excitatory) effect that fosters the development of nociceptive sensitization. The present paper explores how SCI alters GABA function and provides evidence that the loss of descending fibers alters pain transmission to the brain. Prior work has shown that, after SCI, administration of a GABA-A antagonist blocks the development of capsaicin-induced nociceptive sensitization, implying that GABA release plays an essential role. This excitatory effect is linked to serotonergic (5HT) fibers that descend through the dorsolateral funiculus (DLF) and impact spinal function via the 5HT-1A receptor. Supporting this, blocking the 5HT-1A receptor, or lesioning the DLF, emulated the effect of SCI. Conversely, spinal application of a 5HT-1A agonist up-regulated KCC2 and reversed the effect of bicuculline treatment. Finally, lesioning the DLF reversed how a GABA-A antagonist affects a capsaicin-induced aversion in a place conditioning task; in sham operated animals, bicuculline enhanced aversion whereas in DLF-lesioned rats biciculline had an antinociceptive effect. Copyright © 2018 Elsevier Inc. All rights reserved.

Acute sleep deprivation preconditions the heart against ischemia/ reperfusion injury: the role of central GABA-A receptors

PubMed Central

Parsa, Hoda; Imani, Alireza; Faghihi, Mahdieh; Riahi, Esmail; Badavi, Mohammad; Shakoori, Abbas; Rastegar, Tayebeh; Aghajani, Marjan; Rajani, Sulail Fatima

2017-01-01

Objective(s): Central γ-aminobutyric acid (GABA) neurotransmission modulates cardiovascular functions and sleep. Acute sleep deprivation (ASD) affects functions of various body organs via different mechanisms. Here, we evaluated the effect of ASD on cardiac ischemia/reperfusion injury (IRI), and studied the role of GABA-A receptor inhibition in central nucleus of amygdala (CeA) by assessing nitric oxide (NO) and oxidative stress. Materials and Methods: The CeA in sixty male Wistar rats was cannulated for saline or bicuculline (GABA-A receptor antagonist) administration. All animals underwent 30 min of coronary occlusion (ischemia), followed by 2 hr reperfusion (IR). The five experimental groups (n=12) included are as follows: IR: received saline; BIC+IR: received Bicuculline; MLP+IR: received saline, followed by the placement of animals in an aquarium with multiple large platforms; ASD+IR: underwent ASD in an aquarium with multiple small platforms; and BIC+ASD+IR: received bicuculline prior to ASD. Results: Bicuculline administration increased the malondialdehyde levels and infarct size, and decreased the NO metabolites levels and endothelial nitric oxide synthase (eNOS) gene expression in infarcted and non-infarcted areas in comparison to IR group. ASD reduced malondialdehyde levels and infarct size and increased NO metabolites, corticosterone levels and eNOS expression in infarcted and non-infarcted areas as compared to the IR group. Levels of malondialdehyde were increased while levels of NO metabolites, corticosterone and eNOS expression in infarcted and non-infarcted areas were reduced in the BIC+ASD+IR as compared to the ASD+IR group. Conclusion: Blockade of GABA-A receptors in the CeA abolishes ASD-induced cardioprotection by suppressing oxidative stress and NO production. PMID:29299201

Acute sleep deprivation preconditions the heart against ischemia/ reperfusion injury: the role of central GABA-A receptors.

PubMed

Parsa, Hoda; Imani, Alireza; Faghihi, Mahdieh; Riahi, Esmail; Badavi, Mohammad; Shakoori, Abbas; Rastegar, Tayebeh; Aghajani, Marjan; Rajani, Sulail Fatima

2017-11-01

Central γ-aminobutyric acid (GABA) neurotransmission modulates cardiovascular functions and sleep. Acute sleep deprivation (ASD) affects functions of various body organs via different mechanisms. Here, we evaluated the effect of ASD on cardiac ischemia/reperfusion injury (IRI), and studied the role of GABA-A receptor inhibition in central nucleus of amygdala (CeA) by assessing nitric oxide (NO) and oxidative stress. The CeA in sixty male Wistar rats was cannulated for saline or bicuculline (GABA-A receptor antagonist) administration. All animals underwent 30 min of coronary occlusion (ischemia), followed by 2 hr reperfusion (IR). The five experimental groups (n=12) included are as follows: IR: received saline; BIC+IR: received Bicuculline; MLP+IR: received saline, followed by the placement of animals in an aquarium with multiple large platforms; ASD+IR: underwent ASD in an aquarium with multiple small platforms; and BIC+ASD+IR: received bicuculline prior to ASD. Bicuculline administration increased the malondialdehyde levels and infarct size, and decreased the NO metabolites levels and endothelial nitric oxide synthase (eNOS) gene expression in infarcted and non-infarcted areas in comparison to IR group. ASD reduced malondialdehyde levels and infarct size and increased NO metabolites, corticosterone levels and eNOS expression in infarcted and non-infarcted areas as compared to the IR group. Levels of malondialdehyde were increased while levels of NO metabolites, corticosterone and eNOS expression in infarcted and non-infarcted areas were reduced in the BIC+ASD+IR as compared to the ASD+IR group. Blockade of GABA-A receptors in the CeA abolishes ASD-induced cardioprotection by suppressing oxidative stress and NO production.

Chronic Exposure to Anabolic Androgenic Steroids Alters Neuronal Function in the Mammalian Forebrain via Androgen Receptor- and Estrogen Receptor-Mediated Mechanisms

PubMed Central

Penatti, Carlos A A; Porter, Donna M; Henderson, Leslie P

2009-01-01

Anabolic androgenic steroids (AAS) can promote detrimental effects on social behaviors for which γ-aminobutyric acid type A (GABAA) receptor-mediated circuits in the forebrain play a critical role. While all AAS bind to androgen receptors (AR), they may also be aromatized to estrogens and thus potentially impart effects via estrogen receptors (ER). Chronic exposure of wild type male mice to a combination of chemically distinct AAS increased action potential (AP) frequency, selective GABAA receptor subunit mRNAs, and GABAergic synaptic current decay in the medial preoptic area (mPOA). Experiments performed with pharmacological agents and in AR-deficient Tfm mutant mice suggest that the AAS-dependent enhancement of GABAergic transmission in wild type mice is AR-mediated. In AR-deficient mice, the AAS elicited dramatically different effects, decreasing AP frequency, sIPSC amplitude and frequency and the expression of selective GABAA receptor subunit mRNAs. Surprisingly, in the absence of AR signaling, the data indicate that the AAS do not act as ER agonists, but rather suggest a novel in vivo action in which the AAS inhibit aromatase and impair endogenous ER signaling. These results show that the AAS have the capacity to alter neuronal function in the forebrain via multiple steroid signaling mechanisms and suggest that effects of these steroids in the brain will depend not only on the balance of AR- vs. ER-mediated regulation for different target genes, but also on the ability of these drugs to alter steroid metabolism and thus the endogenous steroid milieu. PMID:19812324

Involvement of the GABAergic system in the anxiolytic-like effect of the flavonoid ellagic acid in mice.

PubMed

Girish, Chandrashekaran; Raj, Vishnu; Arya, Jayasree; Balakrishnan, Sadasivam

2013-06-15

Anxiolytic-like effects of dietary flavonoids are relatively well known. Ellagic acid is a naturally occurring flavonoid compound which is abundant in many plants and fruits. The present study was designed to investigate the antianxiety-like effect of ellagic acid in mice using an elevated plus-maze test. The involvement of the GABAergic and serotonergic systems in the antianxiety-like activity of ellagic acid was also studied. Our results showed that ellagic acid treatment (25, 50 and 100 mg/kg, p.o.), produced a significant increase in the percentage of time spent and entry into the open arms, with a profile comparable to that of diazepam (1 mg/kg, p.o.). Unlike diazepam, the anxiolytic doses of ellagic acid did not prolong the duration of sodium thiopental-induced loss of righting reflex, indicating that this flavonoid is non-hypnotic. The anxiolytic effect observed with ellagic acid treatment (25 mg/kg, p.o.) was antagonized by pretreatment with picrotoxin (a non-competitive GABAA receptor antagonist, 1 mg/kg, i.p.) and flumazenil (a benzodiazepine site antagonist, 1 mg/kg, i.p.) but not with p-chlorophenylalanine (a serotonin synthesis inhibitor, 100 mg/kg, i.p.) and pindolol (a β-adrenoceptors blocker/5-HT1A/1B receptor antagonist, 10 mg/kg, i.p.). Taken together, the data demonstrated that acute and chronic administration of ellagic acid to mice has produced antianxiety-like effect when tested in the elevated plus-maze. The experiments with different receptor blockers suggest an involvement of GABAergic system in the anxiolytic action of this bioflavonoid. However, this action is not seems to be mediated through serotonergic system. Copyright © 2013 Elsevier B.V. All rights reserved.

Benzodiazepines modulate the A2 adenosine binding sites on 108CC15 neuroblastoma X glioma hybrid cells.

PubMed Central

Snell, C. R.; Snell, P. H.

1984-01-01

We have demonstrated high affinity diazepam binding sites of the Ro5-4864 benzodiazepine receptor subtype on 108CC15 neuroblastoma X glioma hybrid cells. These cells were previously shown to have purinoceptors of the A2 adenosine subtype and we have now found that [3H]-adenosine can be displaced from this binding site by the benzodiazepines and related compounds that can also bind to the Ro5-4864 site. Diazepam was found to have no intrinsic activity at the A2-receptor as measured by the stimulation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) production in this cell line. At concentrations sufficient to compete for the A2-receptor, diazepam was shown to facilitate, by approximately 2 fold, the stimulation of cyclic AMP by adenosine. These effects are not due to inhibition of adenosine uptake or phosphodiesterase activity, but are probably a consequence of modulation of the coupling of the A2-receptor to cyclic AMP production in this hybrid cell line. PMID:6150742

Aspects of the homeostaic plasticity of GABAA receptor-mediated inhibition

PubMed Central

Mody, Istvan

2005-01-01

Plasticity of ligand-gated ion channels plays a critical role in nervous system development, circuit formation and refinement, and pathological processes. Recent advances have mainly focused on the plasticity of channels gated by excitatory amino acids, including their acclaimed role in learning and memory. These receptors, together with voltage-gated ion channels, have also been known to be subjected to a homeostatic form of plasticity that prevents destabilization of the neurone's function and that of the network during various physiological processes. To date, the plasticity of GABAA receptors has been examined mainly from a developmental and a pathological point of view. Little is known about homeostatic mechanisms governing their plasticity. This review summarizes some of the findings on the homeostatic plasticity of tonic and phasic inhibitory activity. PMID:15528237

An anxiogenic benzodiazepine receptor ligand induces learned helplessness.

PubMed

Drugan, R C; Maier, S F; Skolnick, P; Paul, S M; Crawley, J N

1985-07-31

Rats treated with the anxiogenic beta-carboline, N-methyl-beta-carboline-3-carboxamide (FG-7142), failed to acquire an escape response 24 h after treatment. Administration of FG-7142 resulted in a behavioral effect equivalent to a session of inescapable tailshock in this paradigm of learned helplessness. Pretreatment of rats with the selective benzodiazepine receptor antagonist Ro15-1788 blocked the development of learned helplessness elicited by FG-7142. These findings suggest that 'anxiety' may be a major factor in the development of learned helplessness.

Variation in GABA-A subunit gene copy number in an autistic patient with mosaic 4 p duplication (p12p16).

PubMed

Kakinuma, Hiroaki; Ozaki, Mamoru; Sato, Hitoshi; Takahashi, Hiroaki

2008-09-05

Autism has been associated with chromosomal aberrations, including duplications at chromosome 4, and the identification of genetic factors contributing to the etiology of this disease is the focus of much research. Here we report a Japanese girl with mosaic of chromosome 4p duplication, mos 46,XX,dup(4)(p12p16)[54]/46,XX[6], who was diagnosed with autism at 3 years of age. Fluorescence in situ hybridization (FISH) with probes covering the region spanning a cluster of the gamma aminobutyric acid A (GABA-A) receptor subunit genes in the proximal short arm of chromosome 4 demonstrated total three signals for the GABRG1, GABRA4, and GABRA2 genes, but only two signals for GABRB1. This suggests that aberrant copy number of the GABA-A receptor subunit genes may contribute to the etiology of autism in this patient. 2007 Wiley-Liss, Inc.

Ipsilateral feeding-specific circuits between the nucleus accumbens shell and the lateral hypothalamus: regulation by glutamate and GABA receptor subtypes.

PubMed

Urstadt, Kevin R; Kally, Peter; Zaidi, Sana F; Stanley, B Glenn

2013-04-01

The nucleus accumbens shell (AcbSh) and the lateral hypothalamus (LH) are both involved in the control of food intake. Activation of GABA(A) receptors or blockade of AMPA and kainate receptors within the AcbSh induces feeding, as does blockade of GABA(A) receptors or activation of NMDA receptors in the LH. Further, evidence suggests that feeding induced via the AcbSh can be suppressed by LH inhibition. However, it is unclear if this suppression is specific to feeding. Adult male Sprague-Dawley rats with 3 intracranial guide cannulas, one unilaterally into the AcbSh and two bilaterally into the LH, were used to explore this issue. DNQX (1.25 μg) or muscimol (100 ng) infused into the AcbSh unilaterally elicited feeding, and this elicited intake was suppressed by bilateral LH injection of d-AP5 (2 μg) or muscimol (25 ng). The effectiveness of d-AP5 or muscimol infusion into either the LH site ipsilateral or contralateral to the AcbSh injection was compared. Ipsilateral LH injection of d-AP5 or muscimol was significantly more effective than contralateral injection in suppressing food intake initiated by AcbSh injection of DNQX or muscimol. These results add to the prior evidence that inhibition of the LH through pharmacological modulation of NMDA or GABA(A) receptors specifically suppresses feeding initiated by AcbSh inhibition, and that these two regions communicate via an ipsilateral circuit to specifically regulate feeding. Copyright © 2012 Elsevier Ltd. All rights reserved.

Involvement of prelimbic medial prefrontal cortex in panic-like elaborated defensive behaviour and innate fear-induced antinociception elicited by GABAA receptor blockade in the dorsomedial and ventromedial hypothalamic nuclei: role of the endocannabinoid CB1 receptor.

