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Sample records for acid gabaa receptor

  1. Direct activation of GABAA receptors by substances in the organic acid fraction of Japanese sake.

    PubMed

    Izu, Hanae; Shigemori, Kensuke; Eguchi, Masaya; Kawane, Shuhei; Fujii, Shouko; Kitamura, Yuji; Aoshima, Hitoshi; Yamada, Yasue

    2017-01-01

    We investigated the effect of substances present in Japanese sake on the response of ionotropic γ-aminobutyric acid (GABA)A receptors expressed in Xenopus oocytes. Sake was fractionated by ion-exchange chromatography. The fraction containing organic acids (OA fraction) showed agonist activities on the GABAA receptor. OA fractions from sake were analyzed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Of the 64 compounds identified, 13 compounds showed GABAA receptor agonist activities. Especially, l-lactic acid showed high agonist activity and its EC50 value was 37μM. Intraperitoneal injections of l-lactic acid, gluconic acid, and pyruvic acid (10, 10, and 5mg/kg BW, respectively), which showed agonistic activity on the GABAA receptor, led to significant anxiolytic effects during an elevated plus-maze test in mice. PMID:27507485

  2. Statistical Mechanics Model for the Interaction between the Neurotransmitter γ-Aminobutyric acid and GABAA Receptors

    NASA Astrophysics Data System (ADS)

    Zafar, Sufi; Saxena, Nina C.; Conrad, Kevin A.; Hussain, Arif

    2004-07-01

    Interactions between the neurotransmitter γ-aminobutyric acid (GABA) and GABAA receptor ion channels play an important role in the central nervous system. A statistical mechanics model is proposed for the interaction between GABA and GABAA receptors. The model provides good fits to the electrophysiology data as well as an estimation of receptor activation energies, and predicts the temperature dependence consistent with measurements. In addition, the model provides insights into single channel conductance measurements. This model is also applicable to other ligand-gated ion channels with similar pentameric structures.

  3. γ-Hydroxybutyric acid (GHB) is not an agonist of extrasynaptic GABAA receptors.

    PubMed

    Connelly, William M; Errington, Adam C; Crunelli, Vincenzo

    2013-01-01

    γ-Hydroxybutyric acid (GHB) is an endogenous compound and a drug used clinically to treat the symptoms of narcolepsy. GHB is known to be an agonist of GABAB receptors with millimolar affinity, but also binds with much higher affinity to another site, known as the GHB receptor. While a body of evidence has shown that GHB does not bind to GABAA receptors widely, recent evidence has suggested that the GHB receptor is in fact on extrasynaptic α4β1δ GABAA receptors, where GHB acts as an agonist with an EC50 of 140 nM. We investigated three neuronal cell types that express a tonic GABAA receptor current mediated by extrasynaptic receptors: ventrobasal (VB) thalamic neurons, dentate gyrus granule cells and striatal medium spiny neurons. Using whole-cell voltage clamp in brain slices, we found no evidence that GHB (10 µM) induced any GABAA receptor mediated current in these cell types, nor that it modulated inhibitory synaptic currents. Furthermore, a high concentration of GHB (3 mM) was able to produce a GABAB receptor mediated current, but did not induce any other currents. These results suggest either that GHB is not a high affinity agonist at native α4β1δ receptors, or that these receptors do not exist in classical areas associated with extrasynaptic currents.

  4. Identification of dehydroabietc acid from Boswellia thurifera resin as a positive GABAA receptor modulator.

    PubMed

    Rueda, Diana C; Raith, Melanie; De Mieri, Maria; Schöffmann, Angela; Hering, Steffen; Hamburger, Matthias

    2014-12-01

    In a two-microelectrode voltage clamp assay with Xenopus laevis oocytes, a petroleum ether extract (100 μg/mL) of the resin of Boswellia thurifera (Burseraceae) potentiated GABA-induced chloride currents (IGABA) through receptors of the subtype α₁β₂γ₂s by 319.8% ± 79.8%. With the aid of HPLC-based activity profiling, three known terpenoids, dehydroabietic acid (1), incensole (2), and AKBA (3), were identified in the active fractions of the extract. Structure elucidation was achieved by means of HR-MS and microprobe 1D/2D NMR spectroscopy. Compound 1 induced significant receptor modulation in the oocyte assay, with a maximal potentiation of IGABA of 397.5% ± 34.0%, and EC₅₀ of 8.7 μM ± 1.3 μM. This is the first report of dehydroabietic acid as a positive GABAA receptor modulator. PMID:25200370

  5. Differential regulation of the postsynaptic clustering of γ-aminobutyric acid type A (GABAA) receptors by collybistin isoforms.

    PubMed

    Chiou, Tzu-Ting; Bonhomme, Bevan; Jin, Hongbing; Miralles, Celia P; Xiao, Haiyan; Fu, Zhanyan; Harvey, Robert J; Harvey, Kirsten; Vicini, Stefano; De Blas, Angel L

    2011-06-24

    Collybistin promotes submembrane clustering of gephyrin and is essential for the postsynaptic localization of gephyrin and γ-aminobutyric acid type A (GABA(A)) receptors at GABAergic synapses in hippocampus and amygdala. Four collybistin isoforms are expressed in brain neurons; CB2 and CB3 differ in the C terminus and occur with and without the Src homology 3 (SH3) domain. We have found that in transfected hippocampal neurons, all collybistin isoforms (CB2(SH3+), CB2(SH3-), CB3(SH3+), and CB3(SH3-)) target to and concentrate at GABAergic postsynapses. Moreover, in non-transfected neurons, collybistin concentrates at GABAergic synapses. Hippocampal neurons co-transfected with CB2(SH3-) and gephyrin developed very large postsynaptic gephyrin and GABA(A) receptor clusters (superclusters). This effect was accompanied by a significant increase in the amplitude of miniature inhibitory postsynaptic currents. Co-transfection with CB2(SH3+) and gephyrin induced the formation of many (supernumerary) non-synaptic clusters. Transfection with gephyrin alone did not affect cluster number or size, but gephyrin potentiated the clustering effect of CB2(SH3-) or CB2(SH3+). Co-transfection with CB2(SH3-) or CB2(SH3+) and gephyrin did not affect the density of presynaptic GABAergic terminals contacting the transfected cells, indicating that collybistin is not synaptogenic. Nevertheless, the synaptic superclusters induced by CB2(SH3-) and gephyrin were accompanied by enlarged presynaptic GABAergic terminals. The enhanced clustering of gephyrin and GABA(A) receptors induced by collybistin isoforms was not accompanied by enhanced clustering of neuroligin 2. Moreover, during the development of GABAergic synapses, the clustering of gephyrin and GABA(A) receptors preceded the clustering of neuroligin 2. We propose a model in which the SH3- isoforms play a major role in the postsynaptic accumulation of GABA(A) receptors and in GABAergic synaptic strength.

  6. α4βδ GABA(A) receptors are high-affinity targets for γ-hydroxybutyric acid (GHB).

    PubMed

    Absalom, Nathan; Eghorn, Laura F; Villumsen, Inge S; Karim, Nasiara; Bay, Tina; Olsen, Jesper V; Knudsen, Gitte M; Bräuner-Osborne, Hans; Frølund, Bente; Clausen, Rasmus P; Chebib, Mary; Wellendorph, Petrine

    2012-08-14

    γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABA(A) receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA(A) receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC(50) = 140 nM) over α4β(2/3)δ (EC(50) = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [(3)H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid showed a 39% reduction (P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABA(A) receptors and postulate a role for extrasynaptic α4δ-containing GABA(A) receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.

  7. Action of tremorgenic mycotoxins on GABAA receptor.

    PubMed

    Gant, D B; Cole, R J; Valdes, J J; Eldefrawi, M E; Eldefrawi, A T

    1987-11-01

    The effects of four tremorgenic and one nontremorgenic mycotoxins were studied on gamma-aminobutyric acid (GABAA) receptor binding and function in rat brain and on binding of a voltage-operated Cl- channel in Torpedo electric organ. None of the mycotoxins had significant effect on [3H]muscimol or [3H]flunitrazepam binding to the GABAA receptor. However, only the four tremorgenic mycotoxins inhibited GABA-induced 36Cl- influx and [35S] t-butylbicyclophosphorothionate [( 35S]TBPS) binding in rat brain membranes, while the nontremorgenic verruculotoxin had no effect. Inhibition of [35S]TBPS binding by paspalinine was non-competitive. This suggests that tremorgenic mycotoxins inhibit GABAA receptor function by binding close to the receptor's Cl- channel. On the voltage-operated Cl- channel, only high concentrations of verruculogen and verruculotoxin caused significant inhibition of the channel's binding of [35S]TBPS. The data suggest that the tremorgenic action of these mycotoxins may be due in part to their inhibition of GABAA receptor function. PMID:2444852

  8. GABAA Receptors at Hippocampal Mossy Fibers

    PubMed Central

    Ruiz, Arnaud; Fabian-Fine, Ruth; Scott, Ricardo; Walker, Matthew C.; Rusakov, Dmitri A.; Kullmann, Dimitri M.

    2012-01-01

    Summary Presynaptic GABAA receptors modulate synaptic transmission in several areas of the CNS but are not known to have this action in the cerebral cortex. We report that GABAA receptor activation reduces hippocampal mossy fibers excitability but has the opposite effect when intracellular Cl− is experimentally elevated. Synaptically released GABA mimics the effect of exogenous agonists. GABAA receptors modulating axonal excitability are tonically active in the absence of evoked GABA release or exogenous agonist application. Presynaptic action potential-dependent Ca2+ transients in individual mossy fiber varicosities exhibit a biphasic dependence on membrane potential and are altered by GABAA receptors. Antibodies against the α2 subunit of GABAA receptors stain mossy fibers. Axonal GABAA receptors thus play a potentially important role in tonic and activity-dependent heterosynaptic modulation of information flow to the hippocampus. PMID:12971896

  9. General Anesthetic Actions on GABAA Receptors

    PubMed Central

    Garcia, Paul S; Kolesky, Scott E; Jenkins, Andrew

    2010-01-01

    General anesthetic drugs interact with many receptors in the nervous system, but only a handful of these interactions are critical for producing anesthesia. Over the last 20 years, neuropharmacologists have revealed that one of the most important target sites for general anesthetics is the GABAA receptor. In this review we will discuss what is known about anesthetic – GABAA receptor interactions. PMID:20808541

  10. Analysis of β-Subunit-dependent GABAA Receptor Modulation and Behavioral Effects of Valerenic Acid Derivatives

    PubMed Central

    Hintersteiner, J.; Luger, D.; Haider, M.; Pototschnig, G.; Mihovilovic, M. D.; Schwarzer, C.; Hering, S.

    2016-01-01

    Valerenic acid (VA)—a β2/3-selective GABA type A (GABAA) receptor modulator—displays anxiolytic and anticonvulsive effects in mice devoid of sedation, making VA an interesting drug candidate. Here we analyzed β-subunit-dependent enhancement of GABA-induced chloride currents (IGABA) by a library of VA derivatives and studied their effects on pentylenetetrazole (PTZ)-induced seizure threshold and locomotion. Compound-induced IGABA enhancement was determined in oocytes expressing α1β1γ2S, α1β2γ2S, or α1β3γ2S receptors. Effects on seizure threshold and locomotion were studied using C57BL/6N mice and compared with saline-treated controls. β2/3-selective VA derivatives such as VA-amide (VA-A) modulating α1β3γ2S (VA-A: Emax = 972 ± 69%, n = 6, P < 0.05) and α1β2γ2S receptors (Emax = 1119 ± 72%, n = 6, P < 0.05) more efficaciously than VA (α1β3γ2S: VA: Emax = 632 ± 88%, n = 9 versus α1β2γ2S: VA: Emax = 721 ± 68%, n = 6) displayed significantly more pronounced seizure threshold elevation than VA (saline control: 40.4 ± 1.4 mg/kg PTZ versus VA 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ versus VA-A 3 mg/kg: 57.9 ± 1.9 mg/kg PTZ, P < 0.05). Similarly, VA’s methylamide (VA-MA) enhancing IGABA through β3-containing receptors more efficaciously than VA (Emax = 1043 ± 57%, P < 0.01, n = 6) displayed stronger anticonvulsive effects. Increased potency of IGABA enhancement and anticonvulsive effects at lower doses compared with VA were observed for VA-tetrazole (α1β3γ2S: VA-TET: EC50 = 6.0 ± 1.0 μM, P < 0.05; VA-TET: 0.3 mg/kg: 47.3 ± 0.5 mg/kg PTZ versus VA: 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ, P < 0.05). At higher doses (≥10 mg/kg), VA-A, VA-MA, and VA-TET reduced locomotion. In contrast, unselective VA derivatives induced anticonvulsive effects only at high doses (30 mg/kg) or did not display any behavioral effects. Our data indicate that the β2/3-selective compounds VA-A, VA-MA, and VA-TET induce anticonvulsive effects at low doses (≤10 mg

  11. Axonal GABAA receptors.

    PubMed

    Trigo, Federico F; Marty, Alain; Stell, Brandon M

    2008-09-01

    Type A GABA receptors (GABA(A)Rs) are well established as the main inhibitory receptors in the mature mammalian forebrain. In recent years, evidence has accumulated showing that GABA(A)Rs are prevalent not only in the somatodendritic compartment of CNS neurons, but also in their axonal compartment. Evidence for axonal GABA(A)Rs includes new immunohistochemical and immunogold data: direct recording from single axonal terminals; and effects of local applications of GABA(A)R modulators on action potential generation, on axonal calcium signalling, and on neurotransmitter release. Strikingly, whereas presynaptic GABA(A)Rs have long been considered inhibitory, the new studies in the mammalian brain mostly indicate an excitatory action. Depending on the neuron that is under study, axonal GABA(A)Rs can be activated by ambient GABA, by GABA spillover, or by an autocrine action, to increase either action potential firing and/or transmitter release. In certain neurons, the excitatory effects of axonal GABA(A)Rs persist into adulthood. Altogether, axonal GABA(A)Rs appear as potent neuronal modulators of the mammalian CNS.

  12. Dynamic Regulation of the GABAA Receptor Function by Redox Mechanisms.

    PubMed

    Calvo, Daniel J; González, Andrea N Beltrán

    2016-09-01

    Oxidizing and reducing agents, which are currently involved in cell metabolism and signaling pathways, can regulate fast inhibitory neurotransmission mediated by GABA receptors in the nervous system. A number of in vitro studies have shown that diverse redox compounds, including redox metabolites and reactive oxygen and nitrogen species, modulate phasic and tonic responses mediated by neuronal GABAA receptors through both presynaptic and postsynaptic mechanisms. We review experimental data showing that many redox agents, which are normally present in neurons and glia or are endogenously generated in these cells under physiologic states or during oxidative stress (e.g., hydrogen peroxide, superoxide and hydroxyl radicals, nitric oxide, ascorbic acid, and glutathione), induce potentiating or inhibiting actions on different native and recombinant GABAA receptor subtypes. Based on these results, it is thought that redox signaling might represent a homeostatic mechanism that regulates the function of synaptic and extrasynaptic GABAA receptors in physiologic and pathologic conditions.

  13. Effects of essential amino acid deficiency: down-regulation of KCC2 and the GABAA receptor; disinhibition in the anterior piriform cortex.

    PubMed

    Sharp, James W; Ross-Inta, Catherine M; Baccelli, Irène; Payne, John A; Rudell, John B; Gietzen, Dorothy W

    2013-11-01

    The anterior piriform cortex (APC) is activated by, and is the brain area most sensitive to, essential (indispensable) amino acid (IAA) deficiency. The APC is required for the rapid (20 min) behavioral rejection of IAA deficient diets and increased foraging, both crucial adaptive functions supporting IAA homeostasis in omnivores. The biochemical mechanisms signaling IAA deficiency in the APC block initiation of translation in protein synthesis via uncharged tRNA and the general amino acid control kinase, general control nonderepressing kinase 2. Yet, how inhibition of protein synthesis activates the APC is unknown. The neuronal K(+) Cl(-) cotransporter, neural potassium chloride co-transporter (KCC2), and GABAA receptors are essential inhibitory elements in the APC with short plasmalemmal half-lives that maintain control in this highly excitable circuitry. After a single IAA deficient meal both proteins were reduced (vs. basal diet controls) in western blots of APC (but not neocortex or cerebellum) and in immunohistochemistry of APC. Furthermore, electrophysiological analyses support loss of inhibitory elements such as the GABAA receptor in this model. As the crucial inhibitory function of the GABAA receptor depends on KCC2 and the Cl(-) transmembrane gradient it establishes, these results suggest that loss of such inhibitory elements contributes to disinhibition of the APC in IAA deficiency. The circuitry of the anterior piriform cortex (APC) is finely balanced between excitatory (glutamate, +) and inhibitory (GABA, -) transmission. GABAA receptors use Cl(-), requiring the neural potassium chloride co-transporter (KCC2). Both are rapidly turning-over proteins, dependent on protein synthesis for repletion. In IAA (indispensable amino acid) deficiency, within 20 min, blockade of protein synthesis prevents restoration of these inhibitors; they are diminished; disinhibition ensues. GCN2 = general control non-derepressing kinase 2, eIF2α = α-subunit of the eukaryotic

  14. Mechanisms of neurosteroid interactions with GABAA receptors

    PubMed Central

    Akk, Gustav; Covey, Douglas F.; Evers, Alex S.; Steinbach, Joe Henry; Zorumski, Charles F.; Mennerick, Steven

    2007-01-01

    Neuroactive steroids have some of their most potent actions by augmenting the function of GABAA receptors. Endogenous steroid actions on GABAA receptors may underlie important effects on mood and behavior. Exogenous neuroactive steroids have potential as anesthetics, anticonvulsants, and neuroprotectants. We have taken multiple approaches to understand more completely the interaction of neuroactive steroids with GABAA receptors. We have developed many novel steroid analogues in this effort. Recent work has resulted in synthesis of new enantiomer analogue pairs, novel ligands that probe various properties of the steroid pharmacophore, fluorescent neuroactive steroid analogues, and photoaffinity labels. Using these tools, combined with receptor binding and electrophysiological assays, we have begun to untangle the complexity of steroid actions at this important class of ligand-gated ion channel. PMID:17524487

  15. Luteinizing hormone releasing hormone (LHRH) neurons maintained in nasal explants decrease LHRH messenger ribonucleic acid levels after activation of GABA(A) receptors.

    PubMed

    Fueshko, S M; Key, S; Wray, S

    1998-06-01

    Inhibition of the LHRH system appears to play an important role in preventing precocious activation of the hypothalamic-pituitary-gonadal axis. Evidence points to gamma-aminobutyric acid (GABA) as the major negative regulator of postnatal LHRH neuronal activity. Changes in LHRH messenger RNA (mRNA) levels after alterations of GABAergic activity have been reported in vivo. However, the extent to which GABA acts directly on LHRH neurons to effect LHRH mRNA levels has been difficult to ascertain. The present work evaluates the effect of GABAergic activity, via GABA(A) receptors, on LHRH neuropeptide gene expression in LHRH neurons maintained in olfactory explants generated from E11.5 mouse embryos. These explants maintain large numbers of primary LHRH neurons that migrate from bilateral olfactory pits in a directed manner. Using in situ hybridization histochemistry and single cell analysis, we report dramatic alterations in LHRH mRNA levels. Inhibition of spontaneous synaptic activity by GABA(A) antagonists, bicuculline (10(-5) M) or picrotoxin (10(-4) M), or of electrical activity by tetrodotoxin (TTX, 10(-6) M) significantly increased LHRH mRNA levels. In contrast, LHRH mRNA levels decreased in explants cultured with the GABA(A) receptor agonist, muscimol (10(-4) M), or KCl (50 mM). The observed responses suggest that LHRH neurons possess functional pathways linking GABA(A) receptors to repression of neuropeptide gene expression and indicate that gene expression in embryonic LHRH neurons, outside the CNS, is highly responsive to alterations in neuronal activity.

  16. The central nervous system convulsant pentylenetetrazole stimulates gamma-aminobutyric acid (GABA)-activated current in picrotoxin-resistant GABA(A) receptors in HEK293 cells.

    PubMed

    Dibas, M I; Dillon, G H

    2000-05-19

    We tested the ability of the central nervous system convulsant pentylenetetrazole (PTZ) to inhibit gamma-aminobutyric acid (GABA)-gated current in receptors expressing a mutation that rendered them resistant to picrotoxin. Consistent with previous reports, receptors expressing beta2(T246F), along with alpha3 and gamma2 subunits, resulted in a greatly diminished sensitivity to picrotoxin. Sensitivity to PTZ was completely abolished in the mutant receptor, confirming the hypothesis that PTZ acts at the picrotoxin site. Quite unexpected, however, was our finding that PTZ elicited marked stimulation (up to 400% of control) in the mutated receptors. This stimulatory effect was not mediated via an interaction with the benzodiazepine site, as preincubation with the benzodiazepine antagonist flumazenil did not block the PTZ-induced stimulation. Our results reveal the existence of a novel stimulatory domain of PTZ in GABA(A) receptors.

  17. PKCε and allopregnanolone: functional cross-talk at the GABAA receptor level

    PubMed Central

    Puia, Giulia; Ravazzini, Federica; Castelnovo, Luca Franco; Magnaghi, Valerio

    2015-01-01

    Changes in GABAergic inhibition occur during physiological processes, during response to drugs and in various pathologies. These changes can be achieved through direct allosteric modifications at the γ-amino butyric acid (GABA) type A (GABAA) receptor protein level, or by altering the synthesis, trafficking and stability of the receptor. Neurosteroids (NSs) and protein kinase C (PKC) are potent modulators of GABAA receptors and their effects are presumably intermingled, even though evidence for this hypothesis is only partially explored. However, several PKC isoforms are able to phosphorylate the GABAA receptor, producing different functional effects. We focused on the ε isoform, that has been correlated to the sensitivity of the GABAA receptor to allosteric modulators and whose expression may be regulated in peripheral sensory neurons by NSs. The cross-talk between PKC-ε and NSs, leading to changes in GABAA receptor functionality, is considered and discussed in this perspective. PMID:25852476

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. γ-Aminobutyric acid type A (GABAA) receptor α subunits play a direct role in synaptic versus extrasynaptic targeting.

    PubMed

    Wu, Xia; Wu, Zheng; Ning, Gang; Guo, Yao; Ali, Rashid; Macdonald, Robert L; De Blas, Angel L; Luscher, Bernhard; Chen, Gong

    2012-08-10

    GABA(A) receptors (GABA(A)-Rs) are localized at both synaptic and extrasynaptic sites, mediating phasic and tonic inhibition, respectively. Previous studies suggest an important role of γ2 and δ subunits in synaptic versus extrasynaptic targeting of GABA(A)-Rs. Here, we demonstrate differential function of α2 and α6 subunits in guiding the localization of GABA(A)-Rs. To study the targeting of specific subtypes of GABA(A)-Rs, we used a molecularly engineered GABAergic synapse model to precisely control the GABA(A)-R subunit composition. We found that in neuron-HEK cell heterosynapses, GABAergic events mediated by α2β3γ2 receptors were very fast (rise time ∼2 ms), whereas events mediated by α6β3δ receptors were very slow (rise time ∼20 ms). Such an order of magnitude difference in rise time could not be attributed to the minute differences in receptor kinetics. Interestingly, synaptic events mediated by α6β3 or α6β3γ2 receptors were significantly slower than those mediated by α2β3 or α2β3γ2 receptors, suggesting a differential role of α subunit in receptor targeting. This was confirmed by differential targeting of the same δ-γ2 chimeric subunits to synaptic or extrasynaptic sites, depending on whether it was co-assembled with the α2 or α6 subunit. In addition, insertion of a gephyrin-binding site into the intracellular domain of α6 and δ subunits brought α6β3δ receptors closer to synaptic sites. Therefore, the α subunits, together with the γ2 and δ subunits, play a critical role in governing synaptic versus extrasynaptic targeting of GABA(A)-Rs, possibly through differential interactions with gephyrin.

  20. α5GABAA receptor deficiency causes autism-like behaviors.

    PubMed

    Zurek, Agnieszka A; Kemp, Stephen W P; Aga, Zeenia; Walker, Susan; Milenkovic, Marija; Ramsey, Amy J; Sibille, Etienne; Scherer, Stephen W; Orser, Beverley A

    2016-05-01

    The prevalence of autism spectrum disorders (ASDs), which affect over 1% of the population, has increased twofold in recent years. Reduced expression of GABAA receptors has been observed in postmortem brain tissue and neuroimaging of individuals with ASDs. We found that deletion of the gene for the α5 subunit of the GABAA receptor caused robust autism-like behaviors in mice, including reduced social contacts and vocalizations. Screening of human exome sequencing data from 396 ASD subjects revealed potential missense mutations in GABRA5 and in RDX, the gene for the α5GABAA receptor-anchoring protein radixin, further supporting a α5GABAA receptor deficiency in ASDs. PMID:27231709

  1. Aging of whiskey increases the potentiation of GABA(A) receptor response.

    PubMed

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

    2003-08-27

    It is known that the target of most mood-defining compounds such as ethanol is an ionotropic gamma-aminobutyric acid receptor (GABA(A) receptor). The potentiation of the response of these inhibitory neurotransmitter receptors induces anxiolytic, sedative, and anesthetic activities in the human brain. Because both extracts of whiskey by pentane and fragrant components in whiskey potentiate the GABA(A) receptor-mediated response, GABA(A) receptors were expressed in Xenopus oocyte by injecting cRNAs prepared from the cloned cDNA for the alpha(1) and beta(1) subunits of the bovine receptors in order to study the effects of whiskey itself on the GABA(A) receptor-mediated response. Whiskey itself also potentiated the electrical responses of GABA(A) receptors generally more than ethanol at the same concentration as that of the whiskey. The potentiation of the GABA(A) receptor-mediated response increased with the aging period of the whiskey. Inhalation of whiskey to mice increased the sleeping time induced by pentobarbital more than that of the same concentration of ethanol as the whiskey. These results suggest that not only ethanol but also minor components in whiskey play an important role in the potentiation of GABA(A) receptor-mediated response and possibly the sedative effect of whiskey. Although the minor components are present in extremely small quantities compared with ethanol in alcoholic beverages, they may modulate the mood or consciousness of humans through the potentiation of the GABA(A) receptor response after absorption into the brain, because these hydrophobic compounds are easily absorbed into the brain across the blood-brain barrier and are several thousands times as potent as ethanol in the potentiation of the GABA(A) receptor-mediated response.