PubMed

Freitas, Renato Leonardo de; Salgado-Rohner, Carlos José; Hallak, Jaime Eduardo Cecílio; Crippa, José Alexandre de Souza; Coimbra, Norberto Cysne

2013-09-01

It has been shown that GABAA receptor blockade in the dorsomedial and ventromedial hypothalamic nuclei (DMH and VMH, respectively) induces elaborated defensive behavioural responses accompanied by antinociception, which has been utilized as an experimental model of panic attack. Furthermore, the prelimbic (PL) division of the medial prefrontal cortex (MPFC) has been related to emotional reactions and the processing of nociceptive information. The aim of the present study was to investigate the possible involvement of the PL cortex and the participation of local cannabinoid CB1 receptors in the elaboration of panic-like reactions and in innate fear-induced antinociception. Elaborated fear-induced responses were analysed during a 10-min period in an open-field test arena. Microinjection of the GABAA receptor antagonist bicuculline into the DMH/VMH evoked panic-like behaviour and fear-induced antinociception, which was decreased by microinjection of the non-selective synaptic contact blocker cobalt chloride in the PL cortex. Moreover, microinjection of AM251 (25, 100 or 400 pmol), an endocannabinoid CB1 receptor antagonist, into the PL cortex also attenuated the defensive behavioural responses and the antinociception that follows innate fear behaviour elaborated by DMH/VMH. These data suggest that the PL cortex plays an important role in the organization of elaborated forward escape behaviour and that this cortical area is also involved in the elaboration of innate fear-induced antinociception. Additionally, CB1 receptors in the PL cortex modulate both panic-like behaviours and fear-induced antinociception elicited by disinhibition of the DMH/VMH through microinjection of bicuculline.

GABAA Receptors Containing ρ1 Subunits Contribute to In Vivo Effects of Ethanol in Mice

PubMed Central

Blednov, Yuri A.; Benavidez, Jillian M.; Black, Mendy; Leiter, Courtney R.; Osterndorff-Kahanek, Elizabeth; Johnson, David; Borghese, Cecilia M.; Hanrahan, Jane R.; Johnston, Graham A. R.; Chebib, Mary; Harris, R. Adron

2014-01-01

GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ1” antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ2” antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo. PMID:24454882

Reduced Chrna7 expression in C3H mice is associated with increases in hippocampal parvalbumin and glutamate decarboxylase-67 (GAD67) as well as altered levels of GABAA receptor subunits

PubMed Central

Bates, Ryan C.; Stith, Bradley J.; Stevens, Karen E.; Adams, Catherine E.

2014-01-01

Decreased expression of CHRNA7, the gene encoding the α7* subtype of nicotinic receptor, may contribute to the cognitive dysfunction observed in schizophrenia by disrupting the inhibitory/excitatory balance in the hippocampus. C3H mice with reduced Chrna7 expression have significant reductions in hippocampal α7* receptor density, deficits in hippocampal auditory gating, increased hippocampal activity as well as significant decreases in hippocampal glutamate decarboxylase-65 (GAD65) and γ-aminobutyric acid-A (GABAA) receptor levels. The current study investigated whether altered Chrna7 expression is associated with changes in the levels of parvalbumin, GAD67 and/or GABAA receptor subunits in hippocampus from male and female C3H Chrna7 wildtype, C3H Chrna7 heterozygous and C3H Chrna7 knockout mice using quantitative western immunoblotting. Reduced Chrna7 expression was associated with significant increases in hippocampal parvalbumin and GAD67 and with complex alterations in GABAA receptor subunits. A decrease in α3 subunit protein was seen in both female C3H Chrna7 Het and KO mice while a decrease in α4 subunit protein was also detected in C3H Chrna7 KO mice with no sex difference. In contrast, an increase in δ subunit protein was observed in C3H Chrna7 Het mice while a decrease in this subunit was observed in C3H Chrna7 KO mice, with δ subunit protein levels being greater in males than in females. Finally, an increase in γ2 subunit protein was found in C3H Chrna7 KO mice with the levels of this subunit again being greater in males than in females. The increases in hippocampal parvalbumin and GAD67 observed in C3H Chrna7 mice are contrary to reports of reductions in these proteins in postmortem hippocampus from schizophrenic individuals. We hypothesize that the disparate results may occur because of the influence of factors other than CHRNA7 that have been found to be abnormal in schizophrenia. PMID:24836856

Mechanisms of anabolic androgenic steroid inhibition of mammalian ɛ-subunit-containing GABAA receptors

PubMed Central

Jones, Brian L; Whiting, Paul J; Henderson, Leslie P

2006-01-01

GABAergic transmission regulates the activity of gonadotrophin-releasing hormone (GnRH) neurons in the preoptic area/hypothalamus that control the onset of puberty and the expression of reproductive behaviours. One of the hallmarks of illicit use of anabolic androgenic steroids (AAS) is disruption of behaviours under neuroendocrine control. GnRH neurons are among a limited population of cells that express high levels of the ɛ-subunit of the GABAA receptor. To better understand the actions of AAS on neuroendocrine mechanisms, we have characterized modulation of GABAA receptor-mediated currents in mouse native GnRH neurons and in heterologous cells expressing recombinant α2β3ɛ-receptors. GnRH neurons exhibited robust currents in response to millimolar concentrations of GABA and a picrotoxin (PTX)-sensitive, bicuculline-insensitive current that probably arises from spontaneous openings of GABAA receptors. The AAS 17α-methyltestosterone (17α-MeT) inhibited spontaneous and GABA-evoked currents in GnRH neurons. For recombinant α2β3ɛ-receptors, 17α-MeT inhibited phasic and tonic GABA-elicited responses, accelerated desensitization and slowed paired pulse response recovery. Single channel analysis indicated that GABA-evoked events could be described by three open dwell components and that 17α-MeT enhanced residence in the intermediate dwell state. This AAS also inhibited a PTX-sensitive, spontaneous current (open probability, ∼0.15–0.2) in a concentration-dependent fashion (IC50 ≈ 9 μm). Kinetic modelling indicated that the inhibition induced by 17α-MeT occurs by an allosteric block in which the AAS interacts preferentially with a closed state and promotes accumulation in that state. Finally, studies with a G302S mutant ɛ-subunit suggest that this residue within the transmembrane domain TM2 plays a role in mediating AAS binding and modulation. In sum, our results indicate that inclusion of the ɛ-subunit significantly alters the profile of AAS modulation and that this allosteric inhibition of native GnRH neurons should be considered with regard to AAS disruption of neuroendocrine control. PMID:16543268

Synthesis, anticonvulsant, sedative and anxiolytic activities of novel annulated pyrrolo[1,4]benzodiazepines.

PubMed

Sorra, Kumaraswamy; Chen, Chien-Shu; Chang, Chi-Fen; Pusuluri, Srinivas; Mukkanti, Khagga; Wu, Chi-Rei; Chuang, Ta-Hsien

2014-09-18

Four new pentacyclic benzodiazepine derivatives (PBDTs 13-16) were synthesized by conventional thermal heating and microwave-assisted intramolecular cyclocondensation. Their anticonvulsant, sedative and anxiolytic activities were evaluated by drug-induced convulsion models, a pentobarbital-induced hypnotic model and an elevated plus maze in mice. PBDT 13, a triazolopyrrolo[2,1-c][1,4]benzodiazepin-8-one fused with a thiadiazolone ring, exhibited the best anticonvulsant, sedative and anxiolytic effects in our tests. There was no significant difference in potency between PBDT 13 and diazepam, and we proposed that the action mechanism of PBDT 13 could be similar to that of diazepam via benzodiazepine receptors.

Can a Positive Allosteric Modulation of GABAergic Receptors Improve Motor Symptoms in Patients with Parkinson's Disease? The Potential Role of Zolpidem in the Treatment of Parkinson's Disease

PubMed Central

Daniele, Antonio; Panza, Francesco; Greco, Antonio; Logroscino, Giancarlo; Seripa, Davide

2016-01-01

At present, patients with advanced Parkinson's disease (PD) are unsatisfactorily controlled by currently used anti-Parkinsonian dopaminergic drugs. Various studies suggest that therapeutic strategies based on nondopaminergic drugs might be helpful in PD. Zolpidem, an imidazopyridine widely used as sleep inducer, shows high affinity only for GABAA receptors containing the α-1 subunit and facilitates GABAergic neurotransmission through a positive allosteric modulation of GABAA receptors. Various observations, although preliminary, consistently suggest that in PD patients zolpidem may induce beneficial (and sometimes remarkable) effects on motor symptoms even after single doses and may also improve dyskinesias. Since a high density of zolpidem binding sites is in the two main output structures of the basal ganglia which are abnormally overactive in PD (internal globus pallidus, GPi, and substantia nigra pars reticulata, SNr), it was hypothesized that in PD patients zolpidem may induce through GABAA receptors an inhibition of GPi and SNr (and, possibly, of the subthalamic nucleus also), resulting in an increased activity of motor cortical areas (such as supplementary motor area), which may give rise to improvement of motor symptoms of PD. Randomized clinical trials are needed in order to assess the efficacy, safety, and tolerability of zolpidem in treating motor symptoms of PD. PMID:27293955

Blockade of GABA, type A, receptors in the rat pontine reticular formation induces rapid eye movement sleep that is dependent upon the cholinergic system.

PubMed

Marks, G A; Sachs, O W; Birabil, C G

2008-09-22

The brainstem reticular formation is an area important to the control of rapid eye movement (REM) sleep. The antagonist of GABA-type A (GABA(A)) receptors, bicuculline methiodide (BMI), injected into the rat nucleus pontis oralis (PnO) of the reticular formation resulted in a long-lasting increase in REM sleep. Thus, one factor controlling REM sleep appears to be the number of functional GABA(A) receptors in the PnO. The long-lasting effect produced by BMI may result from secondary influences on other neurotransmitter systems known to have long-lasting effects. To study this question, rats were surgically prepared for chronic sleep recording and additionally implanted with guide cannulas aimed at sites in the PnO. Multiple, 60 nl, unilateral injections were made either singly or in combination. GABA(A) receptor antagonists, BMI and gabazine (GBZ), produced dose-dependent increases in REM sleep with GBZ being approximately 35 times more potent than BMI. GBZ and the cholinergic agonist, carbachol, produced very similar results, both increasing REM sleep for about 8 h, mainly through increased period frequency, with little reduction in REM latency. Pre-injection of the muscarinic antagonist, atropine, completely blocked the REM sleep-increase by GBZ. GABAergic control of REM sleep in the PnO requires the cholinergic system and may be acting through presynaptic modulation of acetylcholine release.

Effects of Post-Training Hippocampal Injections of Midazolam on Fear Conditioning

ERIC Educational Resources Information Center

Gafford, Georgette M.; Parsons, Ryan G.; Helmstetter, Fred J.

2005-01-01

Benzodiazepines have been useful tools for investigating mechanisms underlying learning and memory. The present set of experiments investigates the role of hippocampal GABA[subscript A]/benzodiazepine receptors in memory consolidation using Pavlovian fear conditioning. Rats were prepared with cannulae aimed at the dorsal hippocampus and trained…

Do preclinical seizure models preselect certain adverse effects of antiepileptic drugs.

PubMed

Meldrum, Brian

2002-06-01

Classical screening tests (maximal electroshock, MES, and threshold pentylenetetrazol, PTZ) employ non-epileptic rodents and identify antiepileptic drugs (AEDs) with mechanisms of action associated with significant CNS side effects. Thus MES identifies drugs acting on Na+ channels that produce cerebellar toxicity. It may be possible to produce novel AEDs more selectively targeted at voltage-sensitive (VS) ion channels. There is little specific evidence for the likely success of this strategy with subunit selective agents targeted at the different VS Na+ channels. Drugs targeted at specific VS Ca++ channels (T, N, P/Q types) may be useful in generalised seizures. There are many as yet unexplored possibilities relating to K+ channels. GABA related drugs acting on PTZ clonic seizures tend to induce sedation and muscle hypotonia. Studies in mice, particularly with knock-in mutations, but also with subunit selective agents acting via the GABA(A) benzodiazepine site, suggest that it is possible to produce agents which do or do not induce particular side effects (sedative, hypnotic, anxiolytic, muscle relaxant, amnesia, anaesthesia). Whether these findings transfer to man has yet to be established. Acquired epilepsy in rodents (e.g. kindling or spontaneous seizures following chemically- or electrically-induced status epilepticus) or acquired epilepsy in man (following prolonged febrile seizures or traumatic brain injury) is associated with multiple changes in the function and subunit composition of ion channels and receptor molecules. Optimal screening of novel AEDs, both for efficacy and side effects, requires models with receptor and ion channel changes similar to those in the target human syndrome.

Piracetam and other structurally related nootropics.

PubMed

Gouliaev, A H; Senning, A

1994-05-01

Nearly three decades have now passed since the discovery of the piracetam-like nootropics, compounds which exhibit cognition-enhancing properties, but for which no commonly accepted mechanism of action has been established. This review covers clinical, pharmacokinetic, biochemical and behavioural results presented in the literature from 1965 through 1992 (407 references) of piracetam, oxiracetam, pramiracetam, etiracetam, nefiracetam, aniracetam and rolziracetam and their structural analogues. The piracetam-like nootropics are capable of achieving reversal of amnesia induced by, e.g., scopolamine, electroconvulsive shock and hypoxia. Protection against barbiturate intoxication is observed and some benefit in clinical studies with patients suffering from mild to moderate degrees of dementia has been demonstrated. No affinity for the alpha 1-, alpha 2-, beta-, muscarinic, 5-hydroxytryptamine-, dopamine, adenosine-A1-, mu-opiate, gamma-aminobutyric acid (GABA) (except for nefiracetam (GABAA)), benzodiazepine and glutamate receptors has been found. The racetams possess a very low toxicity and lack serious side effects. Increased turnover of different neurotransmitters has been observed as well as other biochemical findings, e.g., inhibition of enzymes such as prolylendopeptidase. So far, no generally accepted mechanism of action has, however, emerged. We believe that the effect of the racetams is due to a potentiation of already present neurotransmission and that much evidence points in the direction of a modulated ion flux by, e.g., potentiated calcium influx through non-L-type voltage-dependent calcium channels, potentiated sodium influx through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor gated channels or voltage-dependent channels or decreases in potassium efflux. Effects on carrier mediated ion transport are also possible.

GABAA receptor: Positive and negative allosteric modulators.

PubMed

Olsen, Richard W

2018-01-31

gamma-Aminobutyric acid (GABA)-mediated inhibitory neurotransmission and the gene products involved were discovered during the mid-twentieth century. Historically, myriad existing nervous system drugs act as positive and negative allosteric modulators of these proteins, making GABA a major component of modern neuropharmacology, and suggesting that many potential drugs will be found that share these targets. Although some of these drugs act on proteins involved in synthesis, degradation, and membrane transport of GABA, the GABA receptors Type A (GABA A R) and Type B (GABA B R) are the targets of the great majority of GABAergic drugs. This discovery is due in no small part to Professor Norman Bowery. Whereas the topic of GABA B R is appropriately emphasized in this special issue, Norman Bowery also made many insights into GABA A R pharmacology, the topic of this article. GABA A R are members of the ligand-gated ion channel receptor superfamily, a chloride channel family of a dozen or more heteropentameric subtypes containing 19 possible different subunits. These subtypes show different brain regional and subcellular localization, age-dependent expression, and potential for plastic changes with experience including drug exposure. Not only are GABA A R the targets of agonist depressants and antagonist convulsants, but most GABA A R drugs act at other (allosteric) binding sites on the GABA A R proteins. Some anxiolytic and sedative drugs, like benzodiazepine and related drugs, act on GABA A R subtype-dependent extracellular domain sites. General anesthetics including alcohols and neurosteroids act at GABA A R subunit-interface trans-membrane sites. Ethanol at high anesthetic doses acts on GABA A R subtype-dependent trans-membrane domain sites. Ethanol at low intoxicating doses acts at GABA A R subtype-dependent extracellular domain sites. Thus GABA A R subtypes possess pharmacologically specific receptor binding sites for a large group of different chemical classes of clinically important neuropharmacological agents. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparing the Antiseizure and Neuroprotective Efficacy of LY293558, Diazepam, Caramiphen, and LY293558-Caramiphen Combination against Soman in a Rat Model Relevant to the Pediatric Population

PubMed Central

Apland, James P.; Aroniadou-Anderjaska, Vassiliki; Figueiredo, Taiza H.; Pidoplichko, Volodymyr I.; Rossetti, Katia

2018-01-01

The currently Food and Drug Administration–approved anticonvulsant for the treatment of status epilepticus (SE) induced by nerve agents is the benzodiazepine diazepam; however, diazepam does not appear to offer neuroprotective benefits. This is of particular concern with respect to the protection of children because, in the developing brain, synaptic transmission mediated via GABAA receptors, the target of diazepam, is weak. In the present study, we exposed 21-day-old male rats to 1.2 × LD50 soman and compared the antiseizure, antilethality, and neuroprotective efficacy of diazepam (10 mg/kg), LY293558 (an AMPA/GluK1 receptor antagonist; 15 mg/kg), caramiphen (CRM, an antimuscarinic with NMDA receptor-antagonistic properties; 50 mg/kg), and LY293558 (15 mg/kg) + CRM (50 mg/kg), administered 1 hour after exposure. Diazepam, LY293558, and LY293558 + CRM, but not CRM alone, terminated SE; LY293558 + CRM treatment acted significantly faster and produced a survival rate greater than 85%. Thirty days after soman exposure, neurodegeneration in limbic regions was most severe in the CRM-treated group, minimal to severe—depending on the region—in the diazepam group, absent to moderate in the LY293558-treated group, and totally absent in the LY293558 + CRM group. Amygdala and hippocampal atrophy, a severe reduction in spontaneous inhibitory activity in the basolateral amygdala, and increased anxiety-like behavior in the open-field and acoustic startle response tests were present in the diazepam and CRM groups, whereas the LY293558 and LY293558 + CRM groups did not differ from controls. The combined administration of LY293558 and CRM, by blocking mainly AMPA, GluK1, and NMDA receptors, is a very effective anticonvulsant and neuroprotective therapy against soman in young rats. PMID:29467308

Food deprivation modulates gamma-aminobutyric acid receptors and peripheral benzodiazepine binding sites in rats.

PubMed

Weizman, A; Bidder, M; Fares, F; Gavish, M

1990-12-03

The effect of 5 days of food deprivation followed by 5 days of refeeding on gamma-aminobutyric acid (GABA) receptors, central benzodiazepine receptors (CBR), and peripheral benzodiazepine binding sites (PBzS) was studied in female Sprague-Dawley rats. Starvation induced a decrease in the density of PBzS in peripheral organs: adrenal (35%; P less than 0.001), kidney (33%; P less than 0.01), and heart (34%; P less than 0.001). Restoration of [3H]PK 11195 binding to normal values was observed in all three organs after 5 days of refeeding. The density of PBzS in the ovary, pituitary, and hypothalamus was not affected by starvation. Food deprivation resulted in a 35% decrease in cerebellar GABA receptors (P less than 0.01), while CBR in the hypothalamus and cerebral cortex remained unaltered. The changes in PBzS observed in the heart and kidney may be related to the long-term metabolic stress associated with starvation and to the functional changes occurring in these organs. The down-regulation of the adrenal PBzS is attributable to the suppressive effect of hypercortisolemia on pituitary ACTH release. The reduction in cerebellar GABA receptors may be an adaptive response to food deprivation stress and may be relevant to the proaggressive effect of hunger.

Isoflurane modulates excitability in the mouse thalamus via GABA-dependent and GABA-independent mechanisms

PubMed Central

Ying, Shui-Wang; Werner, David F.; Homanics, Gregg E.; Harrison, Neil L.; Goldstein, Peter A.

2009-01-01

Summary GABAergic neurons in the reticular thalamic nucleus (RTN) synapse onto thalamocortical neurons in the ventrobasal (VB) thalamus, and this reticulo-thalamocortical pathway is considered an anatomic target for general anesthetic-induced unconsciousness. A mutant mouse was engineered to harbor two amino acid substitutions (S270H, L277A) in the GABAA receptor (GABAA-R) α1 subunit; this mutation abolished sensitivity to the volatile anesthetic isoflurane in recombinant GABAA-Rs, and reduced in vivo sensitivity to isoflurane in the loss-of-righting-reflex assay. We examined the effects of the double mutation on GABAA-R-mediated synaptic currents and isoflurane sensitivity by recording from thalamic neurons in brain slices. The double mutation accelerated the decay, and decreased the ½ width of, evoked inhibitory postsynaptic currents (eIPSCs) in VB neurons and attenuated isoflurane-induced prolongation of the eIPSC. The hypnotic zolpidem, a selective modulator of GABAA-Rs containing the α1 subunit, prolonged eIPSC duration regardless of genotype, indicating that mutant mice incorporate α1-subunit containing GABAA-Rs into synapses. In RTN neurons, which lack the α1 subunit, eIPSC duration was longer than in VB, regardless of genotype. Isoflurane reduced the efficacy of GABAergic transmission from RTN to VB, independent of genotype, suggesting a presynaptic action in RTN neurons. Consistent with this observation, isoflurane inhibited both tonic action potential and rebound burst firing in the presence of GABAA-R blockade. The suppressed excitability in RTN neurons is likely mediated by isoflurane-enhanced Ba2+-sensitive, but 4-aminopyridine-insenstive, potassium conductances. We conclude that isoflurane enhances inhibition of thalamic neurons in VB via GABAA-R-dependent, but in RTN via GABAA-R-independent, mechanisms. PMID:18948126

Tetrazepam: a benzodiazepine which dissociates sedation from other benzodiazepine activities. II. In vitro and in vivo interactions with benzodiazepine binding sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keane, P.E.; Bachy, A.; Morre, M.

1988-05-01

Tetrazepam is a 1,4-benzodiazepine (BZD) derivative which, in rodents, appears to have very little sedative and ataxic effects. In an attempt to identify the molecular mechanisms underlying this particular pharmacological profile we examined the interaction of tetrazepam with BZD binding sites. Tetrazepam interacted competitively with central and peripheral BZD binding sites and exhibited comparable affinities for both sites. Tetrazepam was approximately one-seventh as potent as diazepam at the central receptor and as potent as diazepam at the peripheral binding site. Tetrazepam did not distinguish type I from type II central BZD receptors, as evidenced by comparable affinities for the cerebellarmore » and hippocampal receptors. In vitro autoradiographic studies showed that tetrazepam displaced (3H)flunitrazepam from rat brain membranes without any clear regional specificity. Like all BZD receptor agonists, tetrazepam exhibited a gamma-aminobutyric acid shift, a photoaffinity shift and potentiated the binding of 35S-t-butyl-bicyclophosphorothionate to rat brain membranes. However, the latter effect was observed at relatively high concentrations of tetrazepam. In vivo, tetrazepam displaced specifically bound (3H)flunitrazepam from mouse brain (ID50, 37 mg/kg p.o. vs 3.5 mg/kg p.o. for diazepam) and from mouse kidney (ID50, 38 mg/kg p.o. vs. 21 mg/kg p.o. for diazepam). It is concluded that tetrazepam is a BZD receptor agonist; the molecular mechanisms which underly the low sedative potential of the drug cannot at present be explained by a particular interaction with either central or peripheral BZD binding sites, but may be related to the drug's relatively weak effect on 35S-t-butyl-bicyclophosphorothionate binding.« less

Methyl pyropheophorbide-a analogues: potential fluorescent probes for the peripheral-type benzodiazepine receptor. Effect of central metal in photosensitizing efficacy.

PubMed

Chen, Yihui; Zheng, Xiang; Dobhal, Mahabeer P; Gryshuk, Amy; Morgan, Janet; Dougherty, Thomas J; Oseroff, Allan; Pandey, Ravindra K

2005-06-02

Pyropheophorbides and their metal complexes were synthesized to investigate their applications as nonradioactive peripheral benzodiazepine receptor (PBR) binding probes and photosensitizers for use in photodynamic therapy. They were found to be localized in mitochondria and showed significant binding to PBR. In some cases, the PBR binding values were similar to that for 17 (PK11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)isoquinoline-3-carboxamide). However, no direct correlation between 17 displacement ability and photosensitizing efficacy of photosensitizers was observed.

Positron emission tomography assessment of effects of benzodiazepines on regional glucose metabolic rate in patients with anxiety disorder

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buchsbaum, M.S.; Wu, J.; Haier, R.

1987-06-22

Patients with generalized anxiety disorder (n = 18) entered a 21-day, double-blind, placebo-controlled random assignment trial of clorazepate. Positron emission tomography with YF-deoxyglucose was carried out before and after treatment. Decreases in glucose metabolic rate in visual cortex and relative increases in the basal ganglia and thalamus were found. A correlation between regional changes in metabolic rate and regional benzodiazepine receptor binding density from other human autopsy studies was observed; brain regions highest in receptor density showed the greatest decrease in rate.

Population patch-clamp electrophysiology analysis of recombinant GABAA alpha1beta3gamma2 channels expressed in HEK-293 cells.

PubMed

Hollands, Emma C; Dale, Tim J; Baxter, Andrew W; Meadows, Helen J; Powell, Andrew J; Clare, Jeff J; Trezise, Derek J

2009-08-01

Gamma-amino butyric acid (GABA)-activated Cl- channels are critical mediators of inhibitory postsynaptic potentials in the CNS. To date, rational design efforts to identify potent and selective GABA(A) subtype ligands have been hampered by the absence of suitable high-throughput screening approaches. The authors describe 384-well population patch-clamp (PPC) planar array electrophysiology methods for the study of GABA(A) receptor pharmacology. In HEK293 cells stably expressing human alpha1beta3gamma2 GABA(A) channels, GABA evoked outward currents at 0 mV of 1.05 +/- 0.08 nA, measured 8 s post GABA addition. The I(GABA) was linear and reversed close to the theoretical E(Cl) (-56 mV). Concentration-response curve analysis yielded a mean pEC(50) value of 5.4 and Hill slope of 1.5, and for a series of agonists, the rank order of potency was muscimol > GABA > isoguvacine. A range of known positive modulators, including diazepam and pentobarbital, produced concentration-dependent augmentation of the GABA EC( 20) response (1 microM). The competitive antagonists bicuculline and gabazine produced concentration-dependent, parallel, rightward displacement of GABA curves with pA(2) and slope values of 5.7 and 1.0 and 6.7 and 1.0, respectively. In contrast, picrotoxin (0.2-150 microM) depressed the maximal GABA response, implying a non-competitive antagonism. Overall, the pharmacology of human alpha1beta3gamma2 GABA(A) determined by PPC was highly similar to that obtained by conventional patch-clamp methods. In small-scale single-shot screens, Z' values of >0.5 were obtained in agonist, modulator, and antagonist formats with hit rates of 0% to 3%. The authors conclude that despite the inability of the method to resolve the peak agonist responses, PPC can rapidly and usefully quantify pharmacology for the alpha1beta3gamma2 GABA(A) isoform. These data suggest that PPC may be a valuable approach for a focused set and secondary screening of GABA(A) receptors and other slow ligand-gated ion channels.

Bottom-Up versus Top-Down Induction of Sleep by Zolpidem Acting on Histaminergic and Neocortex Neurons

PubMed Central

Uygun, David S.; Ye, Zhiwen; Zecharia, Anna Y.; Harding, Edward C.; Yu, Xiao; Yustos, Raquel; Vyssotski, Alexei L.; Brickley, Stephen G.