  2. Aging of whiskey increases the potentiation of GABA(A) receptor response.

    PubMed

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

    2003-08-27

    It is known that the target of most mood-defining compounds such as ethanol is an ionotropic gamma-aminobutyric acid receptor (GABA(A) receptor). The potentiation of the response of these inhibitory neurotransmitter receptors induces anxiolytic, sedative, and anesthetic activities in the human brain. Because both extracts of whiskey by pentane and fragrant components in whiskey potentiate the GABA(A) receptor-mediated response, GABA(A) receptors were expressed in Xenopus oocyte by injecting cRNAs prepared from the cloned cDNA for the alpha(1) and beta(1) subunits of the bovine receptors in order to study the effects of whiskey itself on the GABA(A) receptor-mediated response. Whiskey itself also potentiated the electrical responses of GABA(A) receptors generally more than ethanol at the same concentration as that of the whiskey. The potentiation of the GABA(A) receptor-mediated response increased with the aging period of the whiskey. Inhalation of whiskey to mice increased the sleeping time induced by pentobarbital more than that of the same concentration of ethanol as the whiskey. These results suggest that not only ethanol but also minor components in whiskey play an important role in the potentiation of GABA(A) receptor-mediated response and possibly the sedative effect of whiskey. Although the minor components are present in extremely small quantities compared with ethanol in alcoholic beverages, they may modulate the mood or consciousness of humans through the potentiation of the GABA(A) receptor response after absorption into the brain, because these hydrophobic compounds are easily absorbed into the brain across the blood-brain barrier and are several thousands times as potent as ethanol in the potentiation of the GABA(A) receptor-mediated response. PMID:12926865

  3. gamma-Aminobutyric acidA (GABAA) receptors modulate [3H]GABA release from isolated neuronal growth cones in the rat.

    PubMed

    Lockerbie, R O; Gordon-Weeks, P R

    1985-04-19

    Potassium-induced release of gamma-[3H]aminobutyric acid [( 3H]GABA) from a growth cone-enriched fraction isolated from neonatal rat forebrain was inhibited by the GABA mimetic muscimol in a dose-dependent manner (IC50 15 nM). The GABA antagonist bicuculline completely reversed the effect of muscimol. Bicuculline alone slightly potentiated the K+-induced release of [3H]GABA. Baclofen, a proposed selective agonist for a bicuculline-insensitive GABAB receptor, was found to cause only a slight reduction in the K+-induced release of [3H]GABA. These results are compatible with the presence of a negative feedback mechanism mediated by GABAA receptors for controlling [3H]GABA release from growth cones of the developing rat forebrain.

  4. Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site.

    PubMed

    Snell, Heather D; Gonzales, Eric B

    2015-06-01

    Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in light of how many GABA-A αβγ receptor modulators have different effects in the GABA-A ρ1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A ρ1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A ρ1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride's positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound's potency within the human GABA-A ρ1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A ρ1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ.

  5. Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site

    PubMed Central

    Snell, Heather D.

    2015-01-01

    Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in light of how many GABA-A αβγ receptor modulators have different effects in the GABA-A ρ1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A ρ1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A ρ1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride’s positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound’s potency within the human GABA-A ρ1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A ρ1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ. PMID:25829529

  6. 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

  7. DISC1 Protein Regulates γ-Aminobutyric Acid, Type A (GABAA) Receptor Trafficking and Inhibitory Synaptic Transmission in Cortical Neurons.

    PubMed

    Wei, Jing; Graziane, Nicholas M; Gu, Zhenglin; Yan, Zhen

    2015-11-13

    Association studies have suggested that Disrupted-in-Schizophrenia 1 (DISC1) confers a genetic risk at the level of endophenotypes that underlies many major mental disorders. Despite the progress in understanding the significance of DISC1 at neural development, the mechanisms underlying DISC1 regulation of synaptic functions remain elusive. Because alterations in the cortical GABA system have been strongly linked to the pathophysiology of schizophrenia, one potential target of DISC1 that is critically involved in the regulation of cognition and emotion is the GABAA receptor (GABAAR). We found that cellular knockdown of DISC1 significantly reduced GABAAR-mediated synaptic and whole-cell current, whereas overexpression of wild-type DISC1, but not the C-terminal-truncated DISC1 (a schizophrenia-related mutant), significantly increased GABAAR currents in pyramidal neurons of the prefrontal cortex. These effects were accompanied by DISC1-induced changes in surface GABAAR expression. Moreover, the regulation of GABAARs by DISC1 knockdown or overexpression depends on the microtubule motor protein kinesin 1 (KIF5). Our results suggest that DISC1 exerts an important effect on GABAergic inhibitory transmission by regulating KIF5/microtubule-based GABAAR trafficking in the cortex. The knowledge gained from this study would shed light on how DISC1 and the GABA system are linked mechanistically and how their interactions are critical for maintaining a normal mental state.

  8. DISC1 Protein Regulates γ-Aminobutyric Acid, Type A (GABAA) Receptor Trafficking and Inhibitory Synaptic Transmission in Cortical Neurons.

    PubMed

    Wei, Jing; Graziane, Nicholas M; Gu, Zhenglin; Yan, Zhen

    2015-11-13

    Association studies have suggested that Disrupted-in-Schizophrenia 1 (DISC1) confers a genetic risk at the level of endophenotypes that underlies many major mental disorders. Despite the progress in understanding the significance of DISC1 at neural development, the mechanisms underlying DISC1 regulation of synaptic functions remain elusive. Because alterations in the cortical GABA system have been strongly linked to the pathophysiology of schizophrenia, one potential target of DISC1 that is critically involved in the regulation of cognition and emotion is the GABAA receptor (GABAAR). We found that cellular knockdown of DISC1 significantly reduced GABAAR-mediated synaptic and whole-cell current, whereas overexpression of wild-type DISC1, but not the C-terminal-truncated DISC1 (a schizophrenia-related mutant), significantly increased GABAAR currents in pyramidal neurons of the prefrontal cortex. These effects were accompanied by DISC1-induced changes in surface GABAAR expression. Moreover, the regulation of GABAARs by DISC1 knockdown or overexpression depends on the microtubule motor protein kinesin 1 (KIF5). Our results suggest that DISC1 exerts an important effect on GABAergic inhibitory transmission by regulating KIF5/microtubule-based GABAAR trafficking in the cortex. The knowledge gained from this study would shed light on how DISC1 and the GABA system are linked mechanistically and how their interactions are critical for maintaining a normal mental state. PMID:26424793

  9. Adenosine receptor antagonists alter the stability of human epileptic GABAA receptors

    PubMed Central

    Roseti, Cristina; Martinello, Katiuscia; Fucile, Sergio; Piccari, Vanessa; Mascia, Addolorata; Di Gennaro, Giancarlo; Quarato, Pier Paolo; Manfredi, Mario; Esposito, Vincenzo; Cantore, Gianpaolo; Arcella, Antonella; Simonato, Michele; Fredholm, Bertil B.; Limatola, Cristina; Miledi, Ricardo; Eusebi, Fabrizio

    2008-01-01

    We examined how the endogenous anticonvulsant adenosine might influence γ-aminobutyric acid type A (GABAA) receptor stability and which adenosine receptors (ARs) were involved. Upon repetitive activation (GABA 500 μM), GABAA receptors, microtransplanted into Xenopus oocytes from neurosurgically resected epileptic human nervous tissues, exhibited an obvious GABAA-current (IGABA) run-down, which was consistently and significantly reduced by treatment with the nonselective adenosine receptor antagonist CGS15943 (100 nM) or with adenosine deaminase (ADA) (1 units/ml), that inactivates adenosine. It was also found that selective antagonists of A2B (MRS1706, 10 nM) or A3 (MRS1334, 30 nM) receptors reduced IGABA run-down, whereas treatment with the specific A1 receptor antagonist DPCPX (10 nM) was ineffective. The selective A2A receptor antagonist SCH58261 (10 nM) reduced or potentiated IGABA run-down in ≈40% and ≈20% of tested oocytes, respectively. The ADA-resistant, AR agonist 2-chloroadenosine (2-CA) (10 μM) potentiated IGABA run-down but only in ≈20% of tested oocytes. CGS15943 administration again decreased IGABA run-down in patch-clamped neurons from either human or rat neocortex slices. IGABA run-down in pyramidal neurons was equivalent in A1 receptor-deficient and wt neurons but much larger in neurons from A2A receptor-deficient mice, indicating that, in mouse cortex, GABAA-receptor stability is tonically influenced by A2A but not by A1 receptors. IGABA run-down from wt mice was not affected by 2-CA, suggesting maximal ARs activity by endogenous adenosine. Our findings strongly suggest that cortical A2–A3 receptors alter the stability of GABAA receptors, which could offer therapeutic opportunities. PMID:18809912

  10. GABAA receptor target of tetramethylenedisulfotetramine

    PubMed Central

    Zhao, Chunqing; Hwang, Sung Hee; Buchholz, Bruce A.; Carpenter, Timothy S.; Lightstone, Felice C.; Yang, Jun; Hammock, Bruce D.; Casida, John E.

    2014-01-01

    Use of the highly toxic and easily prepared rodenticide tetramethylenedisulfotetramine (TETS) was banned after thousands of accidental or intentional human poisonings, but it is of continued concern as a chemical threat agent. TETS is a noncompetitive blocker of the GABA type A receptor (GABAAR), but its molecular interaction has not been directly established for lack of a suitable radioligand to localize the binding site. We synthesized [14C]TETS (14 mCi/mmol, radiochemical purity >99%) by reacting sulfamide with H14CHO and s-trioxane then completion of the sequential cyclization with excess HCHO. The outstanding radiocarbon sensitivity of accelerator mass spectrometry (AMS) allowed the use of [14C]TETS in neuroreceptor binding studies with rat brain membranes in comparison with the standard GABAAR radioligand 4′-ethynyl-4-n-[3H]propylbicycloorthobenzoate ([3H]EBOB) (46 Ci/mmol), illustrating the use of AMS for characterizing the binding sites of high-affinity 14C radioligands. Fourteen noncompetitive antagonists of widely diverse chemotypes assayed at 1 or 10 µM inhibited [14C]TETS and [3H]EBOB binding to a similar extent (r2 = 0.71). Molecular dynamics simulations of these 14 toxicants in the pore region of the α1β2γ2 GABAAR predict unique and significant polar interactions for TETS with α1T1′ and γ2S2′, which are not observed for EBOB or the GABAergic insecticides. Several GABAAR modulators similarly inhibited [14C]TETS and [3H]EBOB binding, including midazolam, flurazepam, avermectin Ba1, baclofen, isoguvacine, and propofol, at 1 or 10 μM, providing an in vitro system for recognizing candidate antidotes. PMID:24912155

  11. Evidence for the participation of peripheral α5 subunit-containing GABAA receptors in GABAA agonists-induced nociception in rats.

    PubMed

    Bravo-Hernández, Mariana; Feria-Morales, Luis Alberto; Torres-López, Jorge Elías; Cervantes-Durán, Claudia; Delgado-Lezama, Rodolfo; Granados-Soto, Vinicio; Rocha-González, Héctor Isaac

    2014-07-01

    The activation of GABAA receptor by γ-amino butyric acid (GABA) in primary afferent fibers produces depolarization. In normal conditions this depolarization causes a reduction in the release of neurotransmitters. Therefore, this depolarization remains inhibitory. However, previous studies have suggested that in inflammatory pain, GABA shifts its signaling from inhibition to excitation by an increased GABA-induced depolarization. The contribution of peripheral α5 subunit-containing GABAA receptors to the inflammatory pain is unknown. The purpose of this study was to investigate the possible pronociceptive role of peripheral α5 subunit-containing GABAA receptors in the formalin test. Formalin (0.5%) injection into the dorsum of the right hind paw produced flinching behavior in rats. Ipsilateral local peripheral pre-treatment (-10min) with exogenous GABA (0.003-0.03µg/paw) or common GABAA receptor agonists muscimol (0.003-0.03µg/paw), diazepam (0.017-0.056µg/paw) or phenobarbital (1-100µg/paw) significantly increased 0.5% formalin-induced nociceptive behavior. The pronociceptive effects of GABA (0.03µg/paw), muscimol (0.03µg/paw), diazepam (0.056µg/paw) and phenobarbital (100µg/paw) were prevented by either the GABAA receptor antagonist bicuculline (0.01-0.1µg/paw) or selective α5 subunit-containing GABAA receptor inverse agonist L-655,708 (0.017-0.17µg/paw). The α5 subunit-containing GABAA receptor protein was expressed in dorsal root ganglion (DRG) and dorsal spinal cord of naïve rats. The formalin injection did not modify α5 subunit-containing GABAA receptor expression. Overall, these results suggest that peripheral α5 subunit-containing GABAA receptors play a pronociceptive role in the rat formalin test. PMID:24726872

  12. GABAA Receptors in Normal Development and Seizures: Friends or Foes?

    PubMed Central

    Galanopoulou, Aristea S

    2008-01-01

    GABAA receptors have an age-adapted function in the brain. During early development, they mediate excitatory effects resulting in activation of calcium sensitive signaling processes that are important for the differentiation of the brain. In more mature stages of development and in adults, GABAA receptors transmit inhibitory signals. The maturation of GABAA signaling follows sex-specific patterns, which appear to also be important for the sexual differentiation of the brain. The inhibitory effects of GABAA receptor activation have been widely exploited in the treatment of conditions where neuronal silencing is necessary. For instance, drugs that target GABAA receptors are the mainstay of treatment of seizures. Recent evidence suggests however that the physiology and function of GABAA receptors changes in the brain of a subject that has epilepsy or status epilepticus. This review will summarize the physiology of and the developmental factors regulating the signaling and function of GABAA receptors; how these may change in the brain that has experienced prior seizures; what are the implications for the age and sex specific treatment of seizures and status epilepticus. Finally, the implications of these changes for the treatment of certain forms of medically refractory epilepsies and status epilepticus will be discussed. PMID:19305785

  13. Tolerance to allopregnanolone with focus on the GABA-A receptor

    PubMed Central

    Turkmen, Sahruh; Backstrom, Torbjorn; Wahlstrom, Goran; Andreen, Lotta; Johansson, Inga-Maj

    2011-01-01

    Many studies have suggested a relationship between stress, sex steroids, and negative mental and mood changes in humans. The progesterone metabolite allopregnanolone is a potent endogenous ligand of the γ-amino butyric acid –A (GABA-A) receptor, and the most discussed neuroactive steroid. Variations in the levels of neuroactive steroids that influence the activity of the GABA-A receptor cause a vulnerability to mental and emotional pathology. There are physiological conditions in which allopregnanolone production increases acutely (e.g. stress) or chronically (e.g. menstrual cycle, pregnancy), thus exposing the GABA-A receptor to high and continuous allopregnanolone concentrations. In such conditions, tolerance to allopregnanolone may develop. We have shown that both acute and chronic tolerances can develop to the effects of allopregnanolone. Following the development of acute allopregnanolone tolerance, there is a decrease in the abundance of the GABA-A receptor α4 subunit and the expression of the α4 subunit mRNA in the ventral-posteriomedial nucleus of the thalamus. Little is known about the mechanism behind allopregnanolone tolerance and its effects on assembly of the GABA-A receptor composition. The exact mechanism of the allopregnanolone tolerance phenomena remains unclear. The purpose of this review is to summarize certain aspects of current knowledge concerning allopregnanolone tolerance and changes in the GABA-A receptors. PMID:20883478

  14. 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

  15. 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. PMID:27462076

  16. GABAA receptor modulation by terpenoids from Sideritis extracts

    PubMed Central

    Kessler, Artur; Sahin-Nadeem, Hilal; Lummis, Sarah C R; Weigel, Ingrid; Pischetsrieder, Monika; Buettner, Andrea; Villmann, Carmen

    2014-01-01

    Scope GABAA receptors are modulated by Sideritis extracts. The aim of this study was to identify single substances from Sideritis extracts responsible for GABAA receptor modulation. Methods and results Single volatile substances identified by GC have been tested in two expression systems, Xenopus oocytes and human embryonic kidney cells. Some of these substances, especially carvacrol, were highly potent on GABAA receptors composed of α1β2 and α1β2γ2 subunits. All effects measured were independent from the presence of the γ2 subunit. As Sideritis extracts contain a high amount of terpenes, 13 terpenes with similar structure elements were tested in the same way. Following a prescreening on α1β2 GABAA receptors, a high-throughput method was used for identification of the most effective terpenoid substances on GABA-affinity of α1β2γ2 receptors expressed in transfected cell lines. Isopulegol, pinocarveol, verbenol, and myrtenol were the most potent modifiers of GABAA receptor function. Conclusion Comparing the chemical structures, the action of terpenes on GABAA receptors is most probably due to the presence of hydroxyl groups and a bicyclic character of the substances tested. We propose an allosteric modulation independent from the γ2 subunit and similar to the action of alcohols and anesthetics. PMID:24273211

  17. Extrasynaptic GABA(A) receptors in the brainstem and spinal cord: structure and function.

    PubMed

    Delgado-Lezama, Rodolfo; Loeza-Alcocer, Emanuel; Andrés, Carmen; Aguilar, Justo; Guertin, Pierre A; Felix, Ricardo

    2013-01-01

    γ-aminobutyric acid (GABA) plays many of its key roles in embryonic development and functioning of the central nervous system (CNS) by acting on ligand gated chloride-permeable channels known as GABAA receptors (GABAAR). Classically, GABAARmediated synaptic communication is tailored to allow rapid and precise transmission of information to synchronize the activity of large populations of cells to generate and maintain neuronal networks oscillations. An alternative type of inhibition mediated by GABAA receptors, initially described about 25 years ago, is characterized by a tonic activation of receptors that react to ambient extracellular GABA. The receptors that mediate this action are wide-spread throughout the nerve cells but are located distant from the sites of GABA release, and therefore they have been called extrasynaptic GABAA receptors. The molecular nature of the extrasynaptic GABAA receptors and the tonic inhibitory current they generate have been characterized in many brain structures, and due to its relevance in controlling neuron excitability they have become attractive pharmacological targets for a variety of neurological disorders such as schizophrenia, epilepsy and Parkinson disease. In the spinal cord, early studies have implicated these receptors in anesthesia, chronic pain, motor control, and locomotion. This review highlights past and present developments in the field of extrasynaptic GABAA receptors and emphasizes their subunit containing distribution and physiological role in the spinal cord. PMID:23360278

  18. Differential vesicular sorting of AMPA and GABAA receptors

    PubMed Central

    Gu, Yi; Chiu, Shu-Ling; Liu, Bian; Wu, Pei-Hsun; Delannoy, Michael; Lin, Da-Ting; Wirtz, Denis; Huganir, Richard L.

    2016-01-01

    In mature neurons AMPA receptors cluster at excitatory synapses primarily on dendritic spines, whereas GABAA receptors cluster at inhibitory synapses mainly on the soma and dendritic shafts. The molecular mechanisms underlying the precise sorting of these receptors remain unclear. By directly studying the constitutive exocytic vesicles of AMPA and GABAA receptors in vitro and in vivo, we demonstrate that they are initially sorted into different vesicles in the Golgi apparatus and inserted into distinct domains of the plasma membrane. These insertions are dependent on distinct Rab GTPases and SNARE complexes. The insertion of AMPA receptors requires SNAP25–syntaxin1A/B–VAMP2 complexes, whereas insertion of GABAA receptors relies on SNAP23–syntaxin1A/B–VAMP2 complexes. These SNARE complexes affect surface targeting of AMPA or GABAA receptors and synaptic transmission. Our studies reveal vesicular sorting mechanisms controlling the constitutive exocytosis of AMPA and GABAA receptors, which are critical for the regulation of excitatory and inhibitory responses in neurons. PMID:26839408

  19. Structural Studies of GABAA Receptor Binding Sites: Which Experimental Structure Tells us What?

    PubMed Central

    Puthenkalam, Roshan; Hieckel, Marcel; Simeone, Xenia; Suwattanasophon, Chonticha; Feldbauer, Roman V.; Ecker, Gerhard F.; Ernst, Margot

    2016-01-01

    Atomic resolution structures of cys-loop receptors, including one of a γ-aminobutyric acid type A receptor (GABAA receptor) subtype, allow amazing insights into the structural features and conformational changes that these pentameric ligand-gated ion channels (pLGICs) display. Here we present a comprehensive analysis of more than 30 cys-loop receptor structures of homologous proteins that revealed several allosteric binding sites not previously described in GABAA receptors. These novel binding sites were examined in GABAA receptor homology models and assessed as putative candidate sites for allosteric ligands. Four so far undescribed putative ligand binding sites were proposed for follow up studies based on their presence in the GABAA receptor homology models. A comprehensive analysis of conserved structural features in GABAA and glycine receptors (GlyRs), the glutamate gated ion channel, the bacterial homologs Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus GLIC, and the serotonin type 3 (5-HT3) receptor was performed. The conserved features were integrated into a master alignment that led to improved homology models. The large fragment of the intracellular domain that is present in the structure of the 5-HT3 receptor was utilized to generate GABAA receptor models with a corresponding intracellular domain fragment. Results of mutational and photoaffinity ligand studies in GABAA receptors were analyzed in the light of the model structures. This led to an assignment of candidate ligands to two proposed novel pockets, candidate binding sites for furosemide and neurosteroids in the trans-membrane domain were identified. The homology models can serve as hypotheses generators, and some previously controversial structural interpretations of biochemical data can be resolved in the light of the presented multi-template approach to comparative modeling. Crystal and cryo-EM microscopic structures of the closest homologs that were solved in different conformational

  20. Molecular, pharmacological and functional properties of GABAA receptors in anterior pituitary cells

    PubMed Central

    Zemkova, Hana W; Bjelobaba, Ivana; Tomic, Melanija; Zemkova, Hana; Stojilkovic, Stanko S

    2008-01-01

    Anterior pituitary cells express γ-aminobutyric acid (GABA)-A receptor-channels, but their structure, distribution within the secretory cell types, and nature of action have not been clarified. Here we addressed these questions using cultured anterior pituitary cells from postpubertal female rats and immortalized αT3-1 and GH3 cells. Our results show that mRNAs for all GABAA receptor subunits are expressed in pituitary cells and that α1/β1 subunit proteins are present in all secretory cells. In voltage-clamped gramicidin-perforated cells, GABA induced dose-dependent increases in current amplitude that were inhibited by bicuculline and picrotoxin and facilitated by diazepam and zolpidem in a concentration-dependent manner. In intact cells, GABA and the GABAA receptor agonist muscimol caused a rapid and transient increase in intracellular calcium, whereas the GABAB receptor agonist baclofen was ineffective, suggesting that chloride-mediated depolarization activates voltage-gated calcium channels. Consistent with this finding, RT-PCR analysis indicated high expression of NKCC1, but not KCC2 cation/chloride transporter mRNAs in pituitary cells. Furthermore, the GABAA channel reversal potential for chloride ions was positive to the baseline membrane potential in most cells and the activation of ion channels by GABA resulted in depolarization of cells and modulation of spontaneous electrical activity. These results indicate that secretory pituitary cells express functional GABAA receptor-channels that are depolarizing. PMID:18450776

  1. Beyond classical benzodiazepines: Novel therapeutic potential of GABAA receptor subtypes

    PubMed Central

    Rudolph, Uwe; Knoflach, Frédéric

    2012-01-01

    GABAA receptors are a family of ligand-gated ion channels which are essential for the regulation of central nervous system function. Benzodiazepines – which target GABAA receptors containing the α1, α2, α3, or α5 subunits non-selectively – have been in clinical use for decades and are still among the most widely prescribed drugs for the treatment of insomnia and anxiety disorders. However, their use is limited by side effects and the risk of drug dependence. In the past decade, the identification of separable key functions of GABAA receptor subtypes suggests that receptor subtype-selective compounds could overcome the limitations of classical benzodiazepines and, furthermore, might be valuable for novel indications, such as analgesia, depression, schizophrenia, cognitive enhancement and stroke. PMID:21799515

  2. GABAA receptor regulation of voluntary ethanol drinking requires PKCepsilon.

    PubMed

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

    2006-11-01

    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 epsilon-isoform of PKC (PKCepsilon) self-administer 75% less ethanol and exhibit supersensitivity to acute ethanol and allosteric positive modulators of GABA(A) receptors when compared with wild-type controls. The purpose of the present study was to examine involvement of PKCepsilon in GABA(A) receptor regulation of voluntary ethanol drinking. To address this question, PKCepsilon 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 GABA(A) BZ positive modulator), zolpidem (GABA(A) alpha1 agonist), L-655,708 (BZ-sensitive GABA(A) alpha5 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 PKCepsilon 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 PKCepsilon null mice. Thus, results of the present study show that PKCepsilon null mice do not respond to doses of GABA(A) BZ receptor ligands that regulate ethanol drinking by wild-type control mice. This suggests that PKCepsilon may be required for GABA(A) receptor regulation of chronic ethanol drinking.

  3. Two groups of amino acids interact with GABA-A receptors coupled to t-[35S]butylbicyclophosphorothionate binding sites: possible involvement with seizures associated with hereditary amino acidemias.