2016-01-01

Zolpidem, a GABAA receptor-positive modulator, is the gold-standard drug for treating insomnia. Zolpidem prolongs IPSCs to decrease sleep latency and increase sleep time, effects that depend on α2 and/or α3 subunit-containing receptors. Compared with natural NREM sleep, zolpidem also decreases the EEG power, an effect that depends on α1 subunit-containing receptors, and which may make zolpidem-induced sleep less optimal. In this paper, we investigate whether zolpidem needs to potentiate only particular GABAergic pathways to induce sleep without reducing EEG power. Mice with a knock-in F77I mutation in the GABAA receptor γ2 subunit gene are zolpidem-insensitive. Using these mice, GABAA receptors in the frontal motor neocortex and hypothalamic (tuberomammillary nucleus) histaminergic-neurons of γ2I77 mice were made selectively sensitive to zolpidem by genetically swapping the γ2I77 subunits with γ2F77 subunits. When histamine neurons were made selectively zolpidem-sensitive, systemic administration of zolpidem shortened sleep latency and increased sleep time. But in contrast to the effect of zolpidem on wild-type mice, the power in the EEG spectra of NREM sleep was not decreased, suggesting that these EEG power-reducing effects of zolpidem do not depend on reduced histamine release. Selective potentiation of GABAA receptors in the frontal cortex by systemic zolpidem administration also reduced sleep latency, but less so than for histamine neurons. These results could help with the design of new sedatives that induce a more natural sleep. SIGNIFICANCE STATEMENT Many people who find it hard to get to sleep take sedatives. Zolpidem (Ambien) is the most widely prescribed “sleeping pill.” It makes the inhibitory neurotransmitter GABA work better at its receptors throughout the brain. The sleep induced by zolpidem does not resemble natural sleep because it produces a lower power in the brain waves that occur while we are sleeping. We show using mouse genetics that zolpidem only needs to work on specific parts and cell types of the brain, including histamine neurons in the hypothalamus, to induce sleep but without reducing the power of the sleep. This knowledge could help in the design of sleeping pills that induce a more natural sleep. PMID:27807161

Evidence for involvement of the astrocytic benzodiazepine receptor in the mechanism of action of convulsant and anticonvulsant drugs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bender, A.S.; Hertz, L.

1988-01-01

The anticonvulsant drugs carbamazepine, phenobarbital, trimethadione, valproic acid and ethosuximide at pharmacologically relevant concentrations inhibit (/sup 3/H)diazepam binding to astrocytes in primary cultures but have much less effect on a corresponding preparation of neurons. Phenytoin as well as pentobarbital (which is not used chronically as an anticonvulsant) are equipotent in the two cell types. The convulsants picrotoxinin and pentylenetetrazol, the convulsant benzodiazepine RO 5-3663 and the two convulsant barbiturates DMBB and CHEB similarly inhibit diazepam binding to astrocytes but have little effect on neurons. On the basis of these findings it is suggested that these convulsants and anticonvulsants owe atmore » least part of their effect to an interaction with the astrocytic benzodiazepine receptor, perhaps by interference with a calcium channel.« less

Noradrenaline Triggers GABAA Inhibition of Bed Nucleus of the Stria Terminalis Neurons Projecting to the Ventral Tegmental Area

PubMed Central

Dumont, Éric C.; Williams, John T.

2014-01-01

The lateral part of the ventral bed nucleus of the stria terminalis (vlBNST) is a critical site for the antiaversive effects of noradrenergic drugs during opioid withdrawal. The objective of the present study is to identify the cellular action(s) of noradrenaline in the vlBNST after withdrawal from a 5 d treatment with morphine. The vlBNST is a heterogeneous cell group with multiple efferent projections. Therefore, neurons projecting to the midbrain were identified by retrograde transport of fluorescent microspheres injected in the ventral tegmental area (VTA). Whole-cell voltage clamp recordings of these neurons and of those sharing physiological properties were done in brain slices. Noradrenaline activated α1-adrenergic receptors to increase GABAA-IPSC frequency. Noradrenaline produced a similar increase in GABAA-IPSCs during acute opioid withdrawal, but this increase resulted from activation of β-adrenergic receptors, adenylyl cyclase, and protein kinase A, as well as α1-adrenergic receptors. Given that neurons in the vlBNST send an excitatory projection to the VTA, noradrenaline may reduce excitatory drive to mesolimbic dopamine cells. This mechanism might contribute to the withdrawal-induced inhibition of dopamine neurons and explain how noradrenergic drugs microinjected into the vlBNST reduce aversive aspects of opioid withdrawal. PMID:15385602

Near-future carbon dioxide levels alter fish behaviour by interfering with neurotransmitter function

NASA Astrophysics Data System (ADS)

Nilsson, Göran E.; Dixson, Danielle L.; Domenici, Paolo; McCormick, Mark I.; Sørensen, Christina; Watson, Sue-Ann; Munday, Philip L.

2012-03-01

Predicted future CO2 levels have been found to alter sensory responses and behaviour of marine fishes. Changes include increased boldness and activity, loss of behavioural lateralization, altered auditory preferences and impaired olfactory function. Impaired olfactory function makes larval fish attracted to odours they normally avoid, including ones from predators and unfavourable habitats. These behavioural alterations have significant effects on mortality that may have far-reaching implications for population replenishment, community structure and ecosystem function. However, the underlying mechanism linking high CO2 to these diverse responses has been unknown. Here we show that abnormal olfactory preferences and loss of behavioural lateralization exhibited by two species of larval coral reef fish exposed to high CO2 can be rapidly and effectively reversed by treatment with an antagonist of the GABA-A receptor. GABA-A is a major neurotransmitter receptor in the vertebrate brain. Thus, our results indicate that high CO2 interferes with neurotransmitter function, a hitherto unrecognized threat to marine populations and ecosystems. Given the ubiquity and conserved function of GABA-A receptors, we predict that rising CO2 levels could cause sensory and behavioural impairment in a wide range of marine species, especially those that tightly control their acid-base balance through regulatory changes in HCO3- and Cl- levels.

Recovery from ketamine-induced amnesia by blockade of GABA-A receptor in the medial prefrontal cortex of mice.

PubMed

Farahmandfar, Maryam; Akbarabadi, Ardeshir; Bakhtazad, Atefeh; Zarrindast, Mohammad-Reza

2017-03-06

Ketamine and other noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists are known to induce deficits in learning and cognitive performance sensitive to prefrontal cortex (PFC) functions. The interaction of a glutamatergic and GABAergic systems is essential for many cognitive behaviors. In order to understand the effect of γ-aminobutyric acid (GABA)/glutamate interactions on learning and memory, we investigated the effects of intra medial prefrontal cortex (mPFC) injections of GABAergic agents on ketamine-induced amnesia using a one-trial passive avoidance task in mice. Pre-training systemic administration of ketamine (5, 10 and 15mg/kg, i.p.) dose-dependently decreased the memory acquisition of a one-trial passive avoidance task. Pre-training intra-mPFC injection of muscimol, GABAA receptor agonist (0.05, 0.1 and 0.2μg/mouse) and baclofen GABAB receptor agonist (0.05, 0.1, 0.5 and 1μg/mouse), impaired memory acquisition. However, co-pretreatment of different doses of muscimol and baclofen with a lower dose of ketamine (5mg/kg), which did not induce amnesia by itself, caused inhibition of memory formation. Our data showed that sole pre-training administration of bicuculline, GABA-A receptor antagonist and phaclofen GABA-B receptor antagonist into the mPFC, did not affect memory acquisition. In addition, the amnesia induced by pre-training ketamine (15mg/kg) was significantly decreased by the pretreatment of bicuculline (0.005, 0.1 and 0.5μg/mouse). It can be concluded that GABAergic system of the mPFC is involved in the ketamine-induced impairment of memory acquisition. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

GABAA- and glycine-mediated inhibitory modulation of the cough reflex in the caudal nucleus tractus solitarii of the rabbit.

PubMed

Cinelli, Elenia; Iovino, Ludovica; Bongianni, Fulvia; Pantaleo, Tito; Mutolo, Donatella

2016-09-01

Cough-related sensory inputs from rapidly adapting receptors (RARs) and C fibers are processed by second-order neurons mainly located in the caudal nucleus tractus solitarii (NTS). Both GABAA and glycine receptors have been proven to be involved in the inhibitory control of second-order cells receiving RAR projections. We investigated the role of these receptors within the caudal NTS in the modulation of the cough reflex induced by either mechanical or chemical stimulation of the tracheobronchial tree in pentobarbital sodium-anesthetized, spontaneously breathing rabbits. Bilateral microinjections (30-50 nl) of the receptor antagonists bicuculline and strychnine as well as of the receptor agonists muscimol and glycine were performed. Bicuculline (0.1 mM) and strychnine (1 mM) caused decreases in peak abdominal activity and marked increases in respiratory frequency due to decreases in both inspiratory time (Ti) and expiratory time (Te), without concomitant changes in arterial blood pressure. Noticeably, these microinjections induced potentiation of the cough reflex consisting of increases in the cough number associated with decreases either in cough-related Ti after bicuculline or in both cough-related Ti and Te after strychnine. The effects caused by muscimol (0.1 mM) and glycine (10 mM) were in the opposite direction to those produced by the corresponding antagonists. The results show that both GABAA and glycine receptors within the caudal NTS mediate a potent inhibitory modulation of the pattern of breathing and cough reflex responses. They strongly suggest that disinhibition is one important mechanism underlying cough regulation and possibly provide new hints for novel effective antitussive strategies. Copyright © 2016 the American Physiological Society.

Anesthetic effects changeable in habitual drinkers: Mechanistic drug interactions with neuro-active indoleamine-aldehyde condensation products associated with alcoholic beverage consumption.

PubMed

Tsuchiya, Hironori

2016-07-01

Clinicians often experience the reduced efficacy of general and local anesthetics and anesthesia-related drugs in habitual drinkers and chronic alcoholics. However, the mechanistic background underlying such anesthetic tolerance remains unclear. Biogenic indoleamines condense with alcohol-derived aldehydes during fermentation processes and under physiological conditions to produce neuro-active tetrahydro-β-carbolines and β-carbolines, many of which are contained not only in various alcoholic beverages but also in human tissues and body fluids. These indoleamine-aldehyde condensation products are increased in the human body because of their exogenous and endogenous supply enhanced by alcoholic beverage consumption. Since tetrahydro-β-carbolines and β-carbolines target receptors, ion channels and neuronal membranes which are common to anesthetic agents, we propose a hypothesis that they may pharmacodynamically interact at GABAA receptors, NMDA receptors, voltage-gated Na(+) channels and membrane lipid bilayers to attenuate anesthetics-induced positive allosteric GABAA receptor modulation, NMDA receptor antagonism, ion channel blockade and neuronal membrane modification, thereby affecting anesthetic efficacy. The condensation products may also cooperatively interact with ethanol that induces adaptive changes and cross-tolerance to anesthetics and with dopamine-aldehyde adducts that act on GABAA receptors and membrane lipids. Because tetrahydro-β-carbolines and β-carbolines are metabolized to lose or decrease their neuro-activities, induction of the relevant enzymes by habitual drinking could produce an inter-individual difference of drinkers in susceptibility to anesthetic agents. The present hypothesis would also provide a unified framework for different modes of anesthetic action, which are inhibited by neuro-active indoleamine-aldehyde condensation products associated with alcoholic beverage consumption. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structural determinants for antagonist pharmacology that distinguish the rho1 GABAC receptor from GABAA receptors.

PubMed

Zhang, Jianliang; Xue, Fenqin; Chang, Yongchang

2008-10-01

GABA receptor (GABAR) types C (GABACR) and A (GABAAR) are both GABA-gated chloride channels that are distinguished by their distinct competitive antagonist properties. The structural mechanism underlying these distinct properties is not well understood. In this study, using previously identified binding residues as a guide, we made individual or combined mutations of nine binding residues in the rho1 GABACR subunit to their counterparts in the alpha1beta2gamma2 GABAAR or reverse mutations in alpha1 or beta2 subunits. The mutants were expressed in Xenopus laevis oocytes and tested for sensitivities of GABA-induced currents to the GABAA and GABAC receptor antagonists. The results revealed that bicuculline insensitivity of the rho1 GABACR was mainly determined by Tyr106, Phe138 and Phe240 residues. Gabazine insensitivity of the rho1 GABACR was highly dependent on Tyr102, Tyr106, and Phe138. The sensitivity of the rho1 GABACR to 3-aminopropyl-phosphonic acid and its analog 3-aminopropyl-(methyl)phosphinic acid mainly depended on residues Tyr102, Val140, FYS240-242, and Phe138. Thus, the residues Tyr102, Tyr106, Phe138, and Phe240 in the rho1 GABACR are major determinants for its antagonist properties distinct from those in the GABAAR. In addition, Val140 in the GABACR contributes to 3-APA binding. In conclusion, we have identified the key structural elements underlying distinct antagonist properties for the GABACR. The mechanistic insights were further extended and discussed in the context of antagonists docking to the homology models of GABAA or GABAC receptors.

Gamma-aminobutyric acid (GABA) stimulates pancreatic cancer growth through overexpressing GABAA receptor pi subunit.

PubMed

Takehara, Akio; Hosokawa, Masayo; Eguchi, Hidetoshi; Ohigashi, Hiroaki; Ishikawa, Osamu; Nakamura, Yusuke; Nakagawa, Hidewaki

2007-10-15

Gamma-aminobutyric acid (GABA) functions primarily as an inhibitory neurotransmitter in the mature central nervous system, and GABA/GABA receptors are also present in nonneural tissues, including cancer, but their precise function in nonneuronal or cancerous cells has thus far been poorly defined. Through the genome-wide cDNA microarray analysis of pancreatic ductal adenocarcinoma (PDAC) cells as well as subsequent reverse transcription-PCR and Northern blot analyses, we identified the overexpression of GABA receptor pi subunit (GABRP) in PDAC cells. We also found the expression of this peripheral type GABAA receptor subunit in few adult human organs. Knockdown of endogenous GABRP expression in PDAC cells by small interfering RNA attenuated PDAC cell growth, suggesting its essential role in PDAC cell viability. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRP-expressing PDAC cells, but not GABRP-negative cells, and GABAA receptor antagonists inhibited this growth-promoting effect by GABA. The HEK293 cells constitutively expressing exogenous GABRP revealed the growth-promoting effect of GABA treatment. Furthermore, GABA treatment in GABRP-positive cells increased intracellular Ca2+ levels and activated the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) cascade. Clinical PDAC tissues contained a higher level of GABA than normal pancreas tissues due to the up-regulation of glutamate decarboxylase 1 expression, suggesting their autocrine/paracrine growth-promoting effect in PDACs. These findings imply that GABA and GABRP could play important roles in PDAC development and progression, and that this pathway can be a promising molecular target for the development of new therapeutic strategies for PDAC.

Inhibition of Orexin Signaling Promotes Sleep Yet Preserves Salient Arousability in Monkeys

PubMed Central

Tannenbaum, Pamela L.; Tye, Spencer J.; Stevens, Joanne; Gotter, Anthony L.; Fox, Steven V.; Savitz, Alan T.; Coleman, Paul J.; Uslaner, Jason M.; Kuduk, Scott D.; Hargreaves, Richard; Winrow, Christopher J.; Renger, John J.

2016-01-01

Study Objectives: In addition to enhancing sleep onset and maintenance, a desirable insomnia therapeutic agent would preserve healthy sleep's ability to wake and respond to salient situations while maintaining sleep during irrelevant noise. Dual orexin receptor antagonists (DORAs) promote sleep by selectively inhibiting wake-promoting neuropeptide signaling, unlike global inhibition of central nervous system excitation by gamma-aminobutyric acid (GABA)-A receptor (GABAaR) modulators. We evaluated the effect of DORA versus GABAaR modulators on underlying sleep architecture, ability to waken to emotionally relevant stimuli versus neutral auditory cues, and performance on a sleepiness-sensitive cognitive task upon awakening. Methods: DORA-22 and GABAaR modulators (eszopiclone, diazepam) were evaluated in adult male rhesus monkeys (n = 34) with continuous polysomnography recordings in crossover studies of sleep architecture, arousability to a classically conditioned salient versus neutral acoustical stimulus, and psychomotor vigilance task (PVT) performance if awakened. Results: All compounds decreased wakefulness, but only DORA-22 sleep resembled unmedicated sleep in terms of underlying sleep architecture, preserved ability to awaken to salient-conditioned acoustic stimuli while maintaining sleep during neutral acoustic stimuli, and no congnitive impairment in PVT performance. Although GABAaR modulators induced lighter sleep, monkeys rarely woke to salient stimuli and PVT performance was impaired if monkeys were awakened. Conclusions: In nonhuman primates, DORAs' targeted mechanism for promoting sleep protects the ability to selectively arouse to salient stimuli and perform attentional tasks unimpaired, suggesting meaningful differentiation between a hypnotic agent that works through antagonizing orexin wake signaling versus the sedative hypnotic effects of the GABAaR modulator mechanism of action. Citation: Tannenbaum PL, Tye SJ, Stevens J, Gotter AL, Fox SV, Savitz AT, Coleman PJ, Uslaner JM, Kuduk SD, Hargreaves R, Winrow CJ, Renger JJ. Inhibition of orexin signaling promotes sleep yet preserves salient arousability in monkeys. SLEEP 2016;39(3):603–612. PMID:26943466

GABA(A) receptor modulation during adolescence alters adult ethanol intake and preference in rats.

PubMed

Hulin, Mary W; Amato, Russell J; Winsauer, Peter J

2012-02-01

To address the hypothesis that GABA(A) receptor modulation during adolescence may alter the abuse liability of ethanol during adulthood, the effects of adolescent administration of both a positive and negative GABA(A) receptor modulator on adult alcohol intake and preference were assessed. Three groups of adolescent male rats received 12 injections of lorazepam (3.2 mg/kg), dehydroepiandrosterone (DHEA, 56 mg/kg), or vehicle on alternate days starting on postnatal day (PD) 35. After this time, the doses were increased to 5.6 and 100 mg/kg, respectively, for 3 more injections on alternate days. Subjects had access to 25 to 30 g of food daily, during the period of the first 6 injections, and 18 to 20 g thereafter. Food intake of each group was measured 60 minutes after food presentation, which occurred immediately after drug administration on injection days or at the same time of day on noninjection days. When subjects reached adulthood (PD 88), ethanol preference was determined on 2 separate occasions, an initial 3-day period and a 12-day period, in which increasing concentrations of ethanol were presented. During each preference test, intake of water, saccharin, and an ethanol/saccharin solution was measured after each 23-hour access period. During adolescence, lorazepam increased 60-minute food intake, and this effect was enhanced under the more restrictive feeding schedule. DHEA had the opposite effect on injection days, decreasing food intake compared with noninjection days. In adulthood, the lorazepam-treated group preferred the 2 lowest concentrations of ethanol/saccharin more than saccharin alone compared with vehicle-treated subjects, which showed no preference for any concentration of ethanol/saccharin over saccharin. DHEA-treated subjects showed no preference among the 3 solutions. These data demonstrate that GABA(A) receptor modulation during adolescence can alter intake and preference for ethanol in adulthood and highlights the importance of drug history as an important variable in the liability for alcohol abuse. Copyright © 2011 by the Research Society on Alcoholism.

A review of evidence for GABergic predominance/glutamatergic deficit as a common etiological factor in both schizophrenia and affective psychoses: more support for a continuum hypothesis of "functional" psychosis.

PubMed

Squires, R F; Saederup, E

1991-10-01

Virtually all antidepressant and antipsychotic drugs, including clozapine, rimcazole and lithium ion, are proconvulsants, and convulsive therapy, using metrazol, a known GABA-A antagonist, as well as electro-convulsive therapy, can be effective in treating both schizophrenia and affective psychoses. Many antidepressant and antipsychotic drugs, including clozapine, as well as some of their metabolites, reverse the inhibitory effect of GABA on 35S-TBPS binding, a reliable predictor of GABA-A receptor blockade. A review of relevant literature suggests that 1) "functional" psychoses constitute a continuum of disorders ranging from schizophrenia to affective psychoses with overlap of symptoms, heredity and treatments, 2) a weakening of GABergic inhibitory activity, or potentiation of counterbalancing glutamatergic neurotransmission, in the brain, may be involved in the therapeutic activities of both antidepressant and antipsychotic drugs, and 3) schizophrenia and the affective psychoses may be different expressions of the same underlying defect: GABergic preponderance/glutamatergic deficit. Schizophrenia and affective psychoses share the following: 1) several treatments are effective in both, 2) similar modes of inheritance, 3) congruent seasonal birth excesses, 4) enlarged cerebral ventricles and cerebellar vermian atrophy, 5) dexamethasone non-suppression. Both genetic and environmental factors are involved in both schizophrenia and affective psychoses, and several lines of evidence suggest that important environmental factors are neurotropic pathogens that selectively destroy glutamatergic neurons. One group of genes associated with psychoses may increase vulnerability to attack and destruction, by neurotropic pathogens, of excitatory glutamatergic neurons that counterbalance inhibitory GABergic neurons. A second group of genes may encode subunits of overactive GABA-A receptors, while a third group of genes may encode subunits of hypo-active glutamate receptors. Improved antipsychotic drugs may be found among selective blockers of GABA-A receptor subtypes and/or enhancers of glutamatergic neurotransmission. A mechanism similar to kindling, leading to long-lasting reduction of GABergic inhibition in the brain, may be involved in several treatments of psychoses.

The effect of age on the discriminative stimulus effects of ethanol and its GABA(A) receptor mediation in cynomolgus monkeys.

PubMed

Helms, Christa M; Grant, Kathleen A

2011-08-01

Excessive alcohol consumption is less common among aged compared to young adults, with aged adults showing greater sensitivity to many behavioral effects of ethanol. This study compared the discriminative stimulus effects of ethanol in young and middle-aged adult cynomolgus monkeys (Macaca fascicularis) and its γ-aminobutyric acid (GABA)(A) receptor mediation. Two male and two female monkeys trained to discriminate ethanol (1.0 g/kg, i.g.; 60-min pre-treatment interval) from water at 5-6 years of age (Grant et al. in Psychopharmacology 152:181-188, 2000) were re-trained in the current study more than a decade later (19.3 ± 1.0 years of age) for a within-subjects comparison. Also, four experimentally naïve middle-aged (mean ± SEM, 17.0 ± 1.5 years of age) female monkeys were trained to discriminate ethanol for between-subjects comparison with published data from young adult naïve monkeys. Two of the naïve middle-aged monkeys attained criterion performance, with weak stimulus control and few discrimination tests, despite greater blood-ethanol concentration 60 min after 1.0 g/kg ethanol in middle-aged compared to young adult female monkeys (Green et al. in Alcohol Clin Exp Res 23:611-616, 1999). The efficacy of the GABA(A) receptor positive modulators pentobarbital, midazolam, allopregnanolone, pregnanolone, and androsterone to substitute for the discriminative stimulus effects of 1.0 g/kg ethanol was maintained from young adulthood to middle age. The data suggest that 1.0 g/kg ethanol is a weak discriminative stimulus in naive middle-aged monkeys. Nevertheless, the GABA(A) receptor mechanisms mediating the discriminative stimulus effects of ethanol, when learned as a young adult, appear stable across one third of the primate lifespan.

Neurosteroid Modulators of GABAA Receptors Differentially Modulate Ethanol Intake Patterns in Male C57BL/6J Mice

PubMed Central

Ford, Matthew M.; Nickel, Jeffrey D.; Phillips, Tamara J.; Finn, Deborah A.

2006-01-01

Background Allopregnanolone (ALLO) and structurally related endogenous neurosteroids are potent modulators of GABAA receptor function at physiologically relevant concentrations. Accumulating evidence implicates a modulatory role for ALLO in behavioral processes underlying ethanol self-administration, discrimination and reinstatement. The purpose of this study was to evaluate the impact of exogenous neurosteroid challenges with the agonist ALLO and the partial agonist/antagonist epipregnanolone (EPI) on the microarchitecture of ethanol drinking patterns. Methods Male C57BL/6J mice were initiated to consume an unsweetened 10% v/v ethanol solution (10E) by a saccharin fading procedure during daily 2-hour limited access sessions beginning 1 hour after dark phase onset. Cumulative lick responses were recorded for 10E and water using lickometer circuits. After establishing 10E intake baselines, mice were habituated to vehicle injection (VEH; 20% w/v β-cyclodextrin; i.p.), and then were treated with either VEH or neurosteroid immediately prior to the drinking session. Each mouse received a series of ALLO doses (3.2, 10, 17 and 24 mg/kg) alone and EPI doses (0.15, 1, 3 and 10 mg/kg) alone in a counterbalanced within-group design. Results The GABAA receptor positive modulator, ALLO, dose-dependently modulated overall ethanol intake throughout the 2-hr session with the 3.2 mg/kg dose eliciting a significant increase whereas the 24 mg/kg dose produced a significant suppression of ethanol intake versus vehicle pretreatment. ALLO-evoked alterations in intake corresponded with a significant, dose-dependent alterations in bout frequency and inter-bout interval. ALLO also elicited robust, dose-dependent elevations in 10E licks during the initial 5-minutes of access, but subsequently resulted in a dose-dependent suppression of 10E licks during session minutes 20–80. In contrast, the partial agonist/antagonist neurosteroid, EPI, exhibited no influence on any consumption parameter evaluated. Conclusions The present findings suggest that GABAA receptor-active neurosteroids may modulate the regulatory processes that govern the onset, maintenance, and termination of drinking episodes. The differential influence of ALLO and EPI on ethanol intake patterns may reflect an alteration in GABAergic inhibitory tone that is likely due to each neurosteroid’s pharmacological profile at GABAA receptors. Manipulation of endogenous ALLO may prove a useful strategy for diminishing excessive intake and protecting against the loss of regulatory control over drinking. PMID:16205363

Nootropic α7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators

PubMed Central

Ng, Herman J.; Whittemore, Edward R.; Tran, Minhtam B.; Hogenkamp, Derk J.; Broide, Ron S.; Johnstone, Timothy B.; Zheng, Lijun; Stevens, Karen E.; Gee, Kelvin W.

2007-01-01

Activation of brain α7 nicotinic acetylcholine receptors (α7 nAChRs) has broad therapeutic potential in CNS diseases related to cognitive dysfunction, including Alzheimer's disease and schizophrenia. In contrast to direct agonist activation, positive allosteric modulation of α7 nAChRs would deliver the clinically validated benefits of allosterism to these indications. We have generated a selective α7 nAChR-positive allosteric modulator (PAM) from a library of GABAA receptor PAMs. Compound 6 (N-(4-chlorophenyl)-α-[[(4-chloro-phenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide) evokes robust positive modulation of agonist-induced currents at α7 nAChRs, while preserving the rapid native characteristics of desensitization, and has little to no efficacy at other ligand-gated ion channels. In rodent models, it corrects sensory-gating deficits and improves working memory, effects consistent with cognitive enhancement. Compound 6 represents a chemotype for allosteric activation of α7 nAChRs, with therapeutic potential in CNS diseases with cognitive dysfunction. PMID:17470817

Laminar distribution of GABAA- and glycine-receptor mediated tonic inhibition in the dorsal horn of the rat lumbar spinal cord: effects of picrotoxin and strychnine on expression of Fos-like immunoreactivity.

PubMed

Cronin, John N; Bradbury, Elizabeth J; Lidierth, Malcolm

2004-11-01

Inhibitory mechanisms are essential in suppressing the development of allodynia and hyperalgesia in the normal animal and there is evidence that loss of inhibition can lead to the development of neuropathic pain. We used Fos expression to map the distribution of tonically inhibited cells in the healthy rat lumbar spinal cord. In a control group, Fos-like immunoreactive (Fos-LI) cells were rare, averaging 7.5+/-2.2 cells (mean+/-SEM; N=13 sections) per 20 microm thick section of dorsal horn. This rose to 103+/-11 (mean+/-SEM; N=20) in picrotoxin-treated rats and to 88+/-11 (mean+/-SEM; N=18) in strychnine-treated rats. These changes were significant (ANOVA; P<0.001). There were marked regional variations in the distribution of Fos-LI cells between picrotoxin- and strychnine-treated animals. Picrotoxin induced a significant increase in the number of Fos-LI cells throughout the dorsal horn (lamina I-VI) while strychnine significantly elevated Fos-like immunoreactivity only in deep laminae (III-VI). For both picrotoxin and strychnine, the increase in Fos-like immunoreactivity peaked in lamina V (at 3579+/-319 and 3649+/-375% of control, respectively; mean+/-SEM) but for picrotoxin an additional peak was observed in the outer part of lamina II (1959+/-196%). Intrathecal administration of both GABAA and glycine receptor antagonists has been shown elsewhere to induce tactile allodynia. The present data suggest that this allodynia could arise due to blockade of tonic GABAA and glycine-receptor mediated inhibition in the deep dorsal horn. GABAA antagonists also induce hypersensitivity to noxious inputs. The blockade of tonic inhibition in the superficial dorsal horn shown here may underlie this hyperalgesia.