    PubMed

    Squires, R F; Saederup, E; Lajtha, A

    1988-09-01

    Seven L-amino acids (Trp, Arg, Lys, Met, Ile, Val, and Phe) partially (28-81%) reversed the inhibitory action of 1 microM gamma-aminobutyric acid (GABA) on t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes, with EC50 values ranging from 5 to 120 mM. D-Trp, D-Arg, D-Lys, D-Met, D-Val, and D-Phe were approximately equipotent with their L-isomers. Tyramine, phenethylamine, and tryptamine, the decarboxylation products of the aromatic amino acids (Tyr, Phe, and Trp, respectively), reversed the inhibitory action of 1 microM GABA on [35S]TBPS binding more potently than the parent amino acids (EC50 values = 1.5-3.0 mM). Human hereditary amino acidemias involving Arg, Lys, Ile, Val, and Phe are associated with seizures, and these amino acids and/or their metabolites may block GABA-A receptors. Five other L-amino acids (ornithine, His, Glu, Pro, and Ala) as well as Gly and beta-Ala inhibited [35S]TBPS binding with IC50 values ranging from 0.1 to 37 mM, and these inhibitions were reversed by the GABA-A receptor blocker R 5135 in all cases. The inhibitory effects of L-ornithine, L-Ala, L-Glu, and L-Pro were stereospecific, because the corresponding D-isomers were considerably less inhibitory. L-His, D-His, and L-Glu gave incomplete (plateau) inhibitions. Human hereditary amino acidemias involving L-ornithine, His, Pro, Gly, and beta-Ala are also associated with seizures, and we speculate that these GABA-mimetic amino acids may desensitize GABA-A receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Status Epilepticus Increases the Intracellular Accumulation of GABAA Receptors

    PubMed Central

    Goodkin, Howard P.; Yeh, Jwu-Lai; Kapur, Jaideep

    2010-01-01

    Status epilepticus is a neurological emergency that results in mortality and neurological morbidity. It has been postulated that the reduction of inhibitory transmission during status epilepticus results from a rapid modification of GABAA receptors. However, the mechanism(s) that contributes to this modification has not been elucidated. We report, using an in vitro model of status epilepticus combined with electrophysiological and cellular imaging techniques, that prolonged epileptiform bursting results in a reduction of GABA-mediated synaptic inhibition. Furthermore, we found that constitutive internalization of GABAA receptors is rapid and accelerated by the increased neuronal activity associated with seizures. Inhibition of neuronal activity reduced the rate of internalization. These findings suggest that the rate of GABAA receptor internalization is regulated by neuronal activity and its acceleration contributes to the reduction of inhibitory transmission observed during prolonged seizures. PMID:15944379

  5. Subcellular localization and complements of GABA(A) and GABA(C) receptors on bullfrog retinal bipolar cells.

    PubMed

    Du, J L; Yang, X L

    2000-08-01

    gamma-Aminobutyric acid (GABA) receptors on retinal bipolar cells (BCs) are highly relevant to spatial and temporal integration of visual signals in the outer and inner retina. In the present work, subcellular localization and complements of GABA(A) and GABA(C) receptors on BCs were investigated by whole cell recordings and local drug application via multi-barreled puff pipettes in the bullfrog retinal slice preparation. Four types of the BCs (types 1-4) were identified morphologically by injection of Lucifer yellow. According to the ramification levels of the axon terminals and the responses of these cells to glutamate (or kainate) applied at their dendrites, types 1 and 2 of BCs were supposed to be OFF type, whereas types 3 and 4 of BCs might be ON type. Bicuculline (BIC), a GABA(A) receptor antagonist, and imidazole-4-acetic acid (I4AA), a GABA(C) receptor antagonist, were used to distinguish GABA receptor-mediated responses. In all BCs tested, not only the axon terminals but also the dendrites showed high GABA sensitivity mediated by both GABA(A) and GABA(C) receptors. Subcellular localization and complements of GABA(A) and GABA(C) receptors at the dendrites and axon terminals were highly related to the dichotomy of OFF and ON BCs. In the case of OFF BCs, GABA(A) receptors were rather evenly distributed at the dendrites and axon terminals, but GABA(C) receptors were predominantly expressed at the axon terminals. Moreover, the relative contribution of GABA(C) receptors to the axon terminals was prevalent over that of GABA(A) receptors, while the situation was reversed at the dendrites. In the case of ON BCs, GABA(A) and GABA(C) receptors both preferred to be expressed at the axon terminals; relative contributions of these two GABA receptor subtypes to both the sites were comparable, while GABA(C) receptors were much less expressed than GABA(A) receptors. GABA(A), but not GABA(C) receptors, were expressed clusteringly at axons of a population of BCs. In a

  6. Novel positive allosteric modulators of GABAA receptors with anesthetic activity

    PubMed Central

    Maldifassi, Maria C.; Baur, Roland; Pierce, David; Nourmahnad, Anahita; Forman, Stuart A.; Sigel, Erwin

    2016-01-01

    GABAA receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics and anesthetics. We previously identified novel ligands of the classical benzodiazepine binding pocket in α1β2γ2 GABAA receptors using an experiment-guided virtual screening (EGVS) method. This screen also identified novel ligands for intramembrane low affinity diazepam site(s). In the current study we have further characterized compounds 31 and 132 identified with EGVS as well as 4-O-methylhonokiol. We investigated the site of action of these compounds in α1β2γ2 GABAA receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology combined with a benzodiazepine site antagonist and transmembrane domain mutations. All three compounds act mainly through the two β+/α− subunit transmembrane interfaces of the GABAA receptors. We then used concatenated receptors to dissect the involvement of individual β+/α− interfaces. We further demonstrated that these compounds have anesthetic activity in a small aquatic animal model, Xenopus laevis tadpoles. The newly identified compounds may serve as scaffolds for the development of novel anesthetics. PMID:27198062

  7. History of cigarette smoking is associated with higher limbic GABAA receptor availability.

    PubMed

    Stokes, Paul R A; Benecke, Aaf; Myers, Jim; Erritzoe, David; Watson, Ben J; Kalk, Nicola; Barros, Daniela Riano; Hammers, Alexander; Nutt, David J; Lingford-Hughes, Anne R

    2013-04-01

    Cigarette smoking presents a significant worldwide healthcare challenge. Preclinical, genetic association and clinical trials studies provide considerable evidence for the involvement of the human γ-aminobutyric acid (GABA) system in the neurobiology of nicotine addiction. However there are few human GABA neurochemical imaging studies of nicotine addiction. We investigated limbic GABA(A) receptor availability in volunteers with a history of cigarette smoking using [(11)C]Ro15 4513 positron emission tomography (PET). Eight [(11)C]Ro15 4513 PET scans from volunteers with a history of cigarette smoking were compared to twelve scans from volunteers who were non-smokers. Total, α1 and α5 GABA(A) receptor subtype [(11)C]Ro15 4513 V(T) values were quantified using spectral analysis of limbic regions implicated in nicotine addiction. Spectral analysis allows quantification of the overall [(11)C]Ro15 4513 spectral frequency as well as α1 and α5 GABA(A) receptor subtype specific spectral frequency components. Volunteers with a history of cigarette smoking showed significantly higher total [(11)C]Ro15 4513 V(T) values in the presubgenual cingulate and parahippocampal gyrus, and at a trend level in the insula, nucleus accumbens and subgenual cingulate. In six abstinent previous smokers ('ex-smokers'), total [(11)C]Ro15 4513 binding was significantly higher in all limbic regions studied, with higher α5 availability in the amygdala, anterior cingulate, nucleus accumbens and presubgenual cingulate. These results suggest that limbic GABA(A) receptor availability is higher in volunteers with a history of cigarette smoking which may reflect either higher expression of GABA(A) receptors or lower endogenous GABA levels. The findings in ex-smokers suggest that higher GABA(A) receptor availability continues with abstinence indicating that this may be a trait marker for nicotine addiction or that alterations in GABA function associated with cigarette smoking persist.

  8. The modulation by chlormethiazole of the GABAA-receptor complex in rat brain.

    PubMed Central

    Cross, A. J.; Stirling, J. M.; Robinson, T. N.; Bowen, D. M.; Francis, P. T.; Green, A. R.

    1989-01-01

    1. The interactions of chlormethiazole with gamma-aminobutyric acid (GABA) synthesis and release, and with ligand binding to sites associated with the GABAA-receptor complex and the GABAB-receptor have been studied in the rat. The GABAA-receptor was studied using [3H]-muscimol, [3H]-flunitrazepam was used to label the benzodiazepine modulatory site, and [35S]-butyl-bicyclophosphorothionate ([35S]-TBPS) to label the chloride channel. 2. Chlormethiazole had no effect on GABA synthesis in the cortex, hippocampus and striatum or on GABA release from cortical slices in vitro. Chlormethiazole did not displace [3H]-baclofen binding to the GABAB-receptor. 3. Chlormethiazole (IC50 = 140 microM) and pentobarbitone (IC50 = 95 microM) both inhibited [35S]-TBPS binding by increasing the rate of [35S]-TBPS dissociation. In addition, chlormethiazole caused an apparent decrease in the affinity of [35S]-TBPS binding. 4. Chlormethiazole enhanced the binding of [3H]-muscimol but had no effect on [3H]-flunitrazepam binding. In contrast, the sedative barbiturate pentobarbitone enhanced both [3H]-muscimol and [3H]-flunitrazepam binding. 5. It is concluded that the sedative and anticonvulsant effects of chlormethiazole are probably mediated through an action at the GABAA-receptor. However, chlormethiazole does not interact with the GABAA-receptor complex in an identical manner to the sedative barbiturate pentobarbitone. PMID:2553191

  9. GABAA receptor-mediated neurotransmission: Not so simple after all.

    PubMed

    Knoflach, Frédéric; Hernandez, Maria-Clemencia; Bertrand, Daniel

    2016-09-01

    GABAA receptors are ligand-gated ion channels that form a fundamental component of inhibitory neurotransmission in the central and peripheral nervous systems. However, since the initial recordings of inhibitory electrical activity of neurons in response to GABA, these receptors have been found to play a more complex role and can, under some circumstances, function in an excitatory manner. This has been demonstrated via electrophysiological recordings conducted in both mature and developing neurons from different brain regions, as well as in various subcellular locations such as dendritic and axonal membranes. The balance between the inhibitory and excitatory effects mediated by GABAA receptor activation depends not only on multiple factors that govern the equilibrium of the transmembrane chloride gradient, but also on bicarbonate concentration. Moreover, electrophysiological and fluorescence measurements have revealed that a spatial distribution of the chloride gradient exists within neurons, which locally influences the effects mediated by GABAA receptor activation. In recent years, it has also become apparent that intra-neuronal chloride concentration is partially regulated by cation-chloride co-transporters (CCCs), in particular NKCC1 and KCC2. The aim of the present commentary is to discuss, in light of the latest findings, potential implications of the tight spatial and temporal regulation of chloride equilibrium in health and disease, as well as its relevance for the therapeutic effects of molecules acting at GABAA receptors. PMID:27002180

  10. 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 α1β2γ2 GABAA receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 μM. However, (S)-reticuline behaved as positive allosteric modulator at the α3, α5, and α6 isoforms of GABAA receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by (S)-reticuline at the α3β2γ2 and α5β2γ2 GABAA receptors. Interestingly, α1, α3, and α5 were not significantly affected by (R)-reticuline, 1,2-tetrahydroreticuline, codeine, and morphine—suspected (S)-reticuline metabolites in the rodent brain. PMID:26509084

  11. Glyoxalase 1 increases anxiety by reducing GABAA receptor agonist methylglyoxal

    PubMed Central

    Distler, Margaret G.; Plant, Leigh D.; Sokoloff, Greta; Hawk, Andrew J.; Aneas, Ivy; Wuenschell, Gerald E.; Termini, John; Meredith, Stephen C.; Nobrega, Marcelo A.; Palmer, Abraham A.

    2012-01-01

    Glyoxalase 1 (Glo1) expression has previously been associated with anxiety in mice; however, its role in anxiety is controversial, and the underlying mechanism is unknown. Here, we demonstrate that GLO1 increases anxiety by reducing levels of methylglyoxal (MG), a GABAA receptor agonist. Mice overexpressing Glo1 on a Tg bacterial artificial chromosome displayed increased anxiety-like behavior and reduced brain MG concentrations. Treatment with low doses of MG reduced anxiety-like behavior, while higher doses caused locomotor depression, ataxia, and hypothermia, which are characteristic effects of GABAA receptor activation. Consistent with these data, we found that physiological concentrations of MG selectively activated GABAA receptors in primary neurons. These data indicate that GLO1 increases anxiety by reducing levels of MG, thereby decreasing GABAA receptor activation. More broadly, our findings potentially link metabolic state, neuronal inhibitory tone, and behavior. Finally, we demonstrated that pharmacological inhibition of GLO1 reduced anxiety, suggesting that GLO1 is a possible target for the treatment of anxiety disorders. PMID:22585572

  12. Modulatory Effects of Eschscholzia californica Alkaloids on Recombinant GABAA Receptors.

    PubMed

    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

  13. Assessment of Methods for the Intracellular Blockade of GABAA Receptors.

    PubMed

    Atherton, Laura A; Burnell, Erica S; Mellor, Jack R

    2016-01-01

    Selective blockade of inhibitory synaptic transmission onto specific neurons is a useful tool for dissecting the excitatory and inhibitory synaptic components of ongoing network activity. To achieve this, intracellular recording with a patch solution capable of blocking GABAA receptors has advantages over other manipulations, such as pharmacological application of GABAergic antagonists or optogenetic inhibition of populations of interneurones, in that the majority of inhibitory transmission is unaffected and hence the remaining network activity preserved. Here, we assess three previously described methods to block inhibition: intracellular application of the molecules picrotoxin, 4,4'-dinitro-stilbene-2,2'-disulphonic acid (DNDS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). DNDS and picrotoxin were both found to be ineffective at blocking evoked, monosynaptic inhibitory postsynaptic currents (IPSCs) onto mouse CA1 pyramidal cells. An intracellular solution containing DIDS and caesium fluoride, but lacking nucleotides ATP and GTP, was effective at decreasing the amplitude of IPSCs. However, this effect was found to be independent of DIDS, and the absence of intracellular nucleotides, and was instead due to the presence of fluoride ions in this intracellular solution, which also blocked spontaneously occurring IPSCs during hippocampal sharp waves. Critically, intracellular fluoride ions also caused a decrease in both spontaneous and evoked excitatory synaptic currents and precluded the inclusion of nucleotides in the intracellular solution. Therefore, of the methods tested, only fluoride ions were effective for intracellular blockade of IPSCs but this approach has additional cellular effects reducing its selectivity and utility. PMID:27501143

  14. GABAA receptor modulation by piperine and a non-TRPV1 activating derivative☆

    PubMed Central

    Khom, Sophia; Strommer, Barbara; Schöffmann, Angela; Hintersteiner, Juliane; Baburin, Igor; Erker, Thomas; Schwarz, Thomas; Schwarzer, Christoph; Zaugg, Janine; Hamburger, Matthias; Hering, Steffen

    2013-01-01

    The action of piperine (the pungent component of pepper) and its derivative SCT-66 ((2E,4E)-5-(1,3-benzodioxol-5-yl))-N,N-diisobutyl-2,4-pentadienamide) on different gamma-aminobutyric acid (GABA) type A (GABAA) receptors, transient-receptor-potential-vanilloid-1 (TRPV1) receptors and behavioural effects were investigated. GABAA receptor subtypes and TRPV1 receptors were expressed in Xenopus laevis oocytes. Modulation of GABA-induced chloride currents (IGABA) by piperine and SCT-66 and activation of TRPV1 was studied using the two-microelectrode-voltage-clamp technique and fast perfusion. Their effects on explorative behaviour, thermoregulation and seizure threshold were analysed in mice. Piperine acted with similar potency on all GABAA receptor subtypes (EC50 range: 42.8 ± 7.6 μM (α2β2)–59.6 ± 12.3 μM (α3β2)). IGABA modulation by piperine did not require the presence of a γ2S-subunit, suggesting a binding site involving only α and β subunits. IGABA activation was slightly more efficacious on receptors formed from β2/3 subunits (maximal IGABA stimulation through α1β3 receptors: 332 ± 64% and α1β2: 271 ± 36% vs. α1β1: 171 ± 22%, p < 0.05) and α3-subunits (α3β2: 375 ± 51% vs. α5β2:136 ± 22%, p < 0.05). Replacing the piperidine ring by a N,N-diisobutyl residue (SCT-66) prevents interactions with TRPV1 and simultaneously increases the potency and efficiency of GABAA receptor modulation. SCT-66 displayed greater efficacy on GABAA receptors than piperine, with different subunit-dependence. Both compounds induced anxiolytic, anticonvulsant effects and reduced locomotor activity; however, SCT-66 induced stronger anxiolysis without decreasing body temperature and without the proconvulsive effects of TRPV1 activation and thus may serve as a scaffold for the development of novel GABAA receptor modulators. PMID:23623790

  15. Azogabazine; a photochromic antagonist of the GABAA receptor.

    PubMed

    Huckvale, Rosemary; Mortensen, Martin; Pryde, David; Smart, Trevor G; Baker, James R

    2016-07-12

    The design and synthesis of azogabazine is described, which represents a highly potent (IC50 = 23 nM) photoswitchable antagonist of the GABAA receptor. An azologization strategy is adopted, in which a benzyl phenyl ether in a high affinity gabazine analogue is replaced by an azobenzene, with resultant retention of antagonist potency. We show that cycling from blue to UV light, switching between trans and cis isomeric forms, leads to photochemically controlled antagonism of the GABA ion channel. PMID:27327397

  16. 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.

  17. Assessment of direct gating and allosteric modulatory effects of meprobamate in recombinant GABA(A) receptors.

    PubMed

    Kumar, Manish; Dillon, Glenn H

    2016-03-15

    Meprobamate is a schedule IV anxiolytic and the primary metabolite of the muscle relaxant carisoprodol. Meprobamate modulates GABAA (γ-aminobutyric acid Type A) receptors, and has barbiturate-like activity. To gain insight into its actions, we have conducted a series of studies using recombinant GABAA receptors. In αxβzγ2 GABAA receptors (where x=1-6 and z=1-3), the ability to enhance GABA-mediated current was evident for all α subunit isoforms, with the largest effect observed in α5-expressing receptors. Direct gating was present with all α subunits, although attenuated in α3-expressing receptors. Allosteric and direct effects were comparable in α1β1γ2 and α1β2γ2 receptors, whereas allosteric effects were enhanced in α1β2 compared to α1β2γ2 receptors. In "extrasynaptic" (α1β3δ and α4β3δ) receptors, meprobamate enhanced EC20 and saturating GABA currents, and directly activated these receptors. The barbiturate antagonist bemegride attenuated direct effects of meprobamate. Whereas pentobarbital directly gated homomeric β3 receptors, meprobamate did not, and instead blocked the spontaneously open current present in these receptors. In wild type homomeric ρ1 receptors, pentobarbital and meprobamate were ineffective in direct gating; a mutation known to confer sensitivity to pentobarbital did not confer sensitivity to meprobamate. Our results provide insight into the actions of meprobamate and parent therapeutic agents such as carisoprodol. Whereas in general actions of meprobamate were comparable to those of carisoprodol, differential effects of meprobamate at some receptor subtypes suggest potential advantages of meprobamate may be exploited. A re-assessment of previously synthesized meprobamate-related carbamate molecules for myorelaxant and other therapeutic indications is warranted. PMID:26872987

  18. Positron Emission Tomography (PET) Quantification of GABAA Receptors in the Brain of Fragile X Patients

    PubMed Central

    Van der Aa, Nathalie; Goffin, Karolien; Koole, Michel; Porke, Kathleen; Van De Velde, Marc; Rooms, Liesbeth; Van Paesschen, Wim; Van Esch, Hilde; Van Laere, Koen; Kooy, R. Frank

    2015-01-01

    Over the last several years, evidence has accumulated that the GABAA receptor is compromised in animal models for fragile X syndrome (FXS), a common hereditary form of intellectual disability. In mouse and fly models, agonists of the GABAA receptor were able to rescue specific consequences of the fragile X mutation. Here, we imaged and quantified GABAA receptors in vivo in brain of fragile X patients using Positron Emission Topography (PET) and [11C]flumazenil, a known high-affinity and specific ligand for the benzodiazepine site of GABAA receptors. We measured regional GABAA receptor availability in 10 fragile X patients and 10 control subjects. We found a significant reduction of on average 10% in GABAA receptor binding potential throughout the brain in fragile X patients. In the thalamus, the brain region showing the largest difference, the GABAA receptor availability was even reduced with 17%. This is one of the first reports of a PET study of human fragile X brain and directly demonstrates that the GABAA receptor availability is reduced in fragile X patients. The study reinforces previous hypotheses that the GABAA receptor is a potential target for rational pharmacological treatment of fragile X syndrome. PMID:26222316

  19. γ-Aminobutyric Acid Type A (GABAA) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains*

    PubMed Central

    Brown, Laura E.; Nicholson, Martin W.; Arama, Jessica E.; Thomson, Alex M.

    2016-01-01

    The establishment of cell-cell contacts between presynaptic GABAergic neurons and their postsynaptic targets initiates the process of GABAergic synapse formation. GABAA receptors (GABAARs), the main postsynaptic receptors for GABA, have been recently demonstrated to act as synaptogenic proteins that can single-handedly induce the formation and functional maturation of inhibitory synapses. To establish how the subunit composition of GABAARs influences their ability to induce synaptogenesis, a co-culture model system incorporating GABAergic medium spiny neurons and the HEK293 cells, stably expressing different combinations of receptor subunits, was developed. Analyses of HEK293 cell innervation by medium spiny neuron axons using immunocytochemistry, activity-dependent labeling, and electrophysiology have indicated that the γ2 subunit is required for the formation of active synapses and that its effects are influenced by the type of α/β subunits incorporated into the functional receptor. To further characterize this process, the large N-terminal extracellular domains (ECDs) of α1, α2, β2, and γ2 subunits were purified using the baculovirus/Sf9 cell system. When these proteins were applied to the co-cultures of MSNs and α1/β2/γ2-expressing HEK293 cells, the α1, β2, or γ2 ECD each caused a significant reduction in contact formation, in contrast to the α2 ECD, which had no effect. Together, our experiments indicate that the structural role of GABAARs in synaptic contact formation is determined by their subunit composition, with the N-terminal ECDs of each of the subunits directly participating in interactions between the presynaptic and postsynaptic elements, suggesting the these interactions are multivalent and specific. PMID:27129275

  20. Assessment of Methods for the Intracellular Blockade of GABAA Receptors

    PubMed Central

    Atherton, Laura A.; Burnell, Erica S.; Mellor, Jack R.

    2016-01-01

    Selective blockade of inhibitory synaptic transmission onto specific neurons is a useful tool for dissecting the excitatory and inhibitory synaptic components of ongoing network activity. To achieve this, intracellular recording with a patch solution capable of blocking GABAA receptors has advantages over other manipulations, such as pharmacological application of GABAergic antagonists or optogenetic inhibition of populations of interneurones, in that the majority of inhibitory transmission is unaffected and hence the remaining network activity preserved. Here, we assess three previously described methods to block inhibition: intracellular application of the molecules picrotoxin, 4,4’-dinitro-stilbene-2,2’-disulphonic acid (DNDS) and 4,4’-diisothiocyanostilbene-2,2’-disulphonic acid (DIDS). DNDS and picrotoxin were both found to be ineffective at blocking evoked, monosynaptic inhibitory postsynaptic currents (IPSCs) onto mouse CA1 pyramidal cells. An intracellular solution containing DIDS and caesium fluoride, but lacking nucleotides ATP and GTP, was effective at decreasing the amplitude of IPSCs. However, this effect was found to be independent of DIDS, and the absence of intracellular nucleotides, and was instead due to the presence of fluoride ions in this intracellular solution, which also blocked spontaneously occurring IPSCs during hippocampal sharp waves. Critically, intracellular fluoride ions also caused a decrease in both spontaneous and evoked excitatory synaptic currents and precluded the inclusion of nucleotides in the intracellular solution. Therefore, of the methods tested, only fluoride ions were effective for intracellular blockade of IPSCs but this approach has additional cellular effects reducing its selectivity and utility. PMID:27501143

  1. Functional rundown of GABAA receptors in human hypothalamic hamartomas

    PubMed Central

    Li, Guohui; Yang, Kechun; Zheng, Chao; Liu, Qiang; Chang, Yongchang; Kerrigan, John F.; Wu, Jie

    2010-01-01

    Objective Human hypothalamic hamartomas (HH) are highly associated with treatment-resistant gelastic seizures. HH are intrinsically epileptogenic, although the basic cellular mechanisms responsible for seizure activity are unknown. Altered gamma-aminobutyric acid (GABA) function can contribute to epileptogenesis in humans and animal models. Recently, functional GABAA receptor (GABAAR) rundown has been described in surgically-resected human temporal lobe epilepsy tissue. We asked whether functional GABAAR rundown also occurs in human HH neurons. Methods GABAAR-mediated currents were measured using perforated patch-clamp recordings in single neurons acutely dissociated from surgically-resected HH tissue. In addition, functional GABAARs were expressed in Xenopus oocytes after microinjection with membrane fractions from either HH or control hypothalamus, and were studied with two-electrode voltage-clamp recordings. Results Perforated patch-clamp recordings in dissociated HH neurons showed that repetitive exposure to GABA (5 consecutive exposures to 0.1 mM GABA with 1 sec duration and at 20 sec intervals) induced a time-dependent rundown of whole-cell currents in small HH neurons, while large HH neurons showed much less rundown using the same protocol. Functional rundown was not observed in HH neurons with repetitive exposure to glycine or glutamate. Two-electrode voltage-clamp recordings (6 consecutive exposures to 1 mM GABA with 10 sec duration and at 40 sec intervals) induced GABA-current rundown in Xenopus oocytes microinjected with HH membrane proteins, but not in the oocytes expressing hypothalamic membrane proteins derived from human autopsy controls. Functional rundown of GABA-currents was significantly attenuated by intracellular application of adenosine triphosphate (ATP) or the non-specific phosphatase inhibitor, okadaic acid. Interpretation Neurons from surgically-resected human HH demonstrate functional rundown of GABAAR-mediated transmembrane currents in

  2. Role of the alpha subunit in the modulation of GABA(A) receptors by anabolic androgenic steroids.

    PubMed

    Yang, Paul; Jones, Brian L; Henderson, Leslie P

    2005-09-01

    Neural transmission mediated by circuits expressing alpha2 subunit-containing gamma-aminobutyric acid type A (GABA(A)) receptors is critical for the expression of behaviors known to be altered by anabolic androgenic steroids (AAS). Here we show that micromolar concentrations of AAS, which reflect levels found in steroid abusers, induce positive modulation of currents from alpha2beta3 gamma2L recombinant receptors elicited by pulses of GABA that mimic synaptic conditions in a manner that is mechanistically distinct from modulation induced at alpha1beta3 gamma2L receptors. Specifically, at alpha2-containing receptors, the AAS, 17alpha-methyltestosterone (17alpha-MeT) enhanced peak current, slowed deactivation, diminished desensitization, and promoted entry of receptors into more distal states along the activation pathway. Analysis of GABA(A) receptor-mediated synaptic currents in primary cortical neurons followed by single cell real-time RT-PCR demonstrated that 17alpha-MeT enhancement of synaptic currents is proportional to the ratio of alpha2 to alpha1 subunit mRNA. Finally, we show that the modulation elicited by AAS is not comparable to that produced by micromolar concentrations of other positive allosteric modulators at alpha2-containing receptors. In sum, these data indicate that AAS elicit effects on GABA(A) receptor function that depend significantly on alpha subunit composition and that the mechanism of AAS modulation of GABA(A) receptors is distinct from that of other positive allosteric modulators.