Altered neurotransmitter function in CO2-exposed stickleback (Gasterosteus aculeatus): a temperate model species for ocean acidification research.

PubMed

Lai, Floriana; Jutfelt, Fredrik; Nilsson, Göran E

2015-01-01

Studies on the consequences of ocean acidification for the marine ecosystem have revealed behavioural changes in coral reef fishes exposed to sustained near-future CO2 levels. The changes have been linked to altered function of GABAergic neurotransmitter systems, because the behavioural alterations can be reversed rapidly by treatment with the GABAA receptor antagonist gabazine. Characterization of the molecular mechanisms involved would be greatly aided if these can be examined in a well-characterized model organism with a sequenced genome. It was recently shown that CO2-induced behavioural alterations are not confined to tropical species, but also affect the three-spined stickleback, although an involvement of the GABAA receptor was not examined. Here, we show that loss of lateralization in the stickleback can be restored rapidly and completely by gabazine treatment. This points towards a worrying universality of disturbed GABAA function after high-CO2 exposure in fishes from tropical to temperate marine habitats. Importantly, the stickleback is a model species with a sequenced and annotated genome, which greatly facilitates future studies on underlying molecular mechanisms.

A Balanced Chromosomal Translocation Disrupting ARHGEF9 Is Associated With Epilepsy, Anxiety, Aggression, and Mental Retardation

PubMed Central

Kalscheuer, Vera M.; Musante, Luciana; Fang, Cheng; Hoffmann, Kirsten; Fuchs, Celine; Carta, Eloisa; Deas, Emma; Venkateswarlu, Kanamarlapudi; Menzel, Corinna; Ullmann, Reinhard; Tommerup, Niels; Dalprà, Leda; Tzschach, Andreas; Selicorni, Angelo; Lüscher, Bernhard; Ropers, Hans-Hilger; Harvey, Kirsten; Harvey, Robert J.

2013-01-01

Clustering of inhibitory γ-aminobutyric acidA (GABAA) and glycine receptors at synapses is thought to involve key interactions between the receptors, a “scaffolding” protein known as gephyrin and the RhoGEF collybistin. We report the identification of a balanced chromosomal translocation in a female patient presenting with a disturbed sleep-wake cycle, late-onset epileptic seizures, increased anxiety, aggressive behavior, and mental retardation, but not hyperekplexia. Fine mapping of the breakpoint indicates disruption of the collybistin gene (ARHGEF9) on chromosome Xq11, while the other breakpoint lies in a region of 18q11 that lacks any known or predicted genes. We show that defective collybistin transcripts are synthesized and exons 7–10 are replaced by cryptic exons from chromosomes X and 18. These mRNAs no longer encode the pleckstrin homology (PH) domain of collybistin, which we now show binds phosphatidylinositol-3-phosphate (PI3P/ PtdIns-3-P), a phosphoinositide with an emerging role in membrane trafficking and signal transduction, rather than phosphatidylinositol 3,4,5-trisphosphate (PIP3/PtdIns-3,4,5-P) as previously suggested in the “membrane activation model” of gephyrin clustering. Consistent with this finding, expression of truncated collybistin proteins in cultured neurons interferes with synaptic localization of endogenous gephyrin and GABAA receptors. These results suggest that collybistin has a key role in membrane trafficking of gephyrin and selected GABAA receptor subtypes involved in epilepsy, anxiety, aggression, insomnia, and learning and memory. PMID:18615734

Delta-opioid receptor expression in the ventral tegmental area protects against elevated alcohol consumption.

PubMed

Margolis, Elyssa B; Fields, Howard L; Hjelmstad, Gregory O; Mitchell, Jennifer M

2008-11-26

Alcoholism is a complex and debilitating syndrome affecting approximately 140 million people worldwide. However, not everyone who consumes ethanol develops abuse, raising the possibility that some individuals have a protective mechanism that inhibits elevated alcohol consumption. We tested the hypothesis that the delta-opioid receptor (DOR) plays such a protective role. Here we show that DOR activity in the ventral tegmental area (VTA) robustly decreases ethanol consumption in rats and that these effects depend on baseline ethanol consumption. Intra-VTA microinjection of the DOR agonist DPDPE decreases drinking, particularly in low-drinking animals. Furthermore, VTA microinjection of the DOR selective antagonist TIPP-Psi increases drinking in low, but not high, drinkers and this increase is blocked by comicroinjection of the GABA(A) antagonist bicuculline. Using electrophysiological techniques we found that in VTA brain slices from drinking rats DPDPE presynaptically inhibits GABA(A) receptor mediated IPSCs in low drinkers, but not in high drinkers or naive animals, most likely through activation of DORs on GABA terminals. This DOR-mediated inhibition of IPSCs also correlates inversely with behavioral correlates of anxiety measured in the elevated plus maze. In contrast, presynaptic inhibition of VTA GABA(A) IPSCs by the mu-opioid receptor agonist DAMGO is significantly reduced in both high- and low-drinking rats (<30%) compared with age-matched nondrinking controls (>70%). Together, our findings demonstrate the protective nature of VTA DORs and identify an important new target for therapeutic intervention for alcoholism.

Recruitment of GABA(A) receptors and fearfulness in chicks: modulation by systemic insulin and/or epinephrine.

PubMed

Cid, Mariana Paula; Toledo, Carolina Maribel; Salvatierra, Nancy Alicia

2013-02-01

One-day-old chicks were individually assessed on their latency to peck pebbles, and categorized as low latency (LL) or high latency (HL) according to fear. Interactions between acute stress and systemic insulin and epinephrine on GABA(A) receptor density in the forebrain were studied. At 10 days of life, LL and HL chicks were intraperitoneally injected with insulin, epinephrine or saline, and immediately after stressed by partial water immersion for 15 min and killed by decapitation. Forebrains were dissected and the GABA(A) receptor density was measured ex vivo by the (3)[H]-flunitrazepam binding assay in synaptosomes. In non-stressed chicks, insulin (non-hypoglycemic dose) at 2.50 IU/kg of body weight incremented the Bmax by 40.53% in the HL chicks compared to saline group whereas no significant differences were observed between individuals in the LL subpopulation. Additionally, insulin increased the Bmax (23.48%) in the HL group with respect to the LL ones, indicating that the insulin responses were different according to the anxiety of each category. Epinephrine administration (0.25 and 0.50mg/kg) incremented the Bmax in non-stressed chicks, in the LL group by about 37% and 33%, respectively, compared to ones injected with saline. In the stressed chicks, 0.25mg/kg bw epinephrine increased the Bmax significantly in the HL group by about 24% compared to saline, suggesting that the effect of epinephrine was only observed in the HL group under acute stress conditions. Similarly, the same epinephrine doses co-administered with insulin increased the receptor density in both subpopulations and also showed that the highest dose of epinephrine did not further increase the maximum density of GABA(A)R in HL chicks. These results suggest that systemic epinephrine, perhaps by evoking central norepinephrine release, modulated the increase in the forebrain GABA(A) receptor recruitment induced by both insulin and stress in different ways depending on the subpopulation fearfulness. Copyright © 2013 Elsevier Inc. All rights reserved.

Taurine-induced attenuation of MPP+ neurotoxicity in vitro: a possible role for the GABA(A) subclass of GABA receptors.

PubMed

O'Byrne, M B; Tipton, K F

2000-05-01

Taurine is a sulphur-containing beta-amino acid found in high (millimolar) concentrations in excitable tissues such as brain and heart. Its suggested roles include osmoregulator, thermoregulator, neuromodulator, and potential neurotransmitter. This amino acid has also been shown to be released in large concentrations during ischaemia and excitotoxin-induced neuronal damage. Here we report a protective effect of taurine against MPP(+)-induced neurotoxicity in coronal slices from rat brain. Significant protective effects were observed at taurine concentrations of 20 and 1 mM, suggesting a potential role for taurine in cases of neuronal insult. Studies with the synthetic taurine analogues taurine phosphonate, guanidinoethane sulphonate, and trimethyltaurine suggested the observed effect to be mediated via an extracellular mechanism. The use of GABA receptor ligands muscimol and bicuculline indicated the effect to be mediated through activation of GABA(A) receptors.

GABA, its receptors, and GABAergic inhibition in mouse taste buds

PubMed Central

Dvoryanchikov, Gennady; Huang, Yijen A; Barro-Soria, Rene; Chaudhari, Nirupa; Roper, Stephen D.

2012-01-01

Taste buds consist of at least three principal cell types that have different functions in processing gustatory signals — glial-like Type I cells, Receptor (Type II) cells, and Presynaptic (Type III) cells. Using a combination of Ca2+ imaging, single cell RT-PCR, and immunostaining, we show that γ-amino butyric acid (GABA) is an inhibitory transmitter in mouse taste buds, acting on GABA-A and GABA-B receptors to suppress transmitter (ATP) secretion from Receptor cells during taste stimulation. Specifically, Receptor cells express GABA-A receptor subunits β2, δ, π, as well as GABA-B receptors. In contrast, Presynaptic cells express the GABA-Aβ3 subunit and only occasionally GABA-B receptors. In keeping with the distinct expression pattern of GABA receptors in Presynaptic cells, we detected no GABAergic suppression of transmitter release from Presynaptic cells. We suggest that GABA may serve function(s) in taste buds in addition to synaptic inhibition. Finally, we also defined the source of GABA in taste buds: GABA is synthesized by GAD65 in Type I taste cells as well as by GAD67 in Presynaptic (Type III) taste cells and is stored in both those two cell types. We conclude that GABA is released during taste stimulation and possibly also during growth and differentiation of taste buds. PMID:21490220

Effects of GABA ligands injected into the nucleus accumbens shell on fear/anxiety-like and feeding behaviours in food-deprived rats.

PubMed

Lopes, Ana Paula Fraga; Ganzer, Laís; Borges, Aline Caon; Kochenborger, Larissa; Januário, Ana Cláudia; Faria, Moacir Serralvo; Marino-Neto, José; Paschoalini, Marta Aparecida

2012-03-01

In an attempt to establish a relationship between food intake and fear/anxiety-related behaviours, the goal of this study was to investigate the effect of bilateral injections of GABAA (Muscimol, MUS, doses 25 and 50ng/side) and GABAB (Baclofen, BAC, doses 32 and 64ng/side) receptor agonists in the nucleus accumbens shell (AcbSh) on the level of fear/anxiety-like and feeding behaviours in 24h food-deprived rats. The antagonists of GABAA (Bicuculline, BIC, doses 75 and 150ng/side) and GABAB (Saclofen, SAC, doses 1.5 and 3μg/side) were also tested. The results indicated that the total number of risk assessment behaviour decreased after the injection of both doses of GABAA agonist (MUS) into the AcbSh of 24h food-deprived rats exposed to elevated plus maze. Similar results were obtained after treatment with both doses of GABAB (BAC) agonist in the AcbSh. These data indicated that the activation of both GABAA and GABAB receptors within the AcbSh caused anxiolysis in 24h food-deprived rats. In addition, feeding behaviour (food intake, feeding latency and feeding duration) remained unchanged after treatment with both GABA agonists. In contrast, both food intake and feeding duration decreased after injections of both doses of BIC (GABAA antagonist), while the feeding latency remained unchanged after treatment with both GABA antagonists in the AcbSh of 24h food-deprived rats. The treatment with SAC (GABAB antagonist) did not affect feeding behaviour. Collectively, these data suggest that emotional changes evoked by pharmacological manipulation of the GABA neurotransmission in the AcbSh are not linked with changes in food intake. Copyright © 2011 Elsevier Inc. All rights reserved.

LINKING GABAA RECEPTOR SUBUNITS TO ALCOHOL-INDUCED CONDITIONED TASTE AVERSION AND RECOVERY FROM ACUTE ALCOHOL INTOXICATION

PubMed Central

Blednov, Y.A.; Benavidez, J.M.; Black, M.; Chandra, D.; Homanics, G.E.; Rudolph, U.; Harris, R.A.

2012-01-01

GABA type A receptors (GABAA-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABAA-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011) and indicate this aversive property of ethanol is dependent on ethanol action on α2-containing GABAA-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor-incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABAA-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 and α3 (-/-) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. PMID:23147414

RDX Binds to the GABAA Receptor–Convulsant Site and Blocks GABAA Receptor–Mediated Currents in the Amygdala: A Mechanism for RDX-Induced Seizures

PubMed Central

Williams, Larry R.; Aroniadou-Anderjaska, Vassiliki; Qashu, Felicia; Finne, Huckelberry; Pidoplichko, Volodymyr; Bannon, Desmond I.; Braga, Maria F. M.

2011-01-01

Background Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high-energy, trinitrated cyclic compound that has been used worldwide since World War II as an explosive in both military and civilian applications. RDX can be released in the environment by way of waste streams generated during the manufacture, use, and disposal of RDX-containing munitions and can leach into groundwater from unexploded munitions found on training ranges. For > 60 years, it has been known that exposure to high doses of RDX causes generalized seizures, but the mechanism has remained unknown. Objective We investigated the mechanism by which RDX induces seizures. Methods and results By screening the affinity of RDX for a number of neurotransmitter receptors, we found that RDX binds exclusively to the picrotoxin convulsant site of the γ-aminobutyric acid type A (GABAA) ionophore. Whole-cell in vitro recordings in the rat basolateral amygdala (BLA) showed that RDX reduces the frequency and amplitude of spontaneous GABAA receptor–mediated inhibitory postsynaptic currents and the amplitude of GABA-evoked postsynaptic currents. In extracellular field recordings from the BLA, RDX induced prolonged, seizure-like neuronal discharges. Conclusions These results suggest that binding to the GABAA receptor convulsant site is the primary mechanism of seizure induction by RDX and that reduction of GABAergic inhibitory transmission in the amygdala is involved in the generation of RDX-induced seizures. Knowledge of the molecular site and the mechanism of RDX action with respect to seizure induction can guide therapeutic strategies, allow more accurate development of safe thresholds for exposures, and help prevent the development of new explosives or other munitions that could pose similar health risks. PMID:21362589

Pharmacology of Ramelteon, a Selective MT1/MT2 Receptor Agonist: A Novel Therapeutic Drug for Sleep Disorders

PubMed Central

Miyamoto, Masaomi

2009-01-01

An estimated one-third of the general population is affected by insomnia, and this number is increasing due to more stressful working conditions and the progressive aging of society. However, current treatment of insomnia with hypnotics, gamma-aminobutyric acid A (GABAA) receptor modulators, induces various side effects, including cognitive impairment, motor disturbance, dependence, tolerance, hangover, and rebound insomnia. Ramelteon (Rozerem; Takeda Pharmaceutical Company Limited, Osaka, Japan) is an orally active, highly selective melatonin MT1/MT2 receptor agonist. Unlike the sedative hypnotics that target GABAA receptor complexes, ramelteon is a chronohypnotic that acts on the melatonin MT1 and MT2 receptors, which are primarily located in the suprachiasmatic nucleus, the body's “master clock.” As such, ramelteon possesses the first new therapeutic mechanism of action for a prescription insomnia medication in over three decades. Ramelteon has demonstrated sleep-promoting effects in clinical trials, and coupled with its favorable safety profile and lack of abuse potential or dependence, this chronohypnotic provides an important treatment option for insomnia. PMID:19228178

Modulation of acetylcholine release from rat striatal slices by the GABA/benzodiazepine receptor complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Supavilai, P.; Karobath, M.

1985-02-04

GABA, THIP and muscimol enhance spontaneous and inhibit electrically induced release of tritium labelled compounds from rat striatal slices which have been pre-labelled with /sup 3/H-choline. Baclofen is inactive in this model. Muscimol can inhibit electrically induced release of tritiated material by approximately 75% with half maximal effects at 2 ..mu..M. The response to muscimol can be blocked by the GABA antagonists bicuculline methobromide, picrotoxin, anisatin, R 5135 and CPTBO (cyclopentylbicyclophosphate). Drugs which act on the benzodiazepine receptor (BR) require the presence of muscimol to be effective and they modulate the effects of muscimol in a bidirectional manner. Thus BRmore » agonists enhance and inverse BR agonists attenuate the inhibitory effects of muscimol on electrically induced release. Ro15-1788, a BR antagonist, does not modulate the inhibitory effects of muscimol but antagonizes the actions of clonazepam, a BR agonist, and of DMCM, an inverse BR agonist. These results demonstrate that a GABA/benzodiazepine receptor complex can modulate acetylcholine release from rat striatal slices in vitro. 24 references, 3 figures, 5 table.« less

Convulsions induced by centrally administered NMDA in mice: effects of NMDA antagonists, benzodiazepines, minor tranquilizers and anticonvulsants.

PubMed Central

Moreau, J. L.; Pieri, L.; Prud'hon, B.

1989-01-01

1. Convulsions were induced reproducibly by intracerebroventricular injection of N-methyl-D-aspartic acid (NMDA) to conscious mice. 2. Competitive (carboxypiperazine-propylphosphonic acid, CPP; 2-amino-7-phosphonoheptanoic acid, AP7) and non-competitive (MK801; phencyclidine, PCP; thienylcyclohexylpiperidine, TCP; dextrorphan; dextromethorphan) NMDA antagonists prevented NMDA-induced convulsions. 3. Benzodiazepine receptor agonists and partial agonists (triazolam, diazepam, clonazepam, Ro 16-6028), classical anticonvulsants (diphenylhydantoin, phenobarbitone, sodium valproate) and meprobamate were also found to prevent NMDA-induced convulsions. 4. Flumazenil (a benzodiazepine receptor antagonist) and the GABA agonists THIP and muscimol (up to subtoxic doses) were without effect. 5. Flumazenil reversed the anticonvulsant action of diazepam, but not that of MK801. 6. Results obtained in this model differ somewhat from those described in a seizure model with systemic administration of NMDA. An explanation for this discrepancy is offered. 7. This model is a simple test for assessing the in vivo activity of NMDA antagonists and also expands the battery of chemically-induced seizure models for characterizing anticonvulsants not acting at NMDA receptors. PMID:2574061

GABAergic anxiolytic drug in water increases migration behaviour in salmon

NASA Astrophysics Data System (ADS)

Hellström, Gustav; Klaminder, Jonatan; Finn, Fia; Persson, Lo; Alanärä, Anders; Jonsson, Micael; Fick, Jerker; Brodin, Tomas

2016-12-01

Migration is an important life-history event in a wide range of taxa, yet many migrations are influenced by anthropogenic change. Although migration dynamics are extensively studied, the potential effects of environmental contaminants on migratory physiology are poorly understood. In this study we show that an anxiolytic drug in water can promote downward migratory behaviour of Atlantic salmon (Salmo salar) in both laboratory setting and in a natural river tributary. Exposing salmon smolt to a dilute concentration of a GABAA receptor agonist (oxazepam) increased migration intensity compared with untreated smolt. These results implicate that salmon migration may be affected by human-induced changes in water chemical properties, such as acidification and pharmaceutical residues in wastewater effluent, via alterations in the GABAA receptor function.

Further characterization of benzodiazepine receptor differences in long-sleep and short-sleep mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marley, R.J.; Stinchcomb, A.; Wehner, J.M.

Molecular and conformational characteristics of benzodiazepine (BZ) receptors in cortex and cerebellum from long-sleep and mice were investigated using heat inactivation and beta-carboline competition techniques. To investigate differences in the allosteric coupling between GABA and BZ receptors, the protection of BZ receptors from heat inactivation, by GABA, was also evaluated. The two genotypes do not differ in the affinity or number of BZ receptors in the cortex or cerebellum. They do, however, appear to differ in the molecular structure and/or regulation of the conformational state of the receptor in the cortex, as indicated by a greater sensitivity of LS micemore » to both heat inactivation and beta-carboline competition of /sup 3/H-flunitrazepam (FNZ) binding in this region. Evidence for differences in the nature of coupling between GABA and BZ receptors is provided by the finding in that in both regions, GABA protected BZ receptors from inactivation to a greater degree in LS mice. The relationship between these differences and the multiplicity of expression of BZ receptors is discussed.« less

Functional aspects of early brain development are preserved in tuberous sclerosis complex (TSC) epileptogenic lesions.

PubMed

Ruffolo, Gabriele; Iyer, Anand; Cifelli, Pierangelo; Roseti, Cristina; Mühlebner, Angelika; van Scheppingen, Jackelien; Scholl, Theresa; Hainfellner, Johannes A; Feucht, Martha; Krsek, Pavel; Zamecnik, Josef; Jansen, Floor E; Spliet, Wim G M; Limatola, Cristina; Aronica, Eleonora; Palma, Eleonora

2016-11-01

Tuberous sclerosis complex (TSC) is a rare multi-system genetic disease characterized by several neurological disorders, the most common of which is the refractory epilepsy caused by highly epileptogenic cortical lesions. Previous studies suggest an alteration of GABAergic and glutamatergic transmission in TSC brain indicating an unbalance of excitation/inhibition that can explain, at least in part, the high incidence of epilepsy in these patients. Here we investigate whether TSC cortical tissues could retain GABAA and AMPA receptors at early stages of human brain development thus contributing to the generation and recurrence of seizures. Given the limited availability of pediatric human brain specimens, we used the microtransplantation method of injecting Xenopus oocytes with membranes from TSC cortical tubers and control brain tissues. Moreover, qPCR was performed to investigate the expression of GABAA and AMPA receptor subunits (GABAA α1-5, β3, γ2, δ; GluA1, GluA2) and cation chloride co-transporters NKCC1 and KCC2. The evaluation of nine human cortical brain samples, from 15 gestation weeks to 15years old, showed a progressive shift towards more hyperpolarized GABAA reversal potential (EGABA). This shift was associated with a differential expression of the chloride cotransporters NKCC1 and KCC2. Furthermore, the GluA1/GluA2 mRNA ratio of expression paralleled the development process. On the contrary, in oocytes micro-transplanted with epileptic TSC tuber tissue from seven patients, neither the GABAA reversal potential nor the GluA1/GluA2 expression showed similar developmental changes. Our data indicate for the first time, that in the same cohort of TSC patients, the pattern of both GABAAR and GluA1/GluA2 functions retains features that are typical of an immature brain. These observations support the potential contribution of altered receptor function to the epileptic disorder of TSC and may suggest novel therapeutic approaches. Furthermore, our findings strengthen the novel hypothesis that other developmental brain diseases can share the same hallmarks of immaturity leading to intractable seizures. Copyright © 2016 Elsevier Inc. All rights reserved.

GABA-B receptor activation and conflict behavior

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ketelaars, C.E.J.; Bollen, E.L.; Rigter, H.

1988-01-01

Baclofen and oxazepam enhance extinction of conflict behavior in the Geller-Seifter test while baclofen and diazepam release punished behavior in Vogel's conflict test. In order to investigate the possibility that the effect of the selective GABA-B receptor agonist baclofen is mediated indirectly via the GABA-A/benzodiazepine receptor complex, the effect of pretreatment of rats with baclofen on (/sup 3/H)-diazepam binding to washed and unwashed cortical and cerebellar membranes of rats has been studied. Baclofen pretreatment increase Bmax in washed cerebellar membranes when bicuculline was present in the incubation mixture. No effect was seen in cortical membranes. The present results render itmore » unlikely that the effect of baclofen on extinction of conflict behavior and punished drinking is mediated via the GABA-A/benzodiazepine receptor complex. 50 references, 1 figure, 4 tables.« less

Alterations in food intake elicited by GABA and opioid agonists and antagonists administered into the ventral tegmental area region of rats.

PubMed

Echo, Joyce A; Lamonte, Nicole; Ackerman, Tsippa F; Bodnar, Richard J

2002-05-01

Food intake is significantly increased following administration of mu-selective opioid agonists into the ventral tegmental area (VTA) region acting through multiple local opioid receptor subtypes. Since GABA receptor agonists in the VTA region are capable of eliciting feeding, the present study investigated whether feeding elicited by the mu-selective opioid agonist [D-Ala(2), NMe(4), Gly-ol(5)]-enkephalin (DAMGO) in the VTA region was altered by pretreatment into the same site with equimolar doses of either GABA(A) (bicuculline) or GABA(B) (saclofen) antagonists, and further, whether pretreatment with either general opioid or selective GABA receptor antagonists decreased feeding elicited by GABA(A) (muscimol) or GABA(B) (baclofen) agonists in the VTA region. DAMGO-induced feeding in the VTA region was dose-dependently decreased following pretreatment with either GABA(A) or GABA(B) antagonists in the absence of significant alterations in food intake by the antagonists per se. However, the presence of short-lived seizures following bicuculline in the VTA region suggests that this ingestive effect was caused by nonspecific actions. In contrast, GABA(B) receptors are involved in the full expression of mu-opioid agonist-induced feeding in this region since saclofen failed to elicit either seizure activity or a conditioned taste aversion. Pretreatment with naltrexone in the VTA region reduced intake elicited by baclofen, but not muscimol. Finally, baclofen-induced feeding was significantly reduced by saclofen, but not bicuculline, pretreatment in the VTA region. Therefore, possible coregulation between GABA(B) and opioid receptors in the VTA region, as suggested by immunocytochemical evidence, is supported by these behavioral effects upon ingestion.

Chronic nicotine treatment differentially modifies acute nicotine and alcohol actions on GABA(A) and glutamate receptors in hippocampal brain slices.

PubMed

Proctor, William R; Dobelis, Peter; Moritz, Anna T; Wu, Peter H

2011-03-01

Tobacco and alcohol are often co-abused producing interactive effects in the brain. Although nicotine enhances memory while ethanol impairs it, variable cognitive changes have been reported from concomitant use. This study was designed to determine how nicotine and alcohol interact at synaptic sites to modulate neuronal processes. Acute effects of nicotine, ethanol, and both drugs on synaptic excitatory glutamatergic and inhibitory GABAergic transmission were measured using whole-cell recording in hippocampal CA1 pyramidal neurons from brain slices of mice on control or nicotine-containing diets. Acute nicotine (50 nM) enhanced both GABAergic and glutamatergic synaptic transmission; potentiated GABA(A) receptor currents via activation of α7* and α4β2* nAChRs, and increased N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor currents through α7* receptors. While ethanol (80 mM) also increased GABA(A) currents, it inhibited NMDA currents. Although ethanol had no effect on AMPA currents, it blocked nicotine-induced increases in NMDA and AMPA currents. Following chronic nicotine treatment, acute nicotine or ethanol did not affect NMDA currents, while the effects of GABAergic responses were not altered. Acute ethanol ingestion selectively attenuated nicotine enhancement of excitatory glutamatergic NMDA and AMPA receptor function, suggesting an overall reduction in excitatory output from the hippocampus. It also indicated that ethanol could decrease the beneficial effects of nicotine on memory performance. In addition, chronic nicotine treatment produced tolerance to the effects of nicotine and cross-tolerance to the effects of ethanol on glutamatergic activity, leading to a potential increase in the use of these drugs. British Journal of Pharmacology © 2011 The British Pharmacological Society. No claim to original US government works.

GABA homeostasis contributes to the developmental programming of anxiety-related behavior.

PubMed

Depino, Amaicha Mara; Tsetsenis, Theodoros; Gross, Cornelius

2008-05-19

During development, when inhibitory and excitatory synapses are formed and refined, homeostatic mechanisms act to adjust inhibitory input in order to maintain neural activity within a normal range. As the brain matures, synaptogenesis slows and a relatively stable level of inhibition is achieved. Deficits in inhibitory neurotransmission are associated with increased anxiety-related behavior and drugs that potentiate GABA function, the major inhibitory neurotransmitter in the brain, are effective anxiolytics. These observations raise the possibility that transient perturbations in the activity of neural circuits during development might induce compensatory changes in inhibition that could persist into adulthood and contribute to changes in anxiety-related behavior. To test this hypothesis, we treated mice continuously during the major period of forebrain synaptogenesis (P14-28) with the GABA-A receptor positive modulator diazepam and assessed anxiety-related behavior in adulthood. Control experiments confirmed anxiolytic effects of the drug following one day of treatment and the development of tolerance following two weeks of treatment. When tested in adulthood, one month after the end of treatment, diazepam-treated mice exhibited significantly increased behavioral inhibition in the open-field, elevated-plus maze, and novel object behavioral paradigms. Levels of benzodiazepine binding sites in amygdala and frontal cortex were specifically decreased in diazepam-treated mice demonstrating that homeostatic adjustments in GABA function persist into adulthood. Our results show that increased GABAergic activity can affect the developmental programming of anxiety-related behavior.