  3. Neurosteroid Agonist at GABAA receptor induces persistent neuroplasticity in VTA dopamine neurons.

    PubMed

    Vashchinkina, Elena; Manner, Aino K; Vekovischeva, Olga; den Hollander, Bjørnar; Uusi-Oukari, Mikko; Aitta-Aho, Teemu; Korpi, Esa R

    2014-02-01

    The main fast-acting inhibitory receptors in the mammalian brain are γ-aminobutyric acid type-A (GABAA) receptors for which neurosteroids, a subclass of steroids synthesized de novo in the brain, constitute a group of endogenous ligands with the most potent positive modulatory actions known. Neurosteroids can act on all subtypes of GABAA receptors, with a preference for δ-subunit-containing receptors that mediate extrasynaptic tonic inhibition. Pathological conditions characterized by emotional and motivational disturbances are often associated with perturbation in the levels of endogenous neurosteroids. We studied the effects of ganaxolone (GAN)-a synthetic analog of endogenous allopregnanolone that lacks activity on nuclear steroid receptors-on the mesolimbic dopamine (DA) system involved in emotions and motivation. A single dose of GAN in young mice induced a dose-dependent, long-lasting neuroplasticity of glutamate synapses of DA neurons ex vivo in the ventral tegmental area (VTA). Increased α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/N-methyl-D-aspartate ratio and rectification of AMPA receptor responses even at 6 days after GAN administration suggested persistent synaptic targeting of GluA2-lacking AMPA receptors. This glutamate neuroplasticity was not observed in GABAA receptor δ-subunit-knockout (δ-KO) mice. GAN (500 nM) applied locally to VTA selectively increased tonic inhibition of GABA interneurons and triggered potentiation of DA neurons within 4 h in vitro. Place-conditioning experiments in adult wild-type C57BL/6J and δ-KO mice revealed aversive properties of repeated GAN administration that were dependent on the δ-subunits. Prolonged neuroadaptation to neurosteroids in the VTA might contribute to both the physiology and pathophysiology underlying processes and changes in motivation, mood, cognition, and drug addiction. PMID:24077066

  4. GABAA receptor signaling in the lateral septum regulates maternal aggression in mice

    PubMed Central

    Lee, Grace; Gammie, Stephen C.

    2010-01-01

    Maternal aggression (maternal defense) is a fierce aggression produced by lactating females towards intruders that plays an important role in protection of vulnerable offspring. Enhancement of GABAA receptor signaling by benzodiazepines increases maternal aggression and we recently found indirect evidence that lateral septum (LS) could be a key site where benzodiazepines elevate aggression. In this study, we directly tested the hypothesis that activation of GABAA receptors in LS would promote maternal aggression while inhibition of this receptor would decrease aggression. Site-directed injections to LS were made using the GABAA receptor antagonist, bicuculline (3-30 ng), or the GABAA receptor agonists, chlordiazepoxide, a benzodiazepine (2.5-5 μg), and muscimol (0.05–5 ng). Maternal aggression and other behavioral measures were then evaluated in lactating mice. Neither GABAA receptor agonist elevated aggression, which could reflect a ceiling effect. However, 7 ng of the GABAA receptor antagonist, bicuculline, in LS significantly decreased maternal aggression without altering other maternal behaviors or light-dark box performance, suggesting some GABAA receptor signaling in LS is required for full maternal aggression expression. Together these results confirm a role for GABAA receptor signaling in LS in the regulation of maternal aggression. PMID:20001101

  5. Potentiation of GABAA receptors expressed in Xenopus oocytes by perfume and phytoncid.

    PubMed

    Aoshima, H; Hamamoto, K

    1999-04-01

    To study the effects of perfume and phytoncid on GABAA receptors, ionotropic GABAA receptors were expressed in Xenopus oocytes by injecting mRNAs that had been prepared from rat whole brain. Essential oil, perfume and such phytoncid as leaf alcohol, hinokitiol, pinene, eugenol, citronellol and citronellal potentiated the response in the presence of GABA at low concentrations (10 and 30 microM), possibly because they bound to the potentiation-site in GABAA receptors and increased the affinity of GABA to the receptors. Since it is known that the potentiation of GABAA receptors by benzodiazepine, barbiturate, steroids and anesthetics induces the anxiolytic, anticonvulsant and sedative activity or anesthetic effect, these results suggest the possibility that the intake of perfume or phytoncid through the lungs, the skin or the intestines modulates the neural transmission in the brain through ionotropic GABAA receptors and changes the frame of the human mind, as alcohol or tobacco does.

  6. Modulation of diazepam-insensitive GABA(A) receptors by micromolar concentrations of thyroxine and related compounds in vitro.

    PubMed

    Ishibashi, Hitoshi; Witt, Michael-Robin; Nabekura, Junichi; Nielsen, Mogens

    2013-01-15

    The effects of thyroxine and its related compounds on the benzodiazepine-insensitive γ-aminobutyric acid type A (GABA(A)) receptors were studied. Thyroxine at micromolar concentrations potentiated the (3)H-Ro15-4513 binding to rat brain membranes in-vitro in the thalamus, striatum, cortex and hippocampus, but not in cerebellum. In the thalamus, the rank order of potency was the following: 3,3',5,5'-tetraiodothyroacetic acid (TETRAC)>L-thyroxine>3,5-diiodo-l-thyronine (3,5-T2). TETRAC induced a slight potentiation of flumazenil binding to diazepam-sensitive GABA(A) receptors in the thalamus and striatum while no effect was found in cortex and hippocampus. Consequently, we examined whether these compounds could exert their modulatory effect on the currents mediated by benzodiazepine-insensitive GABA(A) receptors. The diazepam-insensitive GABA(A) receptor-mediated currents were recorded from acutely isolated rat ventrobasal thalamic neurons by applying low concentrations of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). TETRAC and thyroxine at low μM concentrations potentiated the THIP-evoked currents, although 3,5-T2 had no effect on the THIP-induced currents. Ethanol had no effect on the enhancing effects of TETRAC. TETRAC itself evoked GABA(A) receptor-mediated currents at high concentrations beyond 30 μM. Although the effects of TETRAC and thyroxine were observed at non-physiological concentrations of hormones, the present results might lead to new lead structures with specificity to diazepam-insensitive GABA(A) receptor subtypes.

  7. 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

  8. 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

  9. Allopregnanolone, a GABAA receptor agonist, decreases gonadotropin levels in women. A preliminary study.

    PubMed

    Timby, Erika; Hedström, Helena; Bäckström, Torbjörn; Sundström-Poromaa, Inger; Nyberg, Sigrid; Bixo, Marie

    2011-12-01

    Animal studies suggest regulatory effects on the hypothalamic-pituitary-gonad axis by allopregnanolone, an endogenous gamma-aminobutyric acid A (GABA(A)) receptor agonist. Elevated levels of allopregnanolone in women with hypothalamic amenorrhea have been seen. Isoallopregnanolone is an isomer to allopregnanolone, but without GABA(A) receptor effects. The purpose of this study was to investigate effects of allopregnanolone and isoallopregnanolone on gonadotropin levels in healthy women of fertile age. Ten women were given allopregnanolone and five women isoallopregnanolone intravenously in follicular phase. Repeated blood samples were drawn during the test day. Main outcomes were changes in serum levels of follicle-stimulating hormone (FSH), luteinising hormone (LH), oestradiol, and progesterone. Serum-FSH decreased between 5 and 105 min after the allopregnanolone injection (F(16,144)=2.18, p=0.008). Serum-LH was reduced between 5 and 35 min following the allopregnanolone injection (F(16,144)=2.63, p=0.001). Serum-oestradiol and -progesterone were not significantly changed after allopregnanolone injections. No effect on gonadotropin levels were seen after administration of isoallopregnanolone. Allopregnanolone reduces FSH and LH levels in women and the effect might be mediated via a specific GABA(A) receptor activation since isoallopregnanolone lacked this effect. Although the number of women was small, the results suggest a regulatory mechanism on the hypothalamic-pituitary-gonadal axis by allopregnanolon.

  10. Ethanol Increases Mechanical Pain Sensitivity in Rats via Activation of GABAA Receptors in Medial Prefrontal Cortex.

    PubMed

    Geng, Kai-Wen; He, Ting; Wang, Rui-Rui; Li, Chun-Li; Luo, Wen-Jun; Wu, Fang-Fang; Wang, Yan; Li, Zhen; Lu, Yun-Fei; Guan, Su-Min; Chen, Jun

    2016-10-01

    Ethanol is widely known for its ability to cause dramatic changes in emotion, social cognition, and behavior following systemic administration in humans. Human neuroimaging studies suggest that alcohol dependence and chronic pain may share common mechanisms through amygdala-medial prefrontal cortex (mPFC) interactions. However, whether acute administration of ethanol in the mPFC can modulate pain perception is unknown. Here we showed that bilateral microinjections of ethanol into the prelimbic and infralimbic areas of the mPFC lowered the bilateral mechanical pain threshold for 48 h without influencing thermal pain sensitivity in adult rats. However, bilateral microinjections of artificial cerebrospinal fluid into the mPFC or bilateral microinjections of ethanol into the dorsolateral PFC (also termed as motor cortex area 1 in Paxinos and Watson's atlas of The Rat Brain. Elsevier Academic Press, Amsterdam, 2005) failed to do so, suggesting regional selectivity of the effects of ethanol. Moreover, bilateral microinjections of ethanol did not change the expression of either pro-apoptotic (caspase-3 and Bax) or anti-apoptotic (Bcl-2) proteins, suggesting that the dose was safe and validating the method used in the current study. To determine whether γ-aminobutyric acid A (GABAA) receptors are involved in mediating the ethanol effects, muscimol, a selective GABAA receptor agonist, or bicuculline, a selective GABAA receptor antagonist, was administered alone or co-administered with ethanol through the same route into the bilateral mPFC. The results showed that muscimol mimicked the effects of ethanol while bicuculline completely reversed the effects of ethanol and muscimol. In conclusion, ethanol increases mechanical pain sensitivity through activation of GABAA receptors in the mPFC of rats. PMID:27628528

  11. GABAA receptor pharmacology of fluorinated derivatives of the novel sedative-hypnotic pyrazolopyrimidine indiplon.

    PubMed

    Wegner, Florian; Deuther-Conrad, Winnie; Scheunemann, Matthias; Brust, Peter; Fischer, Steffen; Hiller, Achim; Diekers, Michael; Strecker, Karl; Wohlfarth, Kai; Allgaier, Clemens; Steinbach, Jörg; Hoepping, Alexander

    2008-02-01

    The function of gamma-aminobutyric acid type A receptors (GABA(A) receptors) is enhanced by various clinically important drugs including benzodiazepines that act on an allosteric site formed at the interface between the alpha and gamma subunits. In contrast to classical benzodiazepines, the novel pyrazolopyrimidine indiplon (N-methyl-N-{3-[7-(thiophene-2-carbonyl)-1,5,9-triazabicyclo[4.3.0]nona-2,4,6,8-tetraen-2-yl]phenyl}acetamide; N-methyl-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine-7-yl]phenyl}-acetamide) demonstrates relative binding selectivity for the alpha1 subunit containing receptor subtypes, which are the most frequently expressed in the mammalian central nervous system. To investigate the pharmacological properties at GABA(A) receptors and to promote the development of alpha1 subunit selective radiotracers for positron emission tomography imaging, we have started with the evaluation of various fluorinated indiplon derivatives. Binding affinities were determined in homogenates from newborn and adult rats suggesting an alpha1 preference of the reference compounds indiplon, zaleplon as well as for all newly synthesized indiplon derivatives. In homogenated cerebellar tissue obtained from adult rat brain, known to primarily express alpha1 containing GABA(A) receptors, the high affinity of the basic indiplon structure was only slightly affected by an elongation of the alkyl substituent of the amide N from methyl (indiplon; K(i) 3.1 nM) via ethyl (2a, N-(2-fluoro-ethyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine-7-yl]phenyl}-acetamide; K(i) 5.4 nM) to propyl (2b, N-(3-fluoro-propyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine-7-yl]phenyl}-acetamide; K(i) 2.4 nM). Whole cell patch-clamp recordings at neuronal and recombinant GABA(A) receptors indicated that the fluorinated derivatives 2a and 2b have a high potency at alpha1beta3gamma2L isoforms comparable to indiplon (EC(50): 105, 158, and 81 nM, respectively), with 2b

  12. Menthol shares general anesthetic activity and sites of action on the GABA(A) receptor with the intravenous agent, propofol.

    PubMed

    Watt, Erin E; Betts, Brooke A; Kotey, Francesca O; Humbert, Danielle J; Griffith, Theanne N; Kelly, Elizabeth W; Veneskey, Kelley C; Gill, Nikhila; Rowan, Kathleen C; Jenkins, Andrew; Hall, Adam C

    2008-08-20

    Menthol and related compounds were investigated for modulation of recombinant human gamma-aminobutyric acid type A (GABA(A), alpha(1)beta(2)gamma(2s)) receptor currents expressed in Xenopus oocytes. Sub-maximal (EC(20)) GABA currents were typically enhanced by co-applications of 3-300 microM (+)-menthol (e.g. by approximately 2-fold at 50 microM) > isopulegol > isomenthol> alpha-terpineol > cyclohexanol. We studied menthol's actions on GABA(A) receptors compared to sedatives (benzodiazepines) and intravenous anesthetics (barbiturates, steroids, etomidate and propofol). Flumazenil (a benzodiazepine antagonist) did not inhibit menthol enhancements while currents directly activated by 50 microM propofol were significantly inhibited (by 26+/-3%) by 50 microM (+)-menthol. GABA(A) receptors containing beta(2) subunits with either a point mutation in a methionine residue to a tryptophan at the 286 position (in transmembrane domain 3, TM-3) or a tyrosine to a tryptophan at the 444 position (TM-4) are insensitive to modulation by propofol. Enhancements of GABA EC(20) currents by menthol were equally abolished in GABA(A) alpha(1)beta(2)(M286W)gamma(2s) and alpha(1)beta(2)(Y444W)gamma(2s) receptors while positive modulations by benzodiazepines, barbiturates and steroids were unaffected. Menthol may therefore exert its actions on GABA(A) receptors via sites distinct from benzodiazepines, steroids and barbiturates, and via sites important for modulation by propofol. Finally, using an in vivo tadpole assay, addition of (+)-menthol resulted in a loss of righting reflex with an EC(50) of 23.5+/-4.7 microM (approximately10-fold less potent anesthesia than propofol). Thus, menthol and analogs share general anesthetic action with propofol, possibly via action at similar sites on the GABA(A) receptor. PMID:18593637

  13. 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

  14. Zolpidem modulates GABA(A) receptor function in subthalamic nucleus.

    PubMed

    Chen, Lei; Xie, Jun-Xia; Fung, Kam-Shuen; Yung, Wing-Ho

    2007-05-01

    The subthalamic nucleus occupies a position in the indirect pathway of basal ganglia circuit, which plays an important role in the movement regulation. Zolpidem is an imidazopyridine agonist with a high affinity on the benzodiazepine site of GABA(A) receptors containing alpha 1 subunit. Recently, zolpidem has been reported to be useful in treating subgroups of parkinsonian patients. A high density of zolpidem binding sites has been shown in rat subthalamic nucleus. To further investigate the modulation of zolpidem on GABA(A) receptor-mediated inhibitory synaptic current in subthalamic nucleus, whole-cell patch clamp recordings were used in the present study. Zolpidem at 100nM significantly prolonged the decay time and rise time of miniature inhibitory postsynaptic currents, with no effect on the amplitude and frequency. The benzodiazepine antagonist flumazenil could completely block the potentiation induced by zolpidem, confirming the specificity on the benzodiazepine site. At a high concentration of 1 microM, zolpidem significantly increased the decay time, rise time, amplitude and frequency of miniature inhibitory postsynaptic currents. In the behaving rats, unilateral microinjection of zolpidem into subthalamic nucleus induced a significant contralateral rotation. The present findings on the effect of zolpidem in subthalamic nucleus provide a rationale for further investigations into its potential in the treatment of Parkinson's disease. PMID:17337310

  15. Multiple GABAA receptor subtypes regulate hippocampal ripple oscillations.

    PubMed

    Ponomarenko, A A; Korotkova, T M; Sergeeva, O A; Haas, H L

    2004-10-01

    High-frequency oscillations (140-200 Hz) were recorded in behaving rats from the CA1 area of the hippocampus. As generation of these synchronous patterns is assumed to depend on coordinated interneuronal inhibition, we studied the interference of benzodiazepines with the fine structure and occurrence of ripple oscillations. The nonselective GABAA receptor alpha-subunit agonist, diazepam, lowered the frequency of ripple oscillations and reduced their occurrence, amplitude and duration. Zolpidem, an alpha1-subunit selective benzodiazepine elevated ripple duration but acted similar to diazepam in other respects. The nonselective alpha-subunit benzodiazepine antagonist, flumazenil, reduced ripple numbers, amplitude and duration. Wavelet based analysis of the dynamics of intraripple frequency revealed a dramatic decay within a ripple. Only diazepam (1 mg/kg) accelerated this intraripple frequency accommodation. The effects were not due to increased behavioural activity and alertness as evident from vigilance state control. The results suggest a differential role of GABAA receptor subtype specific inhibitory mechanisms in the mediation and fine-tuning of the network synchronization during approximately 200 Hz hippocampal oscillations.

  16. The voltage dependence of GABAA receptor gating depends on extracellular pH.

    PubMed

    Pytel, Maria; Mercik, Katarzyna; Mozrzymas, Jerzy W

    2005-11-28

    Recent studies have indicated that changes in extracellular pH and in membrane voltage affect the gamma-amino-n-butyric acid type A receptor gating mainly by altering desensitization and binding. To test whether the effects of membrane potential and pH are additive, their combined actions were investigated. By analyzing the current responses to rapid gamma-amino-n-butyric acid applications, we found that the current to voltage relationship was close to linear at acid pH but the increasing pH induced an inward rectification. Desensitization was enhanced at depolarizing potentials, but this strongly depended on pH, being weak at acidic and strong at basic pH values. A similar trend was observed for the onset rate of responses to saturating gamma-amino-n-butyric acid concentration. These data provide evidence that the voltage sensitivity of GABAA receptors depends on extracellular pH.

  17. The voltage dependence of GABAA receptor gating depends on extracellular pH

    PubMed Central

    Pytel, Maria; Mercik, Katarzyna; Mozrzymas, Jerzy W.

    2007-01-01

    Recent studies have indicated that changes in extracellular pH and in membrane voltage affect the γ-amino-n-butyric acid type A receptor gating mainly by altering desensitization and binding. To test whether the effects of membrane potential and pH are additive, their combined actions were investigated. By analyzing the current responses to rapid γ-amino-n-butyric acid applications, we found that the current to voltage relationship was close to linear at acid pH but the increasing pH induced an inward rectification. Desensitization was enhanced at depolarizing potentials, but this strongly depended on pH, being weak at acidic and strong at basic pH values. A similar trend was observed for the onset rate of responses to saturating γ-amino-n-butyric acid concentration. These data provide evidence that the voltage sensitivity of GABAA receptors depends on extracellular pH. PMID:16272885

  18. Tobacco smoking interferes with GABAA receptor neuroadaptations during prolonged alcohol withdrawal.

    PubMed

    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

    2014-12-16

    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.

  19. Reduction of α1GABAA receptor mediated by tyrosine kinase C (PKC) phosphorylation in a mouse model of fragile X syndrome

    PubMed Central

    Zhao, Weidong; Wang, Jiaqin; Song, Shunyi; Li, Fang; Yuan, Fangfang

    2015-01-01

    Fragile X syndrome (FXS) caused by lack of fragile X mental retardation protein (Fmr1) is the most common cause of inherited intellectual disability and characterized by many cognitive disturbances like attention deficit, autistic behavior, and audiogenic seizure and have region-specific altered expression of some gamma-aminobutyric acid (GABAA) receptor subunits. Quantitative real-time polymerase chain reaction and western blot experiments were performed in the cultured cortical neurons and forebrain obtained from wild-type (WT) and Fmr1 KO mice demonstrate the reduction in the expression of α1 gamma-aminobutyric acid (α1GABAA) receptor, phospho-α1GABAA receptor, PKC and phosphor-PKC in Fmr1 KO mice comparing with WT mice, both in vivo and in vitro. Furthermore, we found that the phosphorylation of the α1GABAA receptor was mediated by PKC. Our results elucidate that the lower phosphorylation of the α1GABAA receptor mediated by PKC neutralizes the seizure-promoting effects in Fmr1 KO mice and point to the potential therapeutic targets of α1GABAA agonists for the treatment of fragile X syndrome. PMID:26550246

  20. 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

  1. GABAA receptors and benzodiazepines: a role for dendritic resident subunit mRNAs.

    PubMed

    Costa, E; Auta, J; Grayson, D R; Matsumoto, K; Pappas, G D; Zhang, X; Guidotti, A

    2002-11-01

    This review is designed to describe the evolution of the seminal observation made simultaneously in 1975 by Dr. W. Haefely's laboratory (Hoffman La Roche, Basel, Switzerland) and in the Laboratory of Preclinical Pharmacology (NIH, St. Elizabeths Hospital, Washington DC), that benzodiazepine action was mediated by a modulation of GABA action at GABA(A) receptors. In fact, our suggestion was that the benzodiazepine receptor was "a receptor on a receptor" and that this receptor was GABA(A). Needless to say, this suggestion created opposition, but we did not abandon the original idea, in fact, as shown in this review, there is now universal agreement with our hypothesis on the mode of action of benzodiazepines. Hence, this review deals with the allosteric modulation of GABA(A) receptors by benzodiazepines, the role of GABA(A) receptors and benzodiazepine structure diversities in this modulation, and describes the results of our attempts to establish a benzodiazepine (imidazenil) devoid of tolerance, withdrawal symptoms, and changes in the expression of GABA(A) receptor subunits during tolerance. It also deals with the idea that the synthesis of GABA(A) receptor subunits triggered by tolerance resides in dendrites and spines where mRNAs and the apparatus for this translation is located. New analytic procedures may foster progress in the understanding of tolerance to and withdrawal from benzodiazepines.

  2. The GABAA receptor is an FMRP target with therapeutic potential in fragile X syndrome

    PubMed Central

    Braat, Sien; D'Hulst, Charlotte; Heulens, Inge; De Rubeis, Silvia; Mientjes, Edwin; Nelson, David L; Willemsen, Rob; Bagni, Claudia; Van Dam, Debby; De Deyn, Peter P; Kooy, R Frank

    2015-01-01

    Previous research indicates that the GABAAergic system is involved in the pathophysiology of the fragile X syndrome, a frequent form of inherited intellectual disability and associated with autism spectrum disorder. However, the molecular mechanism underlying GABAAergic deficits has remained largely unknown. Here, we demonstrate reduced mRNA expression of GABAA receptor subunits in the cortex and cerebellum of young Fmr1 knockout mice. In addition, we show that the previously reported underexpression of specific subunits of the GABAA receptor can be corrected in YAC transgenic rescue mice, containing the full-length human FMR1 gene in an Fmr1 knockout background. Moreover, we demonstrate that FMRP directly binds several GABAA receptor mRNAs. Finally, positive allosteric modulation of GABAA receptors with the neurosteroid ganaxolone can modulate specific behaviors in Fmr1 knockout mice, emphasizing the therapeutic potential of the receptor. PMID:25790165

  3. Role of spinal GABAA receptors in pudendal inhibition of nociceptive and nonnociceptive bladder reflexes in cats.

    PubMed

    Xiao, Zhiying; Reese, Jeremy; Schwen, Zeyad; Shen, Bing; Wang, Jicheng; Roppolo, James R; de Groat, William C; Tai, Changfeng

    2014-04-01

    Picrotoxin, an antagonist for γ-aminobutyric acid receptor subtype A (GABAA), was used to investigate the role of GABAA receptors in nociceptive and nonnociceptive reflex bladder activities and pudendal inhibition of these activities in cats under α-chloralose anesthesia. Acetic acid (AA; 0.25%) was used to irritate the bladder and induce nociceptive bladder overactivity, while saline was used to distend the bladder and induce nonnociceptive bladder activity. To modulate the bladder reflex, pudendal nerve stimulation (PNS) was applied at multiple threshold (T) intensities for inducing anal sphincter twitching. AA irritation significantly (P < 0.01) reduced bladder capacity to 34.3 ± 7.1% of the saline control capacity, while PNS at 2T and 4T significantly (P < 0.01) increased AA bladder capacity to 84.0 ± 7.8 and 93.2 ± 15.0%, respectively, of the saline control. Picrotoxin (0.4 mg it) did not change AA bladder capacity but completely removed PNS inhibition of AA-induced bladder overactivity. Picrotoxin (iv) only increased AA bladder capacity at a high dose (0.3 mg/kg) but significantly (P < 0.05) reduced 2T PNS inhibition at low doses (0.01-0.1 mg/kg). During saline cystometry, PNS significantly (P < 0.01) increased bladder capacity to 147.0 ± 7.6% at 2T and 172.7 ± 8.9% at 4T of control capacity, and picrotoxin (0.4 mg it or 0.03-0.3 mg/kg iv) also significantly (P < 0.05) increased bladder capacity. However, picrotoxin treatment did not alter PNS inhibition during saline infusion. These results indicate that spinal GABAA receptors have different roles in controlling nociceptive and nonnociceptive reflex bladder activities and in PNS inhibition of these activities. PMID:24523385

  4. Facilitated CA1 hippocampal synaptic plasticity in dystrophin-deficient mice: role for GABAA receptors?

    PubMed

    Vaillend, Cyrille; Billard, Jean-Marie

    2002-01-01

    Duchenne muscular dystrophy (DMD) is associated with cognitive deficits that may result from a deficiency in the brain isoform of the cytoskeletal membrane-associated protein, dystrophin. CA1 hippocampal short-term potentiation (STP) of synaptic transmission is increased in dystrophin-deficient mdx mice, which has been attributed to a facilitated activation of NMDA receptors. In this study, extracellular recordings in the hippocampal slice preparation were used first to determine the consequences of this alteration on short-term depression (STD). STD induction was facilitated in mdx as compared with wild-type mice in a control medium. Because brain dystrophin deficiency results in a decreased number of gamma-aminobutyric acid A (GABAA)-receptor clusters, we tested the hypothesis that neuronal disinhibition contributes to the enhanced synaptic plasticity in mdx mice. We found that the GABAA receptor antagonist, bicuculline, increased basal neurotransmission in wild-type, but not in mdx mice and prevented the enhanced STP and STD in the CA1 area of slices from mdx mice. The possibility that altered GABA mechanisms underlie the facilitation of NMDA receptor-dependent synaptic plasticity in mdx mice is discussed.

  5. 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.

  6. The α5 subunit containing GABAA receptors contribute to chronic pain.

    PubMed

    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

    2016-03-01

    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-induced, complete Freund's adjuvant (CFA)-induced and L5 and L6 spinal nerve ligation-induced long-lasting hypersensitivity. Formalin or CFA injection and L5 and L6 spinal nerve ligation produced long-lasting allodynia and hyperalgesia. Moreover, formalin injection impaired the rate-dependent depression of the Hofmann reflex. Peripheral and intrathecal pretreatment 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 receptor mRNA expression in the spinal cord and dorsal root ganglia (DRG) mainly at 3 days. The α5-GABAA receptors were localized in the dorsal spinal cord and DRG colabeling with NeuN, CGRP, and IB4 which suggests their presence in peptidergic and nonpeptidergic neurons. These receptors were found mainly in small and medium sized neurons. Formalin injection enhanced α5-GABAA receptor 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 rate-dependent depression. 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.

  7. The α5 subunit containing GABAA receptors contribute to chronic pain.

    PubMed

    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

    2016-03-01

    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-induced, complete Freund's adjuvant (CFA)-induced and L5 and L6 spinal nerve ligation-induced long-lasting hypersensitivity. Formalin or CFA injection and L5 and L6 spinal nerve ligation produced long-lasting allodynia and hyperalgesia. Moreover, formalin injection impaired the rate-dependent depression of the Hofmann reflex. Peripheral and intrathecal pretreatment 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 receptor mRNA expression in the spinal cord and dorsal root ganglia (DRG) mainly at 3 days. The α5-GABAA receptors were localized in the dorsal spinal cord and DRG colabeling with NeuN, CGRP, and IB4 which suggests their presence in peptidergic and nonpeptidergic neurons. These receptors were found mainly in small and medium sized neurons. Formalin injection enhanced α5-GABAA receptor 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 rate-dependent depression. 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

  8. GABAA receptor beta subunit heterogeneity: functional expression of cloned cDNAs.

    PubMed Central

    Ymer, S; Schofield, P R; Draguhn, A; Werner, P; Köhler, M; Seeburg, P H

    1989-01-01

    Cloned cDNAs encoding two new beta subunits of the rat and bovine GABAA receptor have been isolated using a degenerate oligonucleotide probe based on a highly conserved peptide sequence in the second transmembrane domain of GABAA receptor subunits. The beta 2 and beta 3 subunits share approximately 72% sequence identity with the previously characterized beta 1 polypeptide. Northern analysis showed that both beta 2 and beta 3 mRNAs are more abundant in the brain than beta 1 mRNA. All three beta subunit encoding cDNAs were also identified in a library constructed from adrenal medulla RNA. Each beta subunit, when co-expressed in Xenopus oocytes with an alpha subunit, forms functional GABAA receptors. These results, together with the known alpha subunit heterogeneity, suggest that a variety of related but functionally distinct GABAA receptor subtypes are generated by different subunit combinations. Images PMID:2548852

  9. The influence of the membrane on neurosteroid actions at GABAA receptors

    PubMed Central

    Akk, Gustav; Covey, Douglas F.; Evers, Alex S.; Steinbach, Joe Henry; Zorumski, Charles F.; Mennerick, Steven

    2009-01-01

    Summary Modern views of anesthetic neurosteroid interaction with the GABAA receptor conceptualize steroid ligands interacting with a protein binding site on the receptor. It has generally been assumed that the steroid interaction/binding site is contained in an extracellular domain of the receptor, and that steroid interactions are of high potency, evidenced by the low aqueous ligand concentrations required to achieve potentiation of channel function. We have been considering implications of the observations that steroids are quite lipophilic and that recently identified putative steroid binding sites are in transmembrane domains of the receptor. Accordingly, we expect that both the effective plasma membrane steroid concentration and steroid pharmacophore properties will contribute to steady-state potency and to the lifetime of steroid actions following removal of free aqueous steroid. Here we review our recent studies that address the evidence that membrane partitioning and intracellular accumulation are non-specific contributors to the effects of anesthetic steroids at GABAA receptors. We compare and contrast the profile of anesthetic steroids with that of sulfated steroids that negatively regulate GABAA receptor function. These studies give rise to the view that the inherent affinity of anesthetic steroid for GABAA receptors is very low; low effective aqueous concentrations are accounted for by lipid partitioning. This yields a very different picture of the interaction of neurosteroids with the GABAA receptor than that of steroid interactions with classical intracellular steroid receptors, which exhibit inherently high affinity. These considerations have practical implications for actions of endogenous neurosteroids. Liphophilicity will tend to promote autocrine actions of neurosteroids at GABAA receptors within cells that synthesize neurosteroids, and lipophilic retention will limit intercellular diffusion from the source of steroid synthesis. Lipophilicity and

  10. Modulation of the input–output function by GABAA receptor-mediated currents in rat oculomotor nucleus motoneurons

    PubMed Central

    Torres-Torrelo, Julio; Torres, Blas; Carrascal, Livia

    2014-01-01

    The neuronal input–output function depends on recruitment threshold and gain of the firing frequency–current (f–I) relationship. These two parameters are positively correlated in ocular motoneurons (MNs) recorded in alert preparation and inhibitory inputs could contribute to this correlation. Phasic inhibition mediated by γ-amino butyric acid (GABA) occurs when a high concentration of GABA at the synaptic cleft activates postsynaptic GABAA receptors, allowing neuronal information transfer. In some neuronal populations, low concentrations of GABA activate non-synaptic GABAA receptors and generate a tonic inhibition, which modulates cell excitability. This study determined how ambient GABA concentrations modulate the input–output relationship of rat oculomotor nucleus MNs. Superfusion of brain slices with GABA (100 μm) produced a GABAA receptor-mediated current that reduced the input resistance, increased the recruitment threshold and shifted the f–I relationship rightward without any change in gain. These modifications did not depend on MN size. In absence of exogenous GABA, gabazine (20 μm; antagonist of GABAA receptors) abolished spontaneous inhibitory postsynaptic currents and revealed a tonic current in MNs. Gabazine increased input resistance and decreased recruitment threshold mainly in larger MNs. The f–I relationship shifted to the left, without any change in gain. Gabazine effects were chiefly due to MN tonic inhibition because tonic current amplitude was five-fold greater than phasic. This study demonstrates a tonic inhibition in ocular MNs that modulates cell excitability depending on cell size. We suggest that GABAA tonic inhibition acting concurrently with glutamate receptors activation could reproduce the positive covariation between threshold and gain reported in alert preparation. PMID:25194049

  11. Modulation of the input-output function by GABAA receptor-mediated currents in rat oculomotor nucleus motoneurons.

    PubMed

    Torres-Torrelo, Julio; Torres, Blas; Carrascal, Livia

    2014-11-15

    The neuronal input-output function depends on recruitment threshold and gain of the firing frequency-current (f-I) relationship. These two parameters are positively correlated in ocular motoneurons (MNs) recorded in alert preparation and inhibitory inputs could contribute to this correlation. Phasic inhibition mediated by γ-amino butyric acid (GABA) occurs when a high concentration of GABA at the synaptic cleft activates postsynaptic GABAA receptors, allowing neuronal information transfer. In some neuronal populations, low concentrations of GABA activate non-synaptic GABAA receptors and generate a tonic inhibition, which modulates cell excitability. This study determined how ambient GABA concentrations modulate the input-output relationship of rat oculomotor nucleus MNs. Superfusion of brain slices with GABA (100 μm) produced a GABAA receptor-mediated current that reduced the input resistance, increased the recruitment threshold and shifted the f-I relationship rightward without any change in gain. These modifications did not depend on MN size. In absence of exogenous GABA, gabazine (20 μm; antagonist of GABAA receptors) abolished spontaneous inhibitory postsynaptic currents and revealed a tonic current in MNs. Gabazine increased input resistance and decreased recruitment threshold mainly in larger MNs. The f-I relationship shifted to the left, without any change in gain. Gabazine effects were chiefly due to MN tonic inhibition because tonic current amplitude was five-fold greater than phasic. This study demonstrates a tonic inhibition in ocular MNs that modulates cell excitability depending on cell size. We suggest that GABAA tonic inhibition acting concurrently with glutamate receptors activation could reproduce the positive covariation between threshold and gain reported in alert preparation.

  12. GABAA receptor signaling in caudal periaqueductal gray regulates maternal aggression and maternal care in mice

    PubMed Central

    Lee, Grace; Gammie, Stephen C.

    2010-01-01

    Maternal aggression (maternal defense) is exhibited by lactating females towards intruders and contributes to the protection of offspring. Enhancement of GABAA receptor signaling by benzodiazepines elevates maternal aggression, and we previously found indirect evidence (via c-Fos immunhistochemistry) that caudal periaqueductal gray (cPAG) and lateral septum (LS) could be sites where benzodiazepines increase aggression. We recently found that GABAA receptor signaling in LS modulates maternal aggression, and in this study, we tested the hypothesis that GABAA receptor signaling in cPAG also regulates this behavior. Site-directed injections to cPAG were made in lactating mice using the GABAA receptor antagonist, bicuculline (3–9 ng) or the GABAA receptor positive modulator, chlordiazepoxide (CDP), a benzodiazepine (2.5–20 µg). Maternal aggression, other maternal behaviors, and anxiety-like measures (using the light-dark box) were then examined. GABAA receptor positive modulator did not increase aggression, which could have resulted from a ceiling effect. However, 8 ng and 9 ng of bicuculline in cPAG significantly decreased maternal aggression without altering other maternal behaviors or light-dark box performance, suggesting some GABAA receptor signaling in cPAG is required for full maternal aggression expression. Additionally, 7 ng of bicuculline significantly increased licking/grooming of pups, and decreased the number of transitions between the light and dark compartments of the light-dark box without affecting aggression. Given these results indicating that antagonizing GABAA-receptor in cPAG dose-dependently promotes offspring grooming behavior while impairing aggression, it is possible that the cPAG represents a key site for decision making (aggression versus other behaviors) in the lactating female. PMID:20457185

  13. Tonic GABAA Receptor-Mediated Inhibition in the Rat Dorsal Motor Nucleus of the Vagus

    PubMed Central

    Gao, Hong

    2010-01-01

    Type A γ-aminobutyric acid (GABAA) receptors expressed in the dorsal motor nucleus of vagus (DMV) critically regulate the activity of vagal motor neurons and, by inference, the gastrointestinal (GI) tract. Two types of GABAA receptor-mediated inhibition have been identified in the brain, represented by phasic (Iphasic) and tonic (Itonic) inhibitory currents. The hypothesis that Itonic regulates neuron activity was tested in the DMV using whole cell patch-clamp recordings in transverse brain stem slices from rats. An Itonic was present in a subset of DMV neurons, which was determined to be mediated by different receptors than those mediating fast, synaptic currents. Preapplication of tetrodotoxin significantly decreased the resting Itonic amplitude in DMV neurons, suggesting that most of the current was due to action potential (AP)–dependent GABA release. Blocking GABA transport enhanced Itonic and multiple GABA transporters cooperated to regulate Itonic. The Itonic was composed of both a gabazine-insensitive component that was nearly saturated under basal conditions and a gabazine-sensitive component that was activated when extracellular GABA concentration was elevated. Perfusion of THIP (10 μM) significantly increased Itonic amplitude without increasing Iphasic amplitude. The Itonic played a major role in determining the overall excitability of DMV neurons by contributing to resting membrane potential and AP frequency. Our results indicate that Itonic contributes to DMV neuron membrane potential and activity and is thus an important regulator of vagally mediated GI function. PMID:20018836

  14. Enhanced neurosteroid potentiation of ternary GABA(A) receptors containing the delta subunit.

    PubMed

    Wohlfarth, Kai M; Bianchi, Matt T; Macdonald, Robert L

    2002-03-01

    Attenuated behavioral sensitivity to neurosteroids has been reported for mice deficient in the GABA(A) receptor delta subunit. We therefore investigated potential subunit-specific neurosteroid pharmacology of the following GABA(A) receptor isoforms in a transient expression system: alpha1beta3gamma2L, alpha1beta3delta, alpha6beta3gamma2L, and alpha6beta3delta. Potentiation of submaximal GABA(A) receptor currents by the neurosteroid tetrahydrodeoxycorticosterone (THDOC) was greatest for the alpha1beta3delta isoform. Whole-cell GABA concentration--response curves performed with and without low concentrations (30 nm) of THDOC revealed enhanced peak GABA(A) receptor currents for isoforms tested without affecting the GABA EC50. Alpha1beta3delta currents were enhanced the most (>150%), whereas the other isoform currents were enhanced 15-50%. At a higher concentration (1 microm), THDOC decreased peak alpha1beta3gamma2L receptor current amplitude evoked by GABA (1 mm) concentration jumps and prolonged deactivation but had little effect on the rate or extent of apparent desensitization. Thus the polarity of THDOC modulation depended on GABA concentration for alpha1beta3gamma2L GABA(A) receptors. However, the same protocol applied to alpha1beta3delta receptors resulted in peak current enhancement by THDOC of >800% and prolonged deactivation. Interestingly, THDOC induced pronounced desensitization in the minimally desensitizing alpha1beta3delta receptors. Single channel recordings obtained from alpha1beta3delta receptors indicated that THDOC increased the channel opening duration, including the introduction of an additional longer duration open state. Our results suggest that the GABA(A) receptor delta subunit confers increased sensitivity to neurosteroid modulation and that the intrinsic gating and desensitization kinetics of alpha1beta3delta GABA(A) receptors are altered by THDOC.

  15. Role of human GABA(A) receptor beta3 subunit in insecticide toxicity.

    PubMed

    Ratra, G S; Kamita, S G; Casida, J E

    2001-05-01

    The gamma-aminobutyric acid type A (GABA(A)) receptor is the target for the major insecticides alpha-endosulfan, lindane, and fipronil and for many analogs. Their action as chloride channel blockers is directly measured by binding studies with [(3)H]ethynylbicycloorthobenzoate ([(3)H]EBOB). This study tests the hypothesis that GABA(A) receptor subunit composition determines the sensitivity and selectivity of insecticide toxicity. Human receptor subtypes were expressed individually (alpha1, alpha6, beta1, beta3, and gamma2) and in combination in insect Sf9 cells. Binding parameters were similar for [(3)H]EBOB in the beta3 homooligomer, alpha1beta3gamma2 heterooligomer, and native brain membranes, but toxicological profiles were very different. Surprisingly, alpha-endosulfan, lindane, and fipronil were all remarkably potent on the recombinant beta3 homooligomeric receptor (IC50 values of 0.5-2.4 nM), whereas they were similar in potency on the alpha1beta3gamma2 subtype (IC50 values of 16-33 nM) and highly selective on the native receptor (IC50 values of 7.3, 306, and 2470 nM, respectively). The selectivity order for 29 insecticides and convulsants as IC50 ratios for native/beta3 or alpha1beta3gamma2/beta3 was as follows: fipronil > lindane > 19 other insecticides including alpha-endosulfan and picrotoxinin > 4 trioxabicyclooctanes and dithianes (almost nonselective) > tetramethylenedisulfotetramine, 4-chlorophenylsilatrane, or alpha-thujone. Specificity between mammals and insects at the target site (fipronil > lindane > alpha-endosulfan) paralleled that for toxicity. Potency at the native receptor is more predictive for inhibition of GABA-stimulated chloride uptake than that at the beta3 or alpha1beta3gamma2 receptors. Therefore, the beta3 subunit contains the insecticide target and other subunits differentially modulate the binding to confer compound-dependent specificity and selective toxicity.

  16. Extrasynaptic α6 Subunit-Containing GABAA Receptors Modulate Excitability in Turtle Spinal Motoneurons

    PubMed Central

    Andres, Carmen; Aguilar, Justo; González-Ramírez, Ricardo; Elias-Viñas, David; Felix, Ricardo; Delgado-Lezama, Rodolfo

    2014-01-01

    Motoneurons are furnished with a vast repertoire of ionotropic and metabotropic receptors as well as ion channels responsible for maintaining the resting membrane potential and involved in the regulation of the mechanisms underlying its membrane excitability and firing properties. Among them, the GABAA receptors, which respond to GABA binding by allowing the flow of Cl− ions across the membrane, mediate two distinct forms of inhibition in the mature nervous system, phasic and tonic, upon activation of synaptic or extrasynaptic receptors, respectively. In a previous work we showed that furosemide facilitates the monosynaptic reflex without affecting the dorsal root potential. Our data also revealed a tonic inhibition mediated by GABAA receptors activated in motoneurons by ambient GABA. These data suggested that the high affinity GABAA extrasynaptic receptors may have an important role in motor control, though the molecular nature of these receptors was not determined. By combining electrophysiological, immunofluorescence and molecular biology techniques with pharmacological tools here we show that GABAA receptors containing the α6 subunit are expressed in adult turtle spinal motoneurons and can function as extrasynaptic receptors responsible for tonic inhibition. These results expand our understanding of the role of GABAA receptors in motoneuron tonic inhibition. PMID:25531288

  17. Synaptic GABAA Receptor Clustering without the γ2 Subunit

    PubMed Central

    Kerti-Szigeti, Katalin

    2014-01-01

    Rapid activation of postsynaptic GABAA receptors (GABAARs) is crucial in many neuronal functions, including the synchronization of neuronal ensembles and controlling the precise timing of action potentials. Although the γ2 subunit is believed to be essential for the postsynaptic clustering of GABAARs, synaptic currents have been detected in neurons obtained from γ2−/− mice. To determine the role of the γ2 subunit in synaptic GABAAR enrichment, we performed a spatially and temporally controlled γ2 subunit deletion by injecting Cre-expressing viral vectors into the neocortex of GABAARγ277Ilox mice. Whole-cell recordings revealed the presence of miniature IPSCs in Cre+ layer 2/3 pyramidal cells (PCs) with unchanged amplitudes and rise times, but significantly prolonged decays. Such slowly decaying currents could be evoked in PCs by action potentials in presynaptic fast-spiking interneurons. Freeze-fracture replica immunogold labeling revealed the presence of the α1 and β3 subunits in perisomatic synapses of cells that lack the γ2 subunit. Miniature IPSCs in Cre+ PCs were insensitive to low concentrations of flurazepam, providing a pharmacological confirmation of the lack of the γ2 subunit. Receptors assembled from only αβ subunits were unlikely because Zn2+ did not block the synaptic currents. Pharmacological experiments indicated that the αβγ3 receptor, rather than the αβδ, αβε, or αβγ1 receptors, was responsible for the slowly decaying IPSCs. Our data demonstrate the presence of IPSCs and the synaptic enrichment of the α1 and β3 subunits and suggest that the γ3 subunit is the most likely candidate for clustering GABAARs at synapses in the absence of the γ2 subunit. PMID:25080584

  18. Low dose acute alcohol effects on GABAA receptor subtypes

    PubMed Central

    Wallner, Martin; Hanchar, H. Jacob; Olsen, Richard W.

    2010-01-01

    GABAA receptors (GABAARs) are the main inhibitory neurotransmitter receptors and have long been implicated in mediating at least part of the acute actions of ethanol. For example, ethanol and GABAergic drugs including barbiturates and benzodiazepines share many pharmacological properties. Besides the prototypical synaptic GABAAR subtypes, nonsynaptic GABAARs have recently emerged as important regulators of neuronal excitability. While high doses (≥100 mM) of ethanol have been reported to enhance activity of most GABAAR subtypes, most abundant synaptic GABAARs are essentially insensitive to ethanol concentrations that occur during social ethanol consumption (<30 mM). However, extrasynaptic δ and β3 subunit-containing GABAARs, associated in the brain with α4or α6 subunits, are sensitive to low millimolar ethanol concentrations, as produced by drinking half a glass of wine. Additionally, we found that a mutation in the cerebellar α6 subunit (α6R100Q), initially reported in rats selectively bred for increased alcohol sensitivity, is sufficient to produce increased alcohol-induced motor impairment and further increases of alcohol sensitivity in recombinant α6β3δ receptors. Furthermore, the behavioral alcohol antagonist Ro15-4513 blocks the low dose alcohol enhancement on α4/6/β3δ receptors, without reducing GABA-induced currents. In binding assays α4β3δ GABAARs bind [3H] Ro15-4513 with high affinity, and this binding is inhibited, in an apparently competitive fashion, by low ethanol concentrations, as well as analogs of Ro15-4513 that are active to antagonize ethanol or Ro15-4513’s block of ethanol. We conclude that most low to moderate dose alcohol effects are mediated by alcohol actions on alcohol/Ro15-4513 binding sites on GABAAR subtypes. PMID:16814864

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

    PubMed

    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 α1β3(S408A,S409A)γ2L 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 α4β3(S408A,S409A)δ receptors were investigated. Neurosteroid potentiation was reduced at both receptors by the kinase inhibitor staurosporine. By contrast, neurosteroid-mediated potentiation at α4(S443A)β3(S408A,S409A)δ 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 β3(S408,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

  20. 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

  1. [The influence of GABAA receptor on the analgesic action of intrathecally injected oxysophoridine].

    PubMed

    Yang, Guang; Gao, Jin-xian; Yi, Zheng-hong; Yan, Lin; Jiang, Yuan-Xu

    2011-05-01

    .This study is to investigate the analgesic effect produced by intrathecal injection (ith) of oxysophoridine (OSR) and the mechanism of GABAA receptor. Warm water tail-flick test was used to detect the analgesic effect of OSR (12.5, 6.25, and 3.13 mg.kg-1 ith) and to observe the influence of GABA (gamma aminobutyric acid) agonist or antagonist on the analgesic effect of OSR in mice. Immunohistochemistry method were used to detect the influence of OSR (12.5 mg.kg-1, ith) on the GABAARalpha1 protein expression in spinal cord. The results obtained covers that OSR (12.5 and 6.25 mg.kg-, ith) alleviates pain significantly with the warm water tail-flick test (P<0.05, P<0.01), the rate of pain threshold increases by 68.45%; GABA and muscimol (MUS) produces analgesic synergism together with the OSR, picrotoxin (PTX) and bicuculline (BIC) antagonize the analgesic effect of OSR; OSR (12.5 mg.kg-1, ith) significantly increase the positive number of GABAARalpha1 nerve cell in spinal cord (P<0.01) and significantly decrease the average grey levels (P<0.01). In conclusion, OSR intrathecal injection has significant analgesic effect. And GABAA receptor in spinal cord is involved in the analgesic mechanism.

  2. 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.

  3. Comparison of γ-Aminobutyric Acid, Type A (GABAA), Receptor αβγ and αβδ Expression Using Flow Cytometry and Electrophysiology: EVIDENCE FOR ALTERNATIVE SUBUNIT STOICHIOMETRIES AND ARRANGEMENTS.