Effect of Mimosa pudica (Linn.) extract on anxiety behaviour and GABAergic regulation of 5-HT neuronal activity in the mouse.

PubMed

Ayissi Mbomo, Rigobert; Gartside, Sasha; Ngo Bum, Elizabeth; Njikam, Njifutie; Okello, Ed; McQuade, Richard

2012-04-01

Mimosa pudica (Linn.) (M. pudica L.) is a plant used in some countries to treat anxiety and depression. In the present study we investigated the effects of an aqueous extract of M. pudica L. on mouse anxiety-like behaviour using the elevated T maze, and on regulation of dorsal raphe nucleus (DRN) 5-hydroxytryptamine (5-HT) neuronal activity using an in-vitro mouse brain slice preparation. Acute treatment with M. pudica L. extract had an anxiolytic effect on behaviour in the elevated T maze, specifically on inhibitory avoidance behaviour. Acute application of the extract alone had no effect on the activity of DRN 5-HT neurones. However, when co-applied with the GABA(A) receptor agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), the extract enhanced the inhibitory effect of the THIP on DRN 5-HT neurones. These observed effects of M. pudica L. on both behaviour and GABA modulation of 5-HT neuronal activity are similar to the effects of diazepam, the established anxiolytic and positive modulator of the GABA(A) receptor. This study suggests that the aqueous extract of M. pudica L. contains a positive modulator of GABA(A) receptor function and provides impetus for further investigation of the neuropharmacologically active constituents of the extract.

Improvement in daytime sleepiness with clarithromycin in patients with GABA-related hypersomnia: Clinical experience.

PubMed

Trotti, Lynn Marie; Saini, Prabhjyot; Freeman, Amanda A; Bliwise, Donald L; García, Paul S; Jenkins, Andrew; Rye, David B

2014-07-01

The macrolide antibiotic clarithromycin can enhance central nervous system excitability, possibly by antagonism of GABA-A receptors. Enhancement of GABA signaling has recently been demonstrated in a significant proportion of patients with central nervous system hypersomnias, so we sought to determine whether clarithromycin might provide symptomatic benefit in these patients. We performed a retrospective review of all patients treated with clarithromycin for hypersomnia, in whom cerebrospinal fluid enhanced GABA-A receptor activity in vitro in excess of controls, excluding those with hypocretin deficiency or definite cataplexy. Subjective reports of benefit and objective measures of psychomotor vigilance were collected to assess clarithromycin's effects. Clinical and demographic characteristics were compared in responders and non-responders. In total, 53 patients (38 women, mean age 35.2 (SD 12.8 years)) were prescribed clarithromycin. Of these, 34 (64%) reported improvement in daytime sleepiness, while 10 (19%) did not tolerate its side effects, and nine (17%) found it tolerable but without symptomatic benefit. In those who reported subjective benefit, objective corroboration of improved vigilance was evident on the psychomotor vigilance task. Twenty patients (38%) elected to continue clarithromycin therapy. Clarithromycin responders were significantly younger than non-responders. Clarithromycin may be useful in the treatment of hypersomnia associated with enhancement of GABA-A receptor function. Further evaluation of this novel therapy is needed. © The Author(s) 2013.

Progressive Cortical Neuronal Damage and Extracranial-Intracranial Bypass Surgery in Patients with Misery Perfusion.

PubMed

Yamauchi, H; Kagawa, S; Kishibe, Y; Takahashi, M; Higashi, T

2017-05-01

Misery perfusion may cause selective neuronal damage in atherosclerotic ICA or MCA disease. Bypass surgery can improve misery perfusion and may prevent neuronal damage. On the other hand, surgery conveys a risk for neuronal damage. The purpose of this retrospective study was to determine whether progression of cortical neuronal damage in surgically treated patients with misery perfusion is larger than that in surgically treated patients without misery perfusion or medically treated patients with misery perfusion. We evaluated the distribution of benzodiazepine receptors twice by using PET and 11 C-labeled flumazenil in 18 surgically treated patients with atherosclerotic ICA or MCA disease (9 with misery perfusion and 9 without) and no perioperative stroke before and after bypass surgery; in 8 medically treated patients with misery perfusion and no intervening ischemic event; and in 7 healthy controls. We quantified abnormal decreases in the benzodiazepine receptors of the cerebral cortex within the MCA distribution and compared changes in the benzodiazepine receptor index among the 3 groups. The change in the benzodiazepine receptor index in surgically treated patients with misery perfusion (27.5 ± 15.6) during 7 ± 5 months was significantly larger than that in surgically treated patients without misery perfusion (-5.2 ± 9.4) during 6 ± 4 months ( P < .001) and in medically treated patients with misery perfusion (3.2 ± 15.4) during 16 ± 6 months ( P < .01). Progression of cortical neuronal damage in surgically treated patients with misery perfusion and no perioperative stroke may occur and may be larger than that in medically treated patients with misery perfusion and no intervening ischemic event. © 2017 by American Journal of Neuroradiology.

GABAergic control of food intake in the meat-type chickens.

PubMed

Jonaidi, H; Babapour, V; Denbow, D M

2002-08-01

This study examined the effects of intracerebroventricular injections of gamma-aminobutyric acid (GABA) agonists on short-term food intake in meat-type cockerels. In Experiment 1, birds were injected with various doses of muscimol, a GABA(A) agonist. In Experiment 2, the birds received bicuculline, a GABA(A) antagonist, prior to injection of muscimol. In Experiment 3, the effect of varying doses of baclofen, a GABA(B) agonist, on food intake was determined. The intracerebroventricular injection of muscimol caused a dose-dependent increase in food intake. This effect was significantly attenuated by pretreatment with bicuculline. Food intake was not affected by the intracerebroventricular injection of baclofen. These results suggest that GABA acts within the brain of broilers at a GABA(A), but not GABA(B), receptor to increase voluntary food intake.

RNA editing of the GABAA receptor α3 subunit alters the functional properties of recombinant receptors

PubMed Central

Nimmich, Mitchell L.; Heidelberg, Laura S.; Fisher, Janet L.

2009-01-01

RNA editing provides a post-transcriptional mechanism to increase structural heterogeneity of gene products. Recently, the α3 subunit of the GABAA receptors has been shown to undergo RNA editing. As a result, a highly conserved isoleucine residue in the third transmembrane domain is replaced with a methionine. To determine the effect of this structural change on receptor function, we compared the GABA sensitivity, pharmacological properties and macroscopic kinetics of recombinant receptors containing either the edited or unedited forms of the α3 subunit along with β3 and γ2L. Editing substantially altered the GABA sensitivity and deactivation rate of the receptors, with the unedited form showing a lower GABA EC50 and slower decay. Comparable effects were observed with a mutation at the homologous location in the α1 subunit, suggesting a common role for this site in regulation of channel gating. Except for the response to GABA, the pharmacological properties of the receptor were unaffected by editing, with similar enhancement by a variety of modulators. Since RNA editing of the α3 subunit increases through development, our findings suggest that GABAergic neurotransmission may be more effective early in development, with greater GABA sensitivity and slower decay rates conferred by the unedited α3 subunit. PMID:19367790

Induction of θ-frequency oscillations in the rat medial septal diagonal band slice by metabotropic glutamate receptor agonists.

PubMed

Lu, C B; Ouyang, G; Henderson, Z; Li, X

2011-03-17

The aim of this study was to examine the role of metabotropic glutamate receptors (mGluR) in the generation of oscillatory field activity at theta frequency (4-12 Hz) in the medial septal slice prepared from rat brain. Bath application of mGluR agonists and antagonists showed that activation of mGluR1-type receptors produces persistent theta frequency oscillations in a dose-responsive manner. This activity, induced by the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG), was reduced by ionotropic glutamate receptor antagonists and abolished by further addition of a GABAA receptor antagonist. However, addition of a GABAA receptor antagonist on its own converted the DHPG-induced oscillations to intermittent episodes of accentuated theta frequency activity following a burst. In a proportion of slices, DHPG induced large amplitude field population spiking activity (100-300 μV) which is correlated linearly with the field theta oscillations and is sensitive to glutamate receptor antagonists, suggesting a role of this type of spikes in theta generation induced by DHPG. These data demonstrate that DHPG-sensitive neuronal networks within medial septum generate theta rhythmic activity and are differentially modulated by excitatory and inhibitory ionotropic neurotransmissions. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Origin and Properties of Striatal Local Field Potential Responses to Cortical Stimulation: Temporal Regulation by Fast Inhibitory Connections

PubMed Central

Galiñanes, Gregorio L.; Braz, Barbara Y.; Murer, Mario Gustavo

2011-01-01

Evoked striatal field potentials are seldom used to study corticostriatal communication in vivo because little is known about their origin and significance. Here we show that striatal field responses evoked by stimulating the prelimbic cortex in mice are reduced by more than 90% after infusing the AMPA receptor antagonist CNQX close to the recording electrode. Moreover, the amplitude of local field responses and dPSPs recorded in striatal medium spiny neurons increase in parallel with increasing stimulating current intensity. Finally, the evoked striatal fields show several of the basic known properties of corticostriatal transmission, including paired pulse facilitation and topographical organization. As a case study, we characterized the effect of local GABAA receptor blockade on striatal field and multiunitary action potential responses to prelimbic cortex stimulation. Striatal activity was recorded through a 24 channel silicon probe at about 600 µm from a microdialysis probe. Intrastriatal administration of the GABAA receptor antagonist bicuculline increased by 65±7% the duration of the evoked field responses. Moreover, the associated action potential responses were markedly enhanced during bicuculline infusion. Bicuculline enhancement took place at all the striatal sites that showed a response to cortical stimulation before drug infusion, but sites showing no field response before bicuculline remained unresponsive during GABAA receptor blockade. Thus, the data demonstrate that fast inhibitory connections exert a marked temporal regulation of input-output transformations within spatially delimited striatal networks responding to a cortical input. Overall, we propose that evoked striatal fields may be a useful tool to study corticostriatal synaptic connectivity in relation to behavior. PMID:22163020

Antagonism of GABA-B but not GABA-A receptors in the VTA prevents stress- and intra-VTA CRF-induced reinstatement of extinguished cocaine seeking in rats

PubMed Central

Blacktop, Jordan M.; Vranjkovic, Oliver; Mayer, Matthieu; Van Hoof, Matthew; Baker, David A.; Mantsch, John R.

2015-01-01

Stress-induced reinstatement of cocaine seeking requires corticotropin releasing factor (CRF) actions in the ventral tegmental area (VTA). However the mechanisms through which CRF regulates VTA function to promote cocaine use are not fully understood. Here we examined the role of GABAergic neurotransmission in the VTA mediated by GABA-A or GABA-B receptors in the reinstatement of extinguished cocaine seeking by a stressor, uncontrollable intermittent footshock, or bilateral intra-VTA administration of CRF. Rats underwent repeated daily cocaine self-administration (1.0 mg/kg/ing; 14 × 6 hrs/day) and extinction and were tested for reinstatement in response to footshock (0.5 mA, 0.5” duration, average every 40 sec; range 10–70 sec) or intra-VTA CRF delivery (500 ng/side) following intra-VTA pretreatment with the GABA-A antagonist, bicuculline, the GABA-B antagonist, 2-hydroxysaclofen or vehicle. Intra-VTA bicuculline (1, 10 or 20 ng/side) failed to block footshock- or CRF-induced cocaine seeking at either dose tested. By contrast, 2-hydroxysaclofen (0.2 or 2 µg/side) prevented reinstatement by both footshock and intra-VTA CRF at a concentration that failed to attenuate food-reinforced lever pressing (45 mg sucrose-sweetened pellets; FR4 schedule) in a separate group of rats. These data suggest that GABA-B receptor-dependent CRF actions in the VTA mediate stress-induced cocaine seeking and that GABA-B receptor antagonists may have utility for the management of stress-induced relapse in cocaine addicts. PMID:26596556

Parasympathetic reflex vasodilation in the cerebral hemodynamics of rats.

PubMed

Ishii, Hisayoshi; Sato, Toshiya; Izumi, Hiroshi

2014-04-01

We investigated the role of parasympathetic reflex vasodilation in the regulation of the cerebral hemodynamics, and whether GABAA receptors modulate the response. We examined the effects of activation of the parasympathetic fibers through trigeminal afferent inputs on blood flow in the internal carotid artery (ICABF) and the cerebral blood vessels (rCBF) in parietal cortex in urethane-anesthetized rats. Electrical stimulation of the central cut end of the lingual nerve (LN) elicited intensity- and frequency-dependent increases in ICABF that were independent of changes in external carotid artery blood flow. Increases in ICABF were elicited by LN stimulation regardless of the presence or absence of sympathetic innervation. The ICABF increases evoked by LN stimulation were almost abolished by the intravenous administration of hexamethonium (10 mg kg(-1)) and were reduced significantly by atropine administration (0.1 mg kg(-1)). Although the LN stimulation alone had no significant effect on rCBF, LN stimulation in combination with a blocker of the GABAA receptor pentylenetetrazole increased the rCBF markedly. This increase in rCBF was reduced significantly by the administration of hexamethonium and atropine. These observations indicate that the increases in both ICABF and rCBF are evoked by parasympathetic activation via the trigeminal-mediated reflex. The rCBF increase evoked by LN stimulation is thought to be limited by the GABAA receptors in the central nervous system. These results suggest that the parasympathetic reflex vasodilation and its modulation mediated by GABA receptors within synaptic transmission in the brainstem are involved in the regulation of the cerebral hemodynamics during trigeminal afferent inputs.