    PubMed

    Botzolakis, Emmanuel J; Gurba, Katharine N; Lagrange, Andre H; Feng, Hua-Jun; Stanic, Aleksandar K; Hu, Ningning; Macdonald, Robert L

    2016-09-23

    The subunit stoichiometry and arrangement of synaptic αβγ GABAA receptors are generally accepted as 2α:2β:1γ with a β-α-γ-β-α counterclockwise configuration, respectively. Whether extrasynaptic αβδ receptors adopt the analogous β-α-δ-β-α subunit configuration remains controversial. Using flow cytometry, we evaluated expression levels of human recombinant γ2 and δ subunits when co-transfected with α1 and/or β2 subunits in HEK293T cells. Nearly identical patterns of γ2 and δ subunit expression were observed as follows: both required co-transfection with α1 and β2 subunits for maximal expression; both were incorporated into receptors primarily at the expense of β2 subunits; and both yielded similar FRET profiles when probed for subunit adjacency, suggesting similar underlying subunit arrangements. However, because of a slower rate of δ subunit degradation, 10-fold less δ subunit cDNA was required to recapitulate γ2 subunit expression patterns and to eliminate the functional signature of α1β2 receptors. Interestingly, titrating γ2 or δ subunit cDNA levels progressively altered GABA-evoked currents, revealing more than one kinetic profile for both αβγ and αβδ receptors. This raised the possibility of alternative receptor isoforms, a hypothesis confirmed using concatameric constructs for αβγ receptors. Taken together, our results suggest a limited cohort of alternative subunit arrangements in addition to canonical β-α-γ/δ-β-α receptors, including β-α-γ/δ-α-α receptors at lower levels of γ2/δ expression and β-α-γ/δ-α-γ/δ receptors at higher levels of expression. These findings provide important insight into the role of GABAA receptor subunit under- or overexpression in disease states such as genetic epilepsies.

  4. 11-TRIFLUOROMETHYL-PHENYLDIAZIRINYL NEUROSTEROID ANALOGUES: POTENT GENERAL ANESTHETICS AND PHOTOLABELING REAGENTS FOR GABAA RECEPTORS

    PubMed Central

    Chen, Zi-Wei; Wang, Cunde; Krishnan, Kathiresan; Manion, Brad D.; Hastings, Randy; Bracamontes, John; Taylor, Amanda; Eaton, Megan M.; Zorumski, Charles F.; Steinbach, Joseph H.; Akk, Gustav; Mennerick, Steven; Covey, Douglas F.; Evers, Alex S.

    2014-01-01

    Rationale While neurosteroids are well-described positive allosteric modulators of GABAA receptors, the binding sites that mediate these actions have not been definitively identified. Objectives To synthesize neurosteroid analogue photolabeling reagents that closely mimic the biological effects of endogenous neurosteroids and have photochemical properties that will facilitate their use as tools for identifying the binding sites for neurosteroids on GABAA receptors. Results Two neurosteroid analogues containing a trifluromethyl-phenyldiazirine group linked to the steroid C11 position were synthesized. These reagents, CW12 and CW14, are analogues of allopregnanolone (5α-reduced steroid) and pregnanolone (5β-reduced steroid), respectively. Both reagents were shown to have favorable photochemical properties with efficient insertion into the C–H bonds of cyclohexane. They also effectively replicated the actions of allopregnanolone and pregnanolone on GABAA receptor functions: they potentiated GABA-induced currents in Xenopus laevis oocytes transfected with α1β2γ2L subunits, modulated [35S]t-butylbicyclophosphorothionate binding in rat brain membranes and were effective anesthetics in Xenopus tadpoles. Studies using [3H]CW12 and [3H]CW14 showed that these reagents covalently label GABAA receptors in both rat brain membranes and in TSA cells expressing either α1 and β2 subunits or β3 subunits of the GABAA receptor. Photolabeling of rat brain GABAA receptors was shown to be both concentration-dependent and stereospecific. Conclusions CW12 and CW14 have the appropriate photochemical and pharmacological properties for use as photolabeling reagents to identify specific neurosteroid binding sites on GABAA receptors. PMID:24756762

  5. Mutations in the GABAA receptor that mimic the allosteric ligand, etomidate

    PubMed Central

    Forman, Stuart A.; Stewart, Deirdre

    2012-01-01

    Etomidate is a hydrophobic molecule, a potent general anesthetic, and the best understood drug in this group. Etomidate’s target molecules are GABAA receptors, its site of action has been identified with photolabeling, and a quantitative allosteric co-agonist model has emerged for etomidate effects on GABAA receptors. We have shown that when methionine residues that are thought to be adjacent to the etomidate site are mutated to tryptophan, that the bulky hydrophobic side-chains alter mutant GABAA receptor function in ways that mimic the effects of etomidate binding to wild-type receptors. Furthermore, these mutations reduce receptor modulation by etomidate. Both of these observations support the hypothesis that these methionine residues form part of the etomidate binding pocket. PMID:22052498

  6. Sequence of a functional invertebrate GABAA receptor subunit which can form a chimeric receptor with a vertebrate alpha subunit.

    PubMed Central

    Harvey, R J; Vreugdenhil, E; Zaman, S H; Bhandal, N S; Usherwood, P N; Barnard, E A; Darlison, M G

    1991-01-01

    The sequence of an invertebrate GABAA receptor subunit is described. This was deduced from a cDNA which was isolated from the mollusc Lymnaea stagnalis and which corresponds to a transcript of extremely low abundance. The cDNA was isolated using short exonic sequences from part of the corresponding gene in combination with a variant of the polymerase chain reaction (PCR) known as RACE (rapid amplification of cDNA ends). The mature polypeptide has a predicted molecular weight of 54,569 Daltons and exhibits approximately 50% identity to vertebrate GABAA receptor beta subunits. The six intron-exon boundaries determined to date in the molluscan gene occur at the same relative positions as those found in vertebrate GABAA receptor genes. Functional expression, in Xenopus oocytes, of the molluscan cDNA alone results in the formation of GABA-activated chloride ion channels that have a finite open probability even in the absence of agonist. These GABA-evoked currents can be reversibly blocked by the vertebrate GABAA receptor antagonist bicuculline. Surprisingly, the molluscan beta subunit is capable of replacing vertebrate beta subunits in co-expression experiments with the bovine GABAA receptor alpha 1 subunit. These findings suggest that invertebrate GABAA receptors exist in vivo as hetero-oligomeric complexes. PMID:1655414

  7. Plasticity of GABAA Receptors during Pregnancy and Postpartum Period: From Gene to Function

    PubMed Central

    Licheri, Valentina; Talani, Giuseppe; Gorule, Ashish A.; Mostallino, Maria Cristina; Biggio, Giovanni; Sanna, Enrico

    2015-01-01

    Pregnancy needs complex pathways that together play a role in proper growth and protection of the fetus preventing its premature loss. Changes during pregnancy and postpartum period include the manifold machinery of neuroactive steroids that plays a crucial role in neuronal excitability by local modulation of specific inhibitory receptors: the GABAA receptors. Marked fluctuations in both blood and brain concentration of neuroactive steroids strongly contribute to GABAA receptor function and plasticity. In this review, we listed several interesting results regarding the regulation and plasticity of GABAA receptor function during pregnancy and postpartum period in rats. The increase in brain levels of neuroactive steroids during pregnancy and their sudden decrease immediately before delivery are causally related to changes in the expression/function of specific GABAA receptor subunits in the hippocampus. These data suggest that alterations in GABAA receptor expression and function may be related to neurological and psychiatric disorders associated with crucial periods in women. These findings could help to provide potential new treatments for these women's disabling syndromes. PMID:26413323

  8. Lifelong ethanol consumption and brain regional GABAA receptor subunit mRNA expression in alcohol-preferring rats.

    PubMed

    Sarviharju, Maija; Hyytiä, Petri; Hervonen, Antti; Jaatinen, Pia; Kiianmaa, Kalervo; Korpi, Esa R

    2006-11-01

    Brain regional gamma-aminobutyric acid type A (GABAA) receptor subunit mRNA expression was studied in ethanol-preferring AA (Alko, Alcohol) rats after moderate ethanol drinking for up to 2 years of age. In situ hybridization with oligonucleotide probes specific for 13 different subunits was used with coronal cryostat sections of the brains. Selective alterations were observed by ethanol exposure and/or aging in signals for several subunits. Most interestingly, the putative highly ethanol-sensitive alpha4 and beta3 subunit mRNAs were significantly decreased in several brain regions. The age-related alterations in alpha4 subunit expression were parallel to those caused by lifelong ethanol drinking, whereas aging had no significant effect on beta3 subunit expression. The results suggest that prolonged ethanol consumption leading to blood concentrations of about 10 mM may downregulate the mRNA expression of selected GABAA receptor subunits and that aging might have partly similar effects.

  9. GABAA receptor drugs and neuronal plasticity in reward and aversion: focus on the ventral tegmental area.

    PubMed

    Vashchinkina, Elena; Panhelainen, Anne; Aitta-Aho, Teemu; Korpi, Esa R

    2014-01-01

    GABAA receptors are the main fast inhibitory neurotransmitter receptors in the mammalian brain, and targets for many clinically important drugs widely used in the treatment of anxiety disorders, insomnia and in anesthesia. Nonetheless, there are significant risks associated with the long-term use of these drugs particularly related to development of tolerance and addiction. Addictive mechanisms of GABAA receptor drugs are poorly known, but recent findings suggest that those drugs may induce aberrant neuroadaptations in the brain reward circuitry. Recently, benzodiazepines, acting on synaptic GABAA receptors, and modulators of extrasynaptic GABAA receptors (THIP and neurosteroids) have been found to induce plasticity in the ventral tegmental area (VTA) dopamine neurons and their main target projections. Furthermore, depending whether synaptic or extrasynaptic GABAA receptor populations are activated, the behavioral outcome of repeated administration seems to correlate with rewarding or aversive behavioral responses, respectively. The VTA dopamine neurons project to forebrain centers such as the nucleus accumbens and medial prefrontal cortex, and receive afferent projections from these brain regions and especially from the extended amygdala and lateral habenula, forming the major part of the reward and aversion circuitry. Both synaptic and extrasynaptic GABAA drugs inhibit the VTA GABAergic interneurons, thus activating the VTA DA neurons by disinhibition and this way inducing glutamatergic synaptic plasticity. However, the GABAA drugs failed to alter synaptic spine numbers as studied from Golgi-Cox-stained VTA dendrites. Since the GABAergic drugs are known to depress the brain metabolism and gene expression, their likely way of inducing neuroplasticity in mature neurons is by disinhibiting the principal neurons, which remains to be rigorously tested for a number of clinically important anxiolytics, sedatives and anesthetics in different parts of the circuitry.

  10. GABAA receptor drugs and neuronal plasticity in reward and aversion: focus on the ventral tegmental area

    PubMed Central

    Vashchinkina, Elena; Panhelainen, Anne; Aitta-aho, Teemu; Korpi, Esa R.

    2014-01-01

    GABAA receptors are the main fast inhibitory neurotransmitter receptors in the mammalian brain, and targets for many clinically important drugs widely used in the treatment of anxiety disorders, insomnia and in anesthesia. Nonetheless, there are significant risks associated with the long-term use of these drugs particularly related to development of tolerance and addiction. Addictive mechanisms of GABAA receptor drugs are poorly known, but recent findings suggest that those drugs may induce aberrant neuroadaptations in the brain reward circuitry. Recently, benzodiazepines, acting on synaptic GABAA receptors, and modulators of extrasynaptic GABAA receptors (THIP and neurosteroids) have been found to induce plasticity in the ventral tegmental area (VTA) dopamine neurons and their main target projections. Furthermore, depending whether synaptic or extrasynaptic GABAA receptor populations are activated, the behavioral outcome of repeated administration seems to correlate with rewarding or aversive behavioral responses, respectively. The VTA dopamine neurons project to forebrain centers such as the nucleus accumbens and medial prefrontal cortex, and receive afferent projections from these brain regions and especially from the extended amygdala and lateral habenula, forming the major part of the reward and aversion circuitry. Both synaptic and extrasynaptic GABAA drugs inhibit the VTA GABAergic interneurons, thus activating the VTA DA neurons by disinhibition and this way inducing glutamatergic synaptic plasticity. However, the GABAA drugs failed to alter synaptic spine numbers as studied from Golgi-Cox-stained VTA dendrites. Since the GABAergic drugs are known to depress the brain metabolism and gene expression, their likely way of inducing neuroplasticity in mature neurons is by disinhibiting the principal neurons, which remains to be rigorously tested for a number of clinically important anxiolytics, sedatives and anesthetics in different parts of the circuitry. PMID

  11. Benzodiazepine modulation of partial agonist efficacy and spontaneously active GABAA receptors supports an allosteric model of modulation

    PubMed Central

    Downing, Scott S; Lee, Yan T; Farb, David H; Gibbs, Terrell T

    2005-01-01

    Benzodiazepines (BZDs) have been used extensively for more than 40 years because of their high therapeutic index and low toxicity. Although BZDs are understood to act primarily as allosteric modulators of GABAA receptors, the mechanism of modulation is not well understood. The applicability of an allosteric model with two binding sites for γ-aminobutyric acid (GABA) and one for a BZD-like modulator was investigated. This model predicts that BZDs should enhance the efficacy of partial agonists. Consistent with this prediction, diazepam increased the efficacy of the GABAA receptor partial agonist kojic amine in chick spinal cord neurons. To further test the validity of the model, the effects of diazepam, flurazepam, and zolpidem were examined using wild-type and spontaneously active mutant α1(L263S)β3γ2 GABAA receptors expressed in HEK-293 cells. In agreement with the predictions of the allosteric model, all three modulators acted as direct agonists for the spontaneously active receptors. The results indicate that BZD-like modulators enhance the amplitude of the GABA response by stabilizing the open channel active state relative to the inactive state by less than 1 kcal, which is similar to the energy of stabilization conferred by a single hydrogen bond. PMID:15912137

  12. Correlation between GABA(A) receptor density and vagus nerve stimulation in individuals with drug-resistant partial epilepsy.

    PubMed

    Marrosu, Francesco; Serra, Alessandra; Maleci, Alberto; Puligheddu, Monica; Biggio, Giovanni; Piga, Mario

    2003-01-01

    Vagus nerve stimulation (VNS) is an important option for the treatment of drug-resistant epilepsy. Through delivery of a battery-supplied intermittent current, VNS protects against seizure development in a manner that correlates experimentally with electrophysiological modifications. However, the mechanism by which VNS inhibits seizures in humans remains unclear. The impairment of gamma-aminobutyric acid (GABA)-mediated neuronal inhibition associated with epilepsy has suggested that GABA(A) receptors might contribute to the therapeutic efficacy of VNS. We have now applied single photon emission computed tomography (SPECT) with the benzodiazepine receptor inverse agonist [123I]iomazenil to examine cortical GABA(A) receptor density (GRD) before and 1 year after implantation of a VNS device in 10 subjects with drug-resistant partial epilepsy. VNS therapeutic responses resulted significantly correlated with the normalization of GRD. Moreover, a comparable control group, scheduled for a possible VNS implant, failed to show significant GRD variations after 1 year of a stable anti-epileptic treatment. These results suggest that VNS may modulate the cortical excitability of brain areas associated with epileptogenesis and that GABA(A) receptor plasticity contributes to this effect.

  13. Abnormal benzodiazepine and zinc modulation of GABAA receptors in an acquired absence epilepsy model.

    PubMed

    Wu, Jie; Ellsworth, Kevin; Ellsworth, Marc; Schroeder, Katherine M; Smith, Kris; Fisher, Robert S

    2004-07-01

    Brain cholesterol synthesis inhibition (CSI) at a young age in rats has been shown to be a faithful model of acquired absence epilepsy, a devastating condition for which few therapies or models exist. We employed the CSI model to study cellular mechanisms of acquired absence epilepsy in Long-Evans Hooded rats. Patch-clamp, whole-cell recordings were compared from neurons acutely dissociated from the nucleus reticularis of thalamus (nRt) treated and untreated with a cholesterol synthesis inhibitor, U18666A. In U18666A-treated animals, 91% of rats developed EEG spike-waves (SWs). Patchclamp results revealed that although there was no remarkable change in GABAA receptor affinity, both a loss of ability of benzodiazepines to enhance GABAA-receptor responses and an increase of Zn2+ inhibition of GABAA-receptor responses of nRt neurons occurred in Long-Evans Hooded rats previously administered U18666A. This change was specific, since no significant changes were found in neurons exposed to the GABA allosteric modulator, pentobarbital. Taken collectively, these findings provide evidence for abnormalities in benzodiazepine and Zn2+ modulation of GABAA receptors in the CSI model, and suggest that decreased gamma2 subunit expression may underlie important aspects of generation of thalamocortical SWs in atypical absence seizures. The present results are also consistent with recent findings that mutation of the gamma2 subunit of the GABAA receptor changes benzodiazepine modulation in families with generalized epilepsy syndromes.

  14. Regionally specific alterations in the low-affinity GABAA receptor following perinatal exposure to diazepam.

    PubMed

    Gruen, R J; Elsworth, J D; Roth, R H

    1990-04-23

    Alterations in a low affinity form of the GABAA receptor were examined with [3H]bicuculline methylchloride in the adult rat following perinatal exposure to diazepam. Perinatal exposure resulted in a significant reduction in [3H]bicuculline binding in the cingulate cortex. A significant decrease in the ability of GABA to displace bound [3H]bicuculline was observed only in the hypothalamus. The results suggest that the effects of perinatal exposure to diazepam are regionally specific and that benzodiazepine receptors and low affinity GABAA receptors are functionally linked during the perinatal period.

  15. Regionally specific alterations in the low-affinity GABAA receptor following perinatal exposure to diazepam.

    PubMed

    Gruen, R J; Elsworth, J D; Roth, R H

    1990-04-23

    Alterations in a low affinity form of the GABAA receptor were examined with [3H]bicuculline methylchloride in the adult rat following perinatal exposure to diazepam. Perinatal exposure resulted in a significant reduction in [3H]bicuculline binding in the cingulate cortex. A significant decrease in the ability of GABA to displace bound [3H]bicuculline was observed only in the hypothalamus. The results suggest that the effects of perinatal exposure to diazepam are regionally specific and that benzodiazepine receptors and low affinity GABAA receptors are functionally linked during the perinatal period. PMID:2162709

  16. 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. PMID:25278838

  17. Subunit-Specific Trafficking of GABAA Receptors during Status Epilepticus

    PubMed Central

    Goodkin, Howard P.; Joshi, Suchitra; Mtchedlishvili, Zakaria; Brar, Jasmit; Kapur, Jaideep

    2010-01-01

    It is proposed that a reduced surface expression of GABAA receptors (GABARs) contributes to the pathogenesis of status epilepticus (SE), a condition characterized by prolonged seizures. This hypothesis was based on the finding that prolonged epileptiform bursting (repetitive bursts of prolonged depolarizations with superimposed action potentials) in cultures of dissociated hippocampal pyramidal neurons (dissociated cultures) results in the increased intracellular accumulation of GABARs. However, it is not known whether this rapid modification in the surface-expressed GABAR pool results from selective, subunit-dependent or nonselective, subunit-independent internalization of GABARs. In hippocampal slices obtained from animals undergoing prolonged SE (SE-treated slices), we found that the surface expression of the GABARβ2/3 and γ2 subunits was reduced, whereas that of the δ subunit was not. Complementary electrophysiological recordings from dentate granule cells in SE-treated slices demonstrated a reduction in GABAR-mediated synaptic inhibition, but not tonic inhibition. A reduction in the surface expression of the γ2 subunit, but not the δ subunit was also observed in dissociated cultures and organotypic hippocampal slice cultures when incubated in an elevated KCl external medium or an elevated KCl external medium supplemented with NMDA, respectively. Additional studies demonstrated that the reduction in the surface expression of the γ2 subunit was independent of direct ligand binding of the GABAR. These findings demonstrate that the regulation of surface-expressed GABAR pool during SE is subunit-specific and occurs independent of ligand binding. The differential modulation of the surface expression of GABARs during SE has potential implications for the treatment of this neurological emergency. PMID:18322097

  18. Involvement of dorsal striatal α1-containing GABAA receptors in methamphetamine-associated rewarding memories.

    PubMed

    Jiao, D-L; Liu, Y; Long, J-D; Du, J; Ju, Y-Y; Zan, G-Y; Liu, J-G; Zhao, M

    2016-04-21

    Rewarding memories induced by addictive drugs may contribute to persistent drug-seeking behaviors, which is an important contributing factor to drug addiction. However, the biological mechanisms underlying drug-associated rewarding memories have not yet been fully understood, especially the new synthetic drugs, such as amphetamine-type stimulants (ATS). In this study, using the rat-conditioned place preference (CPP) model, a classic animal model for the reward-associated effects of addictive drugs, we found that the expression level of GABAA α1 subunits was significantly decreased in the dorsal striatum (Dstr) after conditioned methamphetamine (METH) pairing, and no significant differences were observed in the other four rewarding memory-associated areas (medial prefrontal cortex (mPFC), nucleus accumbens (NAc), amygdala (Amy), and dorsal hippocampus (DH)). Intra-Dstr injection of either the GABAA receptor agonist muscimol or the specific α1GABAA receptor-preferring benzodiazepine (BDZ) agonist zolpidem significantly abolished METH CPP formation. Thus, this study extends previous findings by showing that GABAA receptors, particularly the α1-containing GABAA receptors, may be strongly implicated in METH-associated rewarding memories. This work provides us with a new perspective on the goal of treating ATS addiction. PMID:26868969

  19. Involvement of dorsal striatal α1-containing GABAA receptors in methamphetamine-associated rewarding memories.

    PubMed

    Jiao, D-L; Liu, Y; Long, J-D; Du, J; Ju, Y-Y; Zan, G-Y; Liu, J-G; Zhao, M

    2016-04-21

    Rewarding memories induced by addictive drugs may contribute to persistent drug-seeking behaviors, which is an important contributing factor to drug addiction. However, the biological mechanisms underlying drug-associated rewarding memories have not yet been fully understood, especially the new synthetic drugs, such as amphetamine-type stimulants (ATS). In this study, using the rat-conditioned place preference (CPP) model, a classic animal model for the reward-associated effects of addictive drugs, we found that the expression level of GABAA α1 subunits was significantly decreased in the dorsal striatum (Dstr) after conditioned methamphetamine (METH) pairing, and no significant differences were observed in the other four rewarding memory-associated areas (medial prefrontal cortex (mPFC), nucleus accumbens (NAc), amygdala (Amy), and dorsal hippocampus (DH)). Intra-Dstr injection of either the GABAA receptor agonist muscimol or the specific α1GABAA receptor-preferring benzodiazepine (BDZ) agonist zolpidem significantly abolished METH CPP formation. Thus, this study extends previous findings by showing that GABAA receptors, particularly the α1-containing GABAA receptors, may be strongly implicated in METH-associated rewarding memories. This work provides us with a new perspective on the goal of treating ATS addiction.

  20. Augmentation of Tonic GABAA Inhibition in Absence Epilepsy: Therapeutic Value of Inverse Agonists at Extrasynaptic GABAA Receptors

    PubMed Central

    Errington, Adam C.; Cope, David W.; Crunelli, Vincenzo

    2011-01-01

    It is well established that impaired GABAergic inhibition within neuronal networks can lead to hypersynchronous firing patterns that are the typical cellular hallmark of convulsive epileptic seizures. However, recent findings have highlighted that a pathological enhancement of GABAergic signalling within thalamocortical circuits is a necessary and sufficient condition for nonconvulsive typical absence seizure genesis. In particular, increased activation of extrasynaptic GABAA receptors (eGABAAR) and augmented “tonic” GABAA inhibition in thalamocortical neurons have been demonstrated across a range of genetic and pharmacological models of absence epilepsy. Moreover, evidence from monogenic mouse models (stargazer/lethargic) and the polygenic Genetic Absence Epilepsy Rats from Strasbourg (GAERS) indicate that the mechanism underlying eGABAAR gain of function is nonneuronal in nature and results from a deficiency in astrocytic GABA uptake through the GAT-1 transporter. These results challenge the existing theory that typical absence seizures are underpinned by a widespread loss of GABAergic function in thalamocortical circuits and illustrate a vital role for astrocytes in the pathology of typical absence epilepsy. Moreover, they explain why pharmacological agents that enhance GABA receptor function can initiate or exacerbate absence seizures and suggest a potential therapeutic role for inverse agonists at eGABAARs in absence epilepsy. PMID:21912539

  1. Imidazenil, a partial positive allosteric modulator of GABAA receptors, exhibits low tolerance and dependence liabilities in the rat.

    PubMed

    Auta, J; Giusti, P; Guidotti, A; Costa, E

    1994-09-01

    Long-term treatment of rats with full (triazolam) or selective (diazepam) allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptors rapidly induced tolerance to the protective effect of these drugs against bicuculline-induced convulsion. In contrast, long-term administration of partial allosteric modulators (imidazenil and bretazenil) of GABAA receptors, in doses equipotent to those of diazepam and triazolam that induce anticonvulsant tolerance, failed to elicit such a tolerance. Furthermore, no cross-tolerance was observed between diazepam and imidazenil. Discontinuation of long-term treatment with diazepam or triazolam, but not of long-term treatment with imidazenil or bretazenil, sensitized rats to behavioral inhibition by punishment (electric shock) in a manner that was potentiated by flumazenil. Administration of a single oral dose of [14C]diazepam or [3H] imidazenil to rats treated repeatedly with the corresponding unlabeled drug or vehicle revealed that the brain concentrations of drugs and their metabolites were similar in both groups of animals. This suggests that tolerance to the full or selective allosteric modulators of GABAA receptors may be associated with changes in the efficacy of the allosteric modulation rather than with changes in drug metabolism. Imidazenil has a longer half-life than an equipotent dose of diazepam and protects rats against bicuculline-induced convulsions for a significantly longer time than diazepam or bretazenil.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Subchronic phencyclidine in rats: alterations in locomotor activity, maze performance, and GABA(A) receptor binding.

    PubMed

    Beninger, Richard J; Beuk, Jonathan; Banasikowski, Tomek J; van Adel, Michael; Boivin, Gregory A; Reynolds, James N

    2010-02-01

    Phencyclidine (PCP), an antagonist at the N-methyl-D-aspartate subtype of ionotropic glutamatergic receptors, decreases gamma-aminobutyric acid (GABA)ergic inhibition, suggesting that changes in GABAergic receptor function underlie behavioral and cognitive deficits resulting from repeated administration of PCP. To test this hypothesis, male Sprague-Dawley rats treated with PCP (4.5 mg/kg, intraperitoneal, twice a day for 7 consecutive days) or saline were tested in behavioral and cognitive tasks 7 days after injections. The PCP group showed increased amphetamine (1.5 mg/kg)-stimulated locomotor activity, and exhibited a greater number of errors in the double Y-maze memory task, when compared with controls. Subchronic PCP treatment increased [H]muscimol-binding sites and decreased affinity for [H]muscimol binding in frontal cortex, hippocampus, and striatum in comparison with controls. There were no changes in the expression of glutamic acid decarboxylase or the GABA membrane transporter protein. These data show that subchronic PCP administration induces an impaired performance of a previously learned task and an enhanced response to amphetamine in the rat. The observed changes in GABAA receptors in the rat brain are consistent with the notion that alterations in GABAergic receptor function contribute to the behavioral and cognitive impairments associated with repeated exposure to PCP. PMID:19949321

  3. Proton sensitivity of the GABA(A) receptor is associated with the receptor subunit composition.

    PubMed Central

    Krishek, B J; Amato, A; Connolly, C N; Moss, S J; Smart, T G

    1996-01-01

    1. Modulation of GABA(A) receptors by external H(+) was examined in cultured rat sympathetic neurones, and in Xenopus laevis oocytes and human embryonic kidney (HEK) cells expressing recombinant GABA(A) receptors composed of combinations of alpha 1, beta 1, beta 2, gamma 2S and delta subunits. 2. Changing the external pH from 7.4 reduced GABA-activated currents in sympathetic neurones. pH titration of the GABA-induced current was fitted with a pH model which predicted that H(+) interact with two sites (PK(a) values of 6.4 and 7.2). 3. For alpha 1 beta 1 GABA(A) receptors, low external pH (< 7.4) enhanced responses to GABA. pH titration predicted the existence of two sites with PK(a) values of 6.6 and 7.5. The GABA concentration-response curve was shifted to the left by low pH and non-competitively inhibited at high pH (> 7.4). 4. alpha 1 beta 1 gamma 2S receptor constructs were not affected by external pH, whereas exchanging the beta 1 subunit for beta 2 conferred a sensitivity to pH, with predicted PK(a) values of 5.16 and 9.44. 5. Low pH enhanced the responses to GABA on alpha 1 beta 1 delta subunits, whilst high pH caused an inhibition (PK(a) values of 6.6 and 9.9). The GABA concentration-response curves were enhanced (pH 5.4) or reduced (pH 9.4) with no changes in the GABA EC(50). 6. Immunoprecipitation with subunit and epitope-specific antisera to alpha 1, beta 1 and delta subunits demonstrated that these subunits could co-assemble in cell membranes. 7. Expression of alpha 1 beta 1 gamma 2S delta constructs resulted in a 'bell-shaped' pH titration relationship. Increasing or decreasing external pH inhibited the responses to GABA. 8. The pH sensitivity of recombinant GABA(A) receptors expressed in HEK cells was generally in accordance with data accrued from Xenopus oocytes. However, rapid application of GABA to alpha 1 beta 1 constructs at high pH (> 7.4) caused an increased peak and reduced steady-state current, with a correspondingly increased rate of

  4. GABA(A) receptors implicated in REM sleep control express a benzodiazepine binding site.

    PubMed

    Nguyen, Tin Quang; Liang, Chang-Lin; Marks, Gerald A

    2013-08-21

    It has been reported that non-subtype-selective GABAA receptor antagonists injected into the nucleus pontis oralis (PnO) of rats induced long-lasting increases in REM sleep. Characteristics of these REM sleep increases were identical to those resulting from injection of muscarinic cholinergic agonists. Both actions were blocked by the muscarinic antagonist, atropine. Microdialysis of GABAA receptor antagonists into the PnO resulted in increased acetylcholine levels. These findings were consistent with GABAA receptor antagonists disinhibiting acetylcholine release in the PnO to result in an acetylcholine-mediated REM sleep induction. Direct evidence has been lacking for localization in the PnO of the specific GABAA receptor-subtypes mediating the REM sleep effects. Here, we demonstrated a dose-related, long-lasting increase in REM sleep following injection (60 nl) in the PnO of the inverse benzodiazepine agonist, methyl-6,7-dimethoxy-4-ethyl-β-carboline (DMCM, 10(-2)M). REM sleep increases were greater and more consistently produced than with the non-selective antagonist gabazine, and both were blocked by atropine. Fluorescence immunohistochemistry and laser scanning confocal microscopy, colocalized in PnO vesicular acetylcholine transporter, a presynaptic marker of cholinergic boutons, with the γ2 subunit of the GABAA receptor. These data provide support for the direct action of GABA on mechanisms of acetylcholine release in the PnO. The presence of the γ2 subunit at this locus and the REM sleep induction by DMCM are consistent with binding of benzodiazepines by a GABAA receptor-subtype in control of REM sleep.

  5. Post-depolarization potentiation of GABAA receptors: A novel mechanism regulating tonic conductance in hippocampal neurons

    PubMed Central

    Ransom, Christopher B.; Wu, Yuanming; Richerson, George B.

    2010-01-01

    Ambient GABA in the brain activates GABAA receptors to produce tonic inhibition. Membrane potential influences both GABA transport and GABAA receptors and could thereby regulate tonic inhibition. We investigated the voltage-dependence of tonic currents in cultured rat hippocampal neurons using patch clamp techniques. Tonic GABAA conductance increased with depolarization from 15±3 pS/pF at −80 mV to 29±5 pS/pF at −40 mV. Inhibition of vesicular or nonvesicular GABA release did not prevent voltage-dependent increases of tonic conductance. Currents evoked with exogenous GABA (1 µM) were outwardly-rectifying, similar to tonic currents due to endogenous GABA. These results indicate that the voltage-dependent increase of tonic conductance was due to intrinsic GABAA receptor properties rather than an elevation of ambient GABA. Following transient depolarization to +40 mV, endogenous tonic currents measured at −60 mV were increased by 75±17%. This novel form of tonic current modulation, termed post-depolarization potentiation (PDP), recovered with a time constant of 63 s, was increased by exogenous GABA, and inhibited by GABAA receptor antagonists. Measurements of EGABA showed PDP was due to increased conductance and not a change in the anion gradient. To assess the functional significance of PDP, we used voltage-clamp waveforms that replicated epileptiform activity. PDP was produced by this pathophysiologic depolarization. These data show that depolarization produces prolonged potentiation of tonic conductance due to voltage-dependent properties of GABAA receptors. These properties are well suited to limit excitability during pathophysiologic depolarization accompanied by rises in ambient GABA, such as occur during seizures and ischemia. PMID:20519542

  6. Agonist-dependent endocytosis of γ-aminobutyric acid type A (GABAA) receptors revealed by a γ2(R43Q) epilepsy mutation.

    PubMed

    Chaumont, Severine; André, Caroline; Perrais, David; Boué-Grabot, Eric; Taly, Antoine; Garret, Maurice

    2013-09-27

    GABA-gated chloride channels (GABAARs) trafficking is involved in the regulation of fast inhibitory transmission. Here, we took advantage of a γ2(R43Q) subunit mutation linked to epilepsy in humans that considerably reduces the number of GABAARs on the cell surface to better understand the trafficking of GABAARs. Using recombinant expression in cultured rat hippocampal neurons and COS-7 cells, we showed that receptors containing γ2(R43Q) were addressed to the cell membrane but underwent clathrin-mediated dynamin-dependent endocytosis. The γ2(R43Q)-dependent endocytosis was reduced by GABAAR antagonists. These data, in addition to a new homology model, suggested that a conformational change in the extracellular domain of γ2(R43Q)-containing GABAARs increased their internalization. This led us to show that endogenous and recombinant wild-type GABAAR endocytosis in both cultured neurons and COS-7 cells can be amplified by their agonists. These findings revealed not only a direct relationship between endocytosis of GABAARs and a genetic neurological disorder but also that trafficking of these receptors can be modulated by their agonist.

  7. 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

  8. Selective targeting of the α5-subunit of GABAA receptors relaxes airway smooth muscle and inhibits cellular calcium handling

    PubMed Central

    Yocum, Gene T.; Siviski, Matthew E.; Yim, Peter D.; Fu, Xiao Wen; Poe, Michael M.; Cook, James M.; Harrison, Neil; Perez-Zoghbi, Jose; Emala, Charles W.

    2015-01-01

    The clinical need for novel bronchodilators for the treatment of bronchoconstrictive diseases remains a major medical issue. Modulation of airway smooth muscle (ASM) chloride via GABAA receptor activation to achieve relaxation of precontracted ASM represents a potentially beneficial therapeutic option. Since human ASM GABAA receptors express only the α4- and α5-subunits, there is an opportunity to selectively target ASM GABAA receptors to improve drug efficacy and minimize side effects. Recently, a novel compound (R)-ethyl8-ethynyl-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4] diazepine-3-carboxylate (SH-053-2′F-R-CH3) with allosteric selectivity for α5-subunit containing GABAA receptors has become available. We questioned whether this novel GABAA α5-selective ligand relaxes ASM and affects intracellular calcium concentration ([Ca2+]i) regulation. Immunohistochemical staining localized the GABAA α5-subunit to human ASM. The selective GABAA α5 ligand SH-053-2′F-R-CH3 relaxes precontracted intact ASM; increases GABA-activated chloride currents in human ASM cells in voltage-clamp electrophysiology studies; and attenuates bradykinin-induced increases in [Ca2+]i, store-operated Ca2+ entry, and methacholine-induced Ca2+ oscillations in peripheral murine lung slices. In conclusion, selective subunit targeting of endogenous α5-subunit containing GABAA receptors on ASM may represent a novel therapeutic option to treat severe bronchospasm. PMID:25659897

  9. Etomidate Uniquely Modulates the Desensitization of Recombinant α1β3δ GABAA Receptors

    PubMed Central

    Liu, Kunpeng; Jounaidi, Youssef; Forman, Stuart A.; Feng, Hua-Jun

    2015-01-01

    Central GABAA receptors mediate GABAergic phasic and tonic inhibition. While synaptic αβγ GABAA receptors primarily mediate phasic inhibition, extrasynaptic αβδ receptors play an important role in mediating tonic inhibition. Etomidate is a general anesthetic that produces its effects by enhancing GABAA receptor activity. We previously showed that etomidate modulates the gating of oocyte-expressed αβγ and αβδ receptors with similar overall allosteric impact, but different pharmacological patterns. In αβγ receptors, etomidate enhances apparent GABA sensitivity (reduces GABA EC50), modestly increases maximal GABA efficacy, and slows current deactivation without affecting desensitization (Zhong et al; Anesthesiology 2008; 108:103–12). In αβδ receptors characterized by low GABA efficacy, etomidate dramatically increases responses to both low and maximal GABA. The effects of etomidate on desensitization and deactivation of αβδ receptors are unknown. To investigate the kinetic effects of etomidate on α1β3δ receptors of defined subunit arrangement, we expressed concatenated trimer (β3-α1-δ) and dimer (β3-α1) GABAA receptor subunit assemblies in HEK293T cells and recorded whole-cell voltage-clamp currents during rapid external solution exchanges. As expected, etomidate substantially increased maximal GABA-induced currents and prolonged deactivation. Moreover, desensitization was significantly decreased by etomidate. During prolonged GABA applications, etomidate enhanced steady-state currents more than peak currents. Thus, etomidate enhances tonic GABAergic inhibition through extrasynaptic αβδ receptors by both augmenting gating and reducing desensitization. PMID:26028470

  10. Dibenzo[1,2,5]thiadiazepines are non-competitive GABAA receptor antagonists.

    PubMed

    Ramírez-Martínez, Juan F; González-Chávez, Rodolfo; Guerrero-Alba, Raquel; Reyes-Gutiérrez, Paul E; Martínez, Roberto; Miranda-Morales, Marcela; Espinosa-Luna, Rosa; González-Chávez, Marco M; Barajas-López, Carlos

    2013-01-01

    A new process for obtaining dibenzo[c,f][1,2,5]thiadiazepines (DBTDs) and their effects on GABA(A) receptors of guinea pig myenteric neurons are described. Synthesis of DBTD derivatives began with two commercial aromatic compounds. An azide group was obtained after two sequential reactions, and the central ring was closed via a nitrene to obtain the tricyclic sulfonamides (DBTDs). Whole-cell recordings showed that DBTDs application did not affect the holding current but inhibited the currents induced by GABA (I(GABA)), which are mediated by GABA(A) receptors. These DBTDs effects reached their maximum 3 min after application and were: (i) reversible, (ii) concentration-dependent (with a rank order of potency of 2c = 2d > 2b), (iii) mediated by a non-competitive antagonism, and (iv) only observed when applied extracellularly. Picrotoxin (which binds in the channel mouth) and DBTDs effects were not modified when both substances were simultaneous applied. Our results indicate that DBTD acted on the extracellular domain of GABA(A) channels but independent of the picrotoxin, benzodiazepine, and GABA binding sites. DBTDs used here could be the initial model for synthesizing new GABA(A) receptor inhibitors with a potential to be used as antidotes for positive modulators of these receptors or to induce experimental epilepsy. PMID:23344200

  11. GABAA receptors are located in cholinergic terminals in the nucleus pontis oralis of the rat: implications for REM sleep control.

    PubMed

    Liang, Chang-Lin; Marks, Gerald A

    2014-01-16

    The oral pontine reticular formation (PnO) of rat is one region identified in the brainstem as a rapid eye movement (REM) sleep induction zone. Microinjection of GABA(A) receptor antagonists into PnO induces a long lasting increase in REM sleep, which is similar to that produced by cholinergic agonists. We previously showed that this REM sleep-induction can be completely blocked by a muscarinic antagonist, indicating that the REM sleep-inducing effect of GABA(A) receptor antagonism is dependent upon the local cholinergic system. Consistent with these findings, it has been reported that GABA(A) receptor antagonists microdialyzed into PnO resulted in increased levels of acetylcholine. We hypothesize that GABA(A) receptors located on cholinergic boutons in the PnO are responsible for the REM sleep induction by GABA(A) receptor antagonists through blocking GABA inhibition of acetylcholine release. Cholinergic, varicose axon fibers were studied in the PnO by immunofluorescence and confocal, laser scanning microscopy. Immunoreactive cholinergic boutons were found to be colocalized with GABA(A) receptor subunit protein γ2. This finding implicates a specific subtype and location of GABA(A) receptors in PnO of rat in the control of REM sleep. PMID:24141149

  12. Luteolin inhibits GABAA receptors in HEK cells and brain slices

    PubMed Central

    Shen, Mei-Lin; Wang, Chen-Hung; Chen, Rita Yu-Tzu; Zhou, Ning; Kao, Shung-Te; Wu, Dong Chuan

    2016-01-01

    Modulation of the A type γ-aminobutyric acid receptors (GABAAR) is one of the major drug targets for neurological and psychological diseases. The natural flavonoid compound luteolin (2-(3,4-Dihydroxyphenyl)- 5,7-dihydroxy-4-chromenone) has been reported to have antidepressant, antinociceptive, and anxiolytic-like effects, which possibly involve the mechanisms of modulating GABA signaling. However, as yet detailed studies of the pharmacological effects of luteolin are still lacking, we investigated the effects of luteolin on recombinant and endogenous GABAAR-mediated current responses by electrophysiological approaches. Our results showed that luteolin inhibited GABA-mediated currents and slowed the activation kinetics of recombinant α1β2, α1β2γ2, α5β2, and α5β2γ2 receptors with different degrees of potency and efficacy. The modulatory effect of luteolin was likely dependent on the subunit composition of the receptor complex: the αβ receptors were more sensitive than the αβγ receptors. In hippocampal pyramidal neurons, luteolin significantly reduced the amplitude and slowed the rise time of miniature inhibitory postsynaptic currents (mIPSCs). However, GABAAR-mediated tonic currents were not significantly influenced by luteolin. These data suggested that luteolin has negative modulatory effects on both recombinant and endogenous GABAARs and inhibits phasic rather than tonic inhibition in hippocampus. PMID:27292079

  13. Control of Inhibition by the Direct Action of Cannabinoids on GABAA Receptors.

    PubMed

    Golovko, Tatiana; Min, Rogier; Lozovaya, Natalia; Falconer, Caroline; Yatsenko, Natalia; Tsintsadze, Timur; Tsintsadze, Vera; Ledent, Catherine; Harvey, Robert J; Belelli, Delia; Lambert, Jeremy J; Rozov, Andrei; Burnashev, Nail

    2015-09-01

    Cannabinoids are known to regulate inhibitory synaptic transmission via activation of presynaptic G protein-coupled cannabinoid CB1 receptors (CB1Rs). Additionally, recent studies suggest that cannabinoids can also directly interact with recombinant GABAA receptors (GABAARs), potentiating currents activated by micromolar concentrations of γ-aminobutyric acid (GABA). However, the impact of this direct interaction on GABAergic inhibition in central nervous system is unknown. Here we report that currents mediated by recombinant GABAARs activated by high (synaptic) concentrations of GABA as well as GABAergic inhibitory postsynaptic currents (IPSCs) at neocortical fast spiking (FS) interneuron to pyramidal neuron synapses are suppressed by exogenous and endogenous cannabinoids in a CB1R-independent manner. This IPSC suppression may account for disruption of inhibitory control of pyramidal neurons by FS interneurons. At FS interneuron to pyramidal neuron synapses, endocannabinoids induce synaptic low-pass filtering of GABAAR-mediated currents evoked by high-frequency stimulation. The CB1R-independent suppression of inhibition is synapse specific. It does not occur in CB1R containing hippocampal cholecystokinin-positive interneuron to pyramidal neuron synapses. Furthermore, in contrast to synaptic receptors, the activity of extrasynaptic GABAARs in neocortical pyramidal neurons is enhanced by cannabinoids in a CB1R-independent manner. Thus, cannabinoids directly interact differentially with synaptic and extrasynaptic GABAARs, providing a potent novel context-dependent mechanism for regulation of inhibition.

  14. Chronic anabolic-androgenic steroid treatment affects brain GABA(A) receptor-gated chloride ion transport.

    PubMed

    Bitran, D; Hilvers, R J; Frye, C A; Erskine, M S

    1996-01-01

    Previous research in this laboratory has shown that chronic treatment of adult male rats with an anabolic-androgenic steroid (AAS) produced anxiolytic behavior and increased the functional response of cortical gamma-aminobutyric acid(A) (GABA(A)) receptors. The experiments reported here were aimed at further characterizing the effect of chronic AAS exposure on cerebral cortical GABA(A) receptors. Adult male rats were injected with dianabol (1,4-androstadien-17alpha-methyl-17beta-ol-3-one; 10 mg/kg/day, SC) for 4 weeks. A significant decrease in ventral prostate gland weight was found after 2 weeks of dianabol, and returned to control levels 3 and 10 days after steroid discontinuation. Testicular weights decreased throughout the treatment period but reached statistical significance only during the withdrawal period. Serum 3alpha-androstanediol level was marginally increased afer 2 weeks of dianabol injection, and was significantly decreased at 3 and 10 days after withdrawal. GABA-stimulated 36chloride (Cl-) influx in cortical synaptoneurosomes was increased in animals treated with dianabol for 2 and 4 weeks, and remained elevated 3 days after dianabol withdrawal, returning to control levels at withdrawal day 10. The increase in receptor efficacy was associated with a transient increase in receptor sensitivity (inverse of EC50), apparent after 2 weeks of AAS treatment and at withdrawal day 3. In a follow-up experiment, metabolites of dianabol were tested for the in vitro efficacy in potentiating GABA-stimulated Cl- transport. Only 3alpha-androstanedial and androsterone were found to have potent stimulatory effects. The 3beta-reduced metabolites were inactive, as were metabolites that contained a methyl group at the 17alpha position. These results point to significant facilitative effects of dianabol treatment on brain GABA(A) receptors via the metabolic formation of neuroactive steroids. PMID:8632710

  15. Modulation of the Expression of the GABAA Receptor β1 and β3 Subunits by Pretreatment with Quercetin in the KA Model of Epilepsy in Mice

    PubMed Central

    Moghbelinejad, Sahar; Rashvand, Zahra; Khodabandehloo, Fatemeh; Mohammadi, Ghazaleh

    2016-01-01

    Objectives: Quercetin is a flavonoid and an important dietary constituent of fruits and vegetables. In recent years, several pharmacological activities of quercetin, such as its neuroprotective activity and, more specifically, its anti-convulsant effects in animal models of epilepsy, have been reported. This study evaluated the role of quercetin pretreatment on gene expression of γ-amino butyric acid type A (GABAA) receptor beta subunits in kainic acid (KA)-induced seizures in mice. Methods: The animals were divided into four groups: one saline group, one group in which seizures were induced by using KA (10 mg/kg) without quercetin pretreatment and two groups pretreated with quercetin (50 and 100 mg/kg) prior to seizures being induced by using KA. Next, the messenger ribonucleic acid (mRNA) levels of the GABAA receptor β subunits in the hippocampus of each animal were assessed at 2 hours and 7 days after KA administration. Quantitative real-time polymerase chain reaction (RT-PCR) assay was used to detect mRNA content in hippocampal tissues. Results: Pretreatments with quercetin at doses of 50 and 100 mg/kg prevented significant increases in the mRNA levels of the β1 and the β3 subunits of the GABAA receptor at 2 hours after KA injection. Pretreatment with quercetin (100 mg/kg) significantly inhibited β1 and β3 gene expression in the hippocampus at 7 days after KA injection. But, this inhibitory effect of quercetin at 50 mg/kg on the mRNA levels of the β3 subunit of the GABAA receptor was not observed at 7 days after KA administration. Conclusion: These results suggest that quercetin (100 mg/kg) modulates the expression of the GABAA receptor β1 and β3 subunits in the KA model of epilepsy, most likely to prevent compensatory responses. This may be related to the narrow therapeutic dose range for the anticonvulsant activities of quercetin. PMID:27386150

  16. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin.

    PubMed

    Eghorn, Laura F; Hoestgaard-Jensen, Kirsten; Kongstad, Kenneth T; Bay, Tina; Higgins, David; Frølund, Bente; Wellendorph, Petrine

    2014-10-01

    γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.

  17. BDNF modulates GABAA receptors microtransplanted from the human epileptic brain to Xenopus oocytes

    PubMed Central

    Palma, E.; Torchia, G.; Limatola, C.; Trettel, F.; Arcella, A.; Cantore, G.; Di Gennaro, G.; Manfredi, M.; Esposito, V.; Quarato, P. P.; Miledi, R.; Eusebi, F.

    2005-01-01

    Cell membranes isolated from brain tissues, obtained surgically from six patients afflicted with drug-resistant temporal lobe epilepsy and from one nonepileptic patient afflicted with a cerebral oligodendroglioma, were injected into frog oocytes. By using this approach, the oocytes acquire human GABAA receptors, and we have shown previously that the “epileptic receptors” (receptors transplanted from epileptic brains) display a marked run-down during repetitive applications of GABA. It was found that exposure to the neurotrophin BDNF increased the amplitude of the “GABA currents” (currents elicited by GABA) generated by the epileptic receptors and decreased their run-down; both events being blocked by K252A, a neurotrophin tyrosine kinase receptor B inhibitor. These effects of BDNF were not mimicked by nerve growth factor. In contrast, the GABAA receptors transplanted from the nonepileptic human hippocampal uncus (obtained during surgical resection as part of the nontumoral tissue from the oligodendroglioma margins) or receptors expressed by injecting rat recombinant α1β2γ2 GABAA receptor subunit cDNAs generated GABA currents whose time-course and run-down were not altered by BDNF. Loading the oocytes with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate-acetoxymethyl ester (BAPTA-AM), or treating them with Rp-8-Br-cAMP, an inhibitor of the cAMP-dependent PKA, did not alter the GABA currents. However, staurosporine (a broad spectrum PK inhibitor), bisindolylmaleimide I (a PKC inhibitor), and U73122 (a phospholipase C inhibitor) blocked the BDNF-induced effects on the epileptic GABA currents. Our results indicate that BDNF potentiates the epileptic GABAA currents and antagonizes their use-dependent run-down, thus strengthening GABAergic inhibition, probably by means of activation of tyrosine kinase receptor B receptors and of both PLC and PKC. PMID:15665077

  18. Association between GABAA Receptor Subunit Gene Cluster and Zolpidem-Induced Complex Sleep Behaviors in Han Chinese

    PubMed Central

    Tsai, Jui-Hsiu; Yang, Pinchen; Lin, Hung-Hsun; Cheng, Kuang-hung; Yang, Yi-Hsin; Wu, Ming-Tsang; Chen, Cheng-Chung

    2013-01-01

    Study Objectives: To investigate and elucidate the role of GABAA receptor subunits, specifically the 2 genetic markers at the GABAA α1 and GABAA α6 receptors, in zolpidem-induced complex sleep behaviors (CSBs). Design: Genetic association study. Setting: Kaohsiung Medical University-affiliated hospitals, Kaohsiung, Taiwan. Patients: 30 zolpidem-induced CSB subjects and 37 controls. Interventions: N/A. Measurements and Results: The χ2 test demonstrated an association between the A15G variant at the GABAA α1 receptor subunit gene and zolpidem-induced CSBs (P = 0.007). The adjusted odds ratio of the GABAA α1 receptor subunit genotype for the risk of zolpidem-induced CSBs was approximately 10 (OR = 9.99, 95% CI = 1.82, 74.87; P = 0.013). Conclusions: The finding reveals that the A15G variant at the GABAA α1 receptor subunit gene confers a high risk of zolpidem-induced CSBs and may be considered in clinical services. Citation: Tsai JH; Yang P; Lin HH; Cheng Kh; Yang YH; Wu MT; Chen CC. Association between GABAA receptor subunit gene cluster and zolpidem-induced complex sleep behaviors in Han Chinese. SLEEP 2013;36(2):197–202. PMID:23372267

  19. A novel GABA(A) alpha 5 receptor inhibitor with therapeutic potential.

    PubMed

    Ling, István; Mihalik, Balázs; Etherington, Lori-An; Kapus, Gábor; Pálvölgyi, Adrienn; Gigler, Gábor; Kertész, Szabolcs; Gaál, Attila; Pallagi, Katalin; Kiricsi, Péter; Szabó, Éva; Szénási, Gábor; Papp, Lilla; Hársing, László G; Lévay, György; Spedding, Michael; Lambert, Jeremy J; Belelli, Delia; Barkóczy, József; Volk, Balázs; Simig, Gyula; Gacsályi, István; Antoni, Ferenc A

    2015-10-01

    Novel 2,3-benzodiazepine and related isoquinoline derivatives, substituted at position 1 with a 2-benzothiophenyl moiety, were synthesized to produce compounds that potently inhibited the action of GABA on heterologously expressed GABAA receptors containing the alpha 5 subunit (GABAA α5), with no apparent affinity for the benzodiazepine site. Substitutions of the benzothiophene moiety at position 4 led to compounds with drug-like properties that were putative inhibitors of extra-synaptic GABAA α5 receptors and had substantial blood-brain barrier permeability. Initial characterization in vivo showed that 8-methyl-5-[4-(trifluoromethyl)-1-benzothiophen-2-yl]-1,9-dihydro-2H-[1,3]oxazolo[4,5-h][2,3]benzodiazepin-2-one was devoid of sedative, pro-convulsive or motor side-effects, and enhanced the performance of rats in the object recognition test. In summary, we have discovered a first-in-class GABA-site inhibitor of extra-synaptic GABAA α5 receptors that has promising drug-like properties and warrants further development.

  20. Neurosteroid interactions with synaptic and extrasynaptic GABAa receptors: regulation of subunit plasticity, phasic and tonic inhibition, and neuronal network excitability

    PubMed Central

    Chase Matthew, Carver; Doodipala Samba, Reddy

    2013-01-01

    Rationale Neurosteroids are steroids synthesized within the brain with rapid effects on neuronal excitability. Allopregnanolone, allotetrahydrodeoxycorticosterone, and androstanediol are three widely explored prototype endogenous neurosteroids. They have very different targets and functions compared to conventional steroid hormones. Neuronal GABAa receptors are one of the prime molecular targets of neurosteroids. Objective This review provides a critical appraisal of recent advances in the pharmacology of endogenous neurosteroids that interact with GABAa receptors in the brain. Neurosteroids possess distinct, characteristic effects on the membrane potential and current conductance of the neuron, mainly via potentiation of GABAa receptors at low concentrations and direct activation of receptor chloride channel at higher concentrations. The GABAa receptor mediates two types of inhibition, now characterized as synaptic (phasic) and extrasynaptic (tonic) inhibition. Synaptic release of GABA results in the activation of low-affinity γ2-containing synaptic receptors, while high-affinity δ-containing extrasynaptic receptors are persistently activated by the ambient GABA present in the extracellular fluid. Neurosteroids are potent positive allosteric modulators of synaptic and extrasynaptic GABAa receptors and therefore enhance both phasic and tonic inhibition. Tonic inhibition is specifically more sensitive to neurosteroids. The resulting tonic conductance generates a form of shunting inhibition that controls neuronal network excitability, seizure susceptibility, and behavior. Conclusion The growing understanding of the mechanisms of neurosteroid regulation of the structure and function of the synaptic and extrasynaptic GABAa receptors provide many opportunities to create improved therapies for sleep, anxiety, stress, epilepsy, and other neuropsychiatric conditions. PMID:24071826

  1. The interaction of general anaesthetics with recombinant GABAA and glycine receptors expressed in Xenopus laevis oocytes: a comparative study

    PubMed Central

    Pistis, Marco; Belelli, Delia; Peters, John A; Lambert, Jeremy J

    1997-01-01

    The effects of five structurally dissimilar general anaesthetics were examined in voltage-clamp recordings of agonist-evoked currents mediated by recombinant γ-aminobutyric acid (GABA)A receptors composed of human α1β1 and γ2L subunits expressed in Xenopus laevis oocytes. A quantitative comparison of the effects of these agents was made upon recombinant glycine receptors expressed as a homo-oligomer of human α1 subunits, or as a hetero-oligomer of human α1 and rat β subunits. Complementary RNA-injected oocytes expressing GABAA receptors responded to bath applied GABA with an EC50 of 158±34 μM. Oocytes expressing α1 and α1β glycine receptors subsequent to cDNA injection displayed EC50 values of 76±2 μM and 66±2 μM, respectively, in response to bath applied glycine. Picrotoxin antagonized responses mediated by homo-oligomeric α1 glycine receptors with an IC50 of 4.2±0.8 μM. Hetero-oligomeric α1β glycine receptors were at least 100-fold less sensitive to blockade by picrotoxin. With the appropriate agonist EC10, propofol enhanced GABA and glycine-evoked currents to approximately the maximal response produced by a saturating concentration of either agonist (i.e. Imax). The calculated EC50 values were 2.3±0.2 μM, 16±3 μM and 27±2 μM, for GABAA α1β1γ2L, glycine α1 and α1β receptors, respectively. At relatively high concentrations, propofol was observed to activate directly both GABAA and glycine receptors. Pentobarbitone potentiated GABA-evoked currents to 117±8.5% of Imax with an EC50 of 65±3 μM. The barbiturate also produced a substantial enhancement of the glycine-evoked currents, Imax and EC50 values being 71±2% and 845±66 μM and 51±10% and 757±30 μM for homomeric α1 and heteromeric α1β glycine receptors respectively. At high concentrations, pentobarbitone directly activated GABAA, but not glycine, receptors. The potentiation by propofol or pentobarbitone of currents mediated by α1 homo

  2. Effects of beer and hop on ionotropic gamma-aminobutyric acid receptors.

    PubMed

    Aoshima, Hitoshi; Takeda, Katsuichi; Okita, Yoichi; Hossain, Sheikh Julfikar; Koda, Hirofumi; Kiso, Yoshinobu

    2006-04-01

    Beer induced the response of the ionotropic gamma-aminobutyric acid receptors (GABA(A) receptors) expressed in Xenopus oocytes, indicating the presence of gamma-aminobutyric acid (GABA)-like activity. Furthermore, the pentane extract of the beer, hop (Humulus lupulus L.) oil, and myrcenol potentiated the GABA(A) receptor response elicited by GABA. The GABA(A) receptor responses were also potentiated by the addition of aliphatic esters, most of which are reported to be present in beer flavor. Aliphatic esters showed the tendency to decrease in the potentiation of the GABA(A) receptor response with an increase in their carbon chain length. When myrcenol was injected to mice prior to intraperitoneal administration of pentobarbital, the pentobarbital-induced sleeping time of mice increased additionally. Therefore, the beer contained not only GABA-like activity but also the modulator(s) of the GABA(A) receptor response.

  3. GABAergic innervation organizes synaptic and extrasynaptic GABAA receptor clustering in cultured hippocampal neurons.

    PubMed

    Christie, Sean B; Miralles, Celia P; De Blas, Angel L

    2002-02-01

    We have studied the effects of GABAergic innervation on the clustering of GABA(A) receptors (GABA(A)Rs) in cultured hippocampal neurons. In the absence of GABAergic innervation, pyramidal cells form small (0.36 +/- 0.01 micrometer diameter) GABA(A)R clusters at their surface in the dendrites and soma. When receiving GABAergic innervation from glutamic acid decarboxylase-containing interneurons, pyramidal cells form large (1.62 +/- 0.08 micrometer breadth) GABA(A)R clusters at GABAergic synapses. This is accompanied by a disappearance of the small GABA(A)R clusters in the local area surrounding each GABAergic synapse. Although the large synaptic GABA(A)R clusters of any neuron contained all GABA(A)R subunits and isoforms expressed by that neuron, the small clusters not localized at GABAergic synapses showed significant heterogeneity in subunit and isoform composition. Another difference between large GABAergic and small non-GABAergic GABA(A)R clusters was that a significant proportion of the latter was juxtaposed to postsynaptic markers of glutamatergic synapses such as PSD-95 and AMPA receptor GluR1 subunit. The densities of both the glutamate receptor-associated and non-associated small GABA(A)R clusters were decreased in areas surrounding GABAergic synapses. However, no effect on the density or distribution of glutamate receptor clusters was observed. The results suggest that there are local signals generated at GABAergic synapses that induce both assembly of large synaptic GABA(A)R clusters at the synapse and disappearance of the small GABA(A)R clusters in the surrounding area. In the absence of GABAergic innervation, weaker GABA(A)R-clustering signals, generated at glutamatergic synapses, induce the formation of small postsynaptic GABA(A)R clusters that remain juxtaposed to glutamate receptors at glutamatergic synapses.

  4. 5-(N, N-Hexamethylene) amiloride is a GABA-A ρ1 receptor positive allosteric modulator.

    PubMed

    Snell, Heather D; Gonzales, Eric B

    2016-11-01

    Guanidine compounds act as ion channel modulators. In the case of Cys-loop receptors, the guanidine compound amiloride antagonized the heteromeric GABA-A, glycine, and nicotinic acetylcholine receptors. However, amiloride exhibits characteristics consistent with a positive allosteric modulator for the human GABA-A (hGABA-A) ρ1 receptor. Site-directed mutagenesis revealed that the positive allosteric modulation was influenced by the GABA-A ρ1 second transmembrane domain 15' position, a site implicated in ligand allosteric modulation of Cys-loop receptors. There are a variety of amiloride derivatives that provide opportunities to assess the significance of amiloride functional groups (e.g., the guanidine group, the pyrazine ring, etc.) in the modulation of the GABA-A ρ1 receptor activity. We utilized 3 amiloride derivatives (benzamil, phenamil, and 5-(N, N-Hexamethylene) amiloride) to assess the contribution of these groups toward the potentiation of the GABA-A ρ1 receptor. Benzamil and phenamil failed to potentiate on the wild type GABA-A ρ1 GABA-mediated current while HMA demonstrated efficacy only at the highest concentration studied. The hGABA-A ρ1 (I15'N) mutant receptor activity was potentiated by lower HMA concentrations compared to the wild type receptor. Our findings suggest that an exposed guanidine group on amiloride and amiloride derivatives is critical for modulating the GABA-A ρ1 receptor. The present study provides a conceptual framework for predicting which amiloride derivatives will demonstrate positive allosteric modulation of the GABA-A ρ1 receptor.

  5. 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.

  6. Regulation of Extrasynaptic GABAA α4 Receptors by Ethanol-Induced Protein Kinase A, but Not Protein Kinase C Activation in Cultured Rat Cerebral Cortical Neurons.

    PubMed

    Carlson, Stephen L; Bohnsack, J Peyton; Patel, Vraj; Morrow, A Leslie

    2016-01-01

    Ethanol produces changes in GABAA receptor trafficking and function that contribute to ethanol dependence symptomatology. Extrasynaptic γ-aminobutyric acid A receptors (GABAA-R) mediate inhibitory tonic current and are of particular interest because they are potentiated by physiologically relevant doses of ethanol. Here, we isolate GABAA α4δ receptors by western blotting in subsynaptic fractions to investigate protein kinase A (PKA) and protein kinase C (PKC) modulation of ethanol-induced receptor trafficking, while extrasynaptic receptor function is determined by measurement of tonic inhibition and responses evoked by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or PKA/PKC modulators. Ethanol exposure (1 hour) did not alter GABAA α4 receptor abundance, but it increased tonic current amplitude, an effect that was prevented by inhibiting PKA, but not PKC. Direct activation of PKA, but not PKC, increased the abundance and tonic current of extrasynaptic α4δ receptors. In contrast, prolonged ethanol exposure (4 hours) reduced α4δ receptor abundance as well as tonic current, and this effect was also PKA dependent. Finally, PKC activation by ethanol or phorbol-12,13-dibutyrate (PdBu) had no effect on extrasynaptic α4δ subunit abundance or activity. We conclude that ethanol alters extrasynaptic α4δ receptor function and expression in cortical neurons in a PKA-dependent manner, but ethanol activation of PKC does not influence these receptors. These results could have clinical relevance for therapeutic strategies to restore normal GABAergic functioning for the treatment of alcohol use disorders.

  7. Lotus Leaf Alkaloid Extract Displays Sedative-Hypnotic and Anxiolytic Effects through GABAA Receptor.

    PubMed

    Yan, Ming-Zhu; Chang, Qi; Zhong, Yu; Xiao, Bing-Xin; Feng, Li; Cao, Fang-Rui; Pan, Rei-Le; Zhang, Ze-Sheng; Liao, Yong-Hong; Liu, Xin-Min

    2015-10-28

    Lotus leaves have been used traditionally as both food and herbal medicine in Asia. Open-field, sodium pentobarbital-induced sleeping and light/dark box tests were used to evaluate sedative-hypnotic and anxiolytic effects of the total alkaloids (TA) extracted from the herb, and the neurotransmitter levels in the brain were determined by ultrafast liquid chromatography-tandem mass spectrometry. The effects of picrotoxin, flumazenil, and bicuculline on the hypnotic activity of TA, as well as the influence of TA on Cl(-) influx in cerebellar granule cells, were also investigated. TA showed a sedative-hypnotic effect by increasing the brain level of γ-aminobutyric acid (GABA), and the hypnotic effect could be blocked by picrotoxin and bicuculline, but could not be antagonized by flumazenil. Additionally, TA could increase Cl(-) influx in cerebellar granule cells. TA at 20 mg/kg induced anxiolytic-like effects and significantly increased the concentrations of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and dopamine (DA). These data demonstrated that TA exerts sedative-hypnotic and anxiolytic effects via binding to the GABAA receptor and activating the monoaminergic system. PMID:26448283

  8. Protein kinase C regulates tonic GABA(A) receptor-mediated inhibition in the hippocampus and thalamus.

    PubMed

    Bright, Damian P; Smart, Trevor G

    2013-11-01

    Tonic inhibition mediated by extrasynaptic GABA(A) receptors (GABA(A) Rs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABA(A) Rs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole-cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC-mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABA(A) R-mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABA(A) R activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABA(A) Rs, which represent a key extrasynaptic GABA(A) R isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABA(A) Rs. The inhibitory effects of PKC activation on α4β2δ GABA(A) R activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC-mediated phosphorylation can be an important physiological regulator of tonic GABA(A) R-mediated inhibition.

  9. Cyclohexanol analogues are positive modulators of GABA(A) receptor currents and act as general anaesthetics in vivo.

    PubMed

    Hall, Adam C; Griffith, Theanne N; Tsikolia, Maia; Kotey, Francesca O; Gill, Nikhila; Humbert, Danielle J; Watt, Erin E; Yermolina, Yuliya A; Goel, Shikha; El-Ghendy, Bahaa; Hall, C Dennis

    2011-09-30

    GABA(A) 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 cyclohexanols were investigated on recombinant human γ-aminobutyric acid (GABA(A), α(1)β(2)γ(2s)) receptors expressed in Xenopus oocytes, and compared to the modulatory effects on GABA currents observed with exposures to the intravenous anaesthetic agent, propofol. Submaximal EC(20) GABA currents were typically enhanced by co-applications of 3-300 μM cyclohexanols. For instance, at 30 μM 2,6-diisopropylcyclohexanol (a novel compound) GABA responses were increased ~3-fold (although similar enhancements were achieved at 3 μM propofol). As regards rank order for modulation by the cyclohexanol analogues at 30 μM, the % enhancements for 2,6-dimethylcyclohexanol~2,6-diethylcyclohexanol~2,6-diisopropylcyclohexanol~2,6-di-sec-butylcyclohexanol ≫2,6-di-tert-butylcyclohexanol~4-tert-butylcyclohexanol>cyclohexanol~cyclopentanol~2-methylcyclohexanol. We further tested the potencies of the cyclohexanol analogues as general anaesthetics using a tadpole in vivo assay. Both 2,6-diisopropylcyclohexanol and 2,6-dimethylcyclohexanol were effective as anaesthetics with EC(50)s of 14.0 μM and 13.1 μM respectively, while other cyclohexanols with bulkier side chains were less potent. In conclusion, our data indicate that cyclohexanols are both positive modulators of GABA(A) receptors currents and anaesthetics. The positioning and size of the alkyl groups at the 2 and 6 positions on the cyclohexanol ring were critical determinants of activity.

  10. The non-competitive blockade of GABAA receptors by an aqueous extract of water hemlock (Cicuta douglasii) tubers.

    PubMed

    Green, Benedict T; Goulart, Camila; Welch, Kevin D; Pfister, James A; McCollum, Isabelle; Gardner, Dale R

    2015-12-15

    Water hemlocks (Cicuta spp.) are acutely toxic members of the Umbellierae family; the toxicity is due to the presence of C17-polyacetylenes such as cicutoxin. There is only limited evidence of noncompetitive antagonism by C17-polyacetylenes at GABAA receptors. In this work with WSS-1 cells, we documented the noncompetitive blockade of GABAA receptors by an aqueous extract of water hemlock (Cicuta douglasii) and modulated the actions of the extract with a pretreatment of 10 μM midazolam. PMID:26415905

  11. 6,3'-Dinitroflavone is a low efficacy modulator of GABA(A) receptors.

    PubMed

    Furtmueller, Roman; Furtmueller, Birgit; Ramerstorfer, Joachim; Paladini, Alejandro C; Wasowski, Cristina; Marder, Mariel; Huck, Sigismund; Sieghart, Werner

    2008-09-01

    6,3'-Dinitroflavone (6,3'-DNF) is a synthetic flavone derivative that exerts anxiolytic effects in the elevated plus maze. Based on the finding that this effect is blocked by Ro15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate) which is a specific antagonist at the benzodiazepine binding site of GABA(A) receptors we investigated the interaction of 6,3'-DNF with several recombinant GABA(A) receptor subtypes. Inhibition of [(3)H]flunitrazepam binding to recombinant GABA(A) receptors in transiently transfected HEK293 cells indicated that 6,3'-DNF exhibited the highest affinity for GABA(A) receptors composed of alpha1beta2gamma2 subunits and a 2-20 fold lower affinity for homologous receptors containing alpha2, alpha3, or alpha5 subunits. Two-electrode voltage-clamp experiments in Xenopus oocytes indicated that 6,3'-DNF does not induce chloride flux in the absence of GABA, but exerts low efficacy inverse agonistic modulatory effects on GABA-elicited currents in the GABA(A) receptor subtypes alpha1beta2gamma2 and alpha5beta2gamma2. In the subtypes alpha2beta2gamma2, alpha3beta2gamma2, alpha4beta2gamma2, alpha6beta2gamma2 or alpha4beta2delta and alpha4beta3delta, 6,3'-DNF exerts either none or very low efficacy positive modulatory effects. In contrast, 100 nM Ro15-1788 exhibited weak to moderate partial agonistic effects on each receptor investigated. These data indicate that Ro15-1788 only can antagonize the weak inverse agonist effects of 6,3'-DNF on alpha1beta2gamma2 and alpha5beta2gamma2 receptors, but will enhance the weak agonistic effects on the other receptor subtypes investigated. The possible mechanism of the Ro15-1788 sensitive anxiolytic effect of 6,3'-DNF is discussed.

  12. 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

  13. GABA-A receptor inhibition of local calcium signaling in spines and dendrites.

    PubMed

    Marlin, Joseph J; Carter, Adam G

    2014-11-26

    Cortical interneurons activate GABA-A receptors to rapidly control electrical and biochemical signaling at pyramidal neurons. Different populations of interneurons are known to uniquely target the soma and dendrites of pyramidal neurons. However, the ability of these interneurons to inhibit Ca(2+) signaling at spines and dendrites is largely unexplored. Here we use whole-cell recordings, two-photon microscopy, GABA uncaging and optogenetics to study dendritic inhibition at layer 5 (L5) pyramidal neurons in slices of mouse PFC. We first show that GABA-A receptors strongly inhibit action potential (AP)-evoked Ca(2+) signals at both spines and dendrites. We find robust inhibition over tens of milliseconds that spreads along the dendritic branch. However, we observe no difference in the amount of inhibition at neighboring spines and dendrites. We then examine the influence of interneurons expressing parvalbumin (PV), somatostatin (SOM), or 5HT3a receptors. We determine that these populations of interneurons make unique contacts onto the apical and basal dendrites of L5 pyramidal neurons. We also show that SOM and 5HT3a but not PV interneurons potently inhibit AP Ca(2+) signals via GABA-A receptors at both spines and dendrites. These findings reveal how multiple interneurons regulate local Ca(2+) signaling in pyramidal neurons, with implications for cortical function and disease.

  14. Proteomic and Systems Biology Analysis of Monocytes Exposed to Securinine, a GABAA Receptor Antagonist and Immune Adjuvant

    PubMed Central

    Shipman, Matt; Lubick, Kirk; Fouchard, David; Guram, Rajani; Grieco, Paul; Jutila, Mark; Dratz, Edward A.

    2012-01-01

    Securinine, a GABAA receptor antagonist, has been reported to enhance monocyte cell killing of Coxiella burnetii without obvious adverse effects in vivo. We employed multiplex 2D gel electrophoresis using Zdyes, a new generation of covalently linked fluorescent differential protein detection dyes to analyze changes in the monocyte proteome in response to Securinine. Securinine antagonism of GABAA receptors triggers the activation of p38. We used the differential protein expression results to guide a search of the literature and network analysis software to construct a systems biology model of the effect of Securinine on monocytes. The model suggests that various metabolic modulators (fatty acid binding protein 5, inosine 5′-monophosphate dehydrogenase, and thioredoxin) are at least partially reshaping the metabolic landscape within the monocytes. The actin bundling protein L-plastin, and the Ca2+ binding protein S100A4 also appear to have important roles in the immune response stimulated by Securinine. Fatty acid binding protein 5 (FABP5) may be involved in effecting lipid raft composition, inflammation, and hormonal regulation of monocytes, and the model suggests that FABP5 may be a central regulator of metabolism in activated monocytes. The model also suggests that the heat shock proteins have a significant impact on the monocyte immune response. The model provides a framework to guide future investigations into the mechanisms of Securinine action and with elaboration may help guide development of new types of immune adjuvants. PMID:23028424

  15. Flunitrazepam rapidly reduces GABAA receptor subunit protein expression via a protein kinase C-dependent mechanism

    PubMed Central

    Johnston, Jonathan D; Price, Sally A; Bristow, David R

    1998-01-01

    Acute flunitrazepam (1 μM) exposure for 1 h reduced GABAA receptor α1 (22±4%, mean±s.e.mean) and β2/3 (21±4%) subunit protein levels in cultured rat cerebellar granule cells. This rapid decrease in subunit proteins was completely prevented by bisindolymaleimide 1 (1 μM), an inhibitor of protein kinase C, but not by N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89, 4.8 μM), an inhibitor of protein kinases A and G. These results suggest the existence of a benzodiazepine-induced mechanism to rapidly alter GABAA receptor protein expression, that appears to be dependent on protein kinase C activity. PMID:9723942

  16. Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry.

    PubMed

    Yamaura, Kei; Kiyonaka, Shigeki; Numata, Tomohiro; Inoue, Ryuji; Hamachi, Itaru

    2016-10-01

    The fast inhibitory actions of γ-aminobutyric acid (GABA) are mainly mediated by GABAA receptors (GABAARs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABAARs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABAAR ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABAARs. Electrophysiological measurements revealed that one hit, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABAAR-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins.

  17. Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry.

    PubMed

    Yamaura, Kei; Kiyonaka, Shigeki; Numata, Tomohiro; Inoue, Ryuji; Hamachi, Itaru

    2016-10-01

    The fast inhibitory actions of γ-aminobutyric acid (GABA) are mainly mediated by GABAA receptors (GABAARs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABAARs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABAAR ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABAARs. Electrophysiological measurements revealed that one hit, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABAAR-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins. PMID:27526031

  18. GABAA receptor-mediated input change on orexin neurons following sleep deprivation in mice.

    PubMed

    Matsuki, T; Takasu, M; Hirose, Y; Murakoshi, N; Sinton, C M; Motoike, T; Yanagisawa, M

    2015-01-22

    Orexins are bioactive peptides, which have been shown to play a pivotal role in vigilance state transitions: the loss of orexin-producing neurons (orexin neurons) leads to narcolepsy with cataplexy in the human. However, the effect of the need for sleep (i.e., sleep pressure) on orexin neurons remains largely unknown. Here, we found that immunostaining intensities of the α1 subunit of the GABAA receptor and neuroligin 2, which is involved in inhibitory synapse specialization, on orexin neurons of mouse brain were significantly increased by 6-h sleep deprivation. In contrast, we noted that immunostaining intensities of the α2, γ2, and β2/3 subunits of the GABAA receptor and Huntingtin-associated protein 1, which is involved in GABAAR trafficking, were not changed by 6-h sleep deprivation. Using a slice patch recording, orexin neurons demonstrated increased sensitivity to a GABAA receptor agonist together with synaptic plasticity changes after sleep deprivation when compared with an ad lib sleep condition. In summary, the GABAergic input property of orexin neurons responds rapidly to sleep deprivation. This molecular response of orexin neurons may thus play a role in the changes that accompany the need for sleep following prolonged wakefulness, in particular the decreased probability of a transition to wakefulness once recovery sleep has begun.

  19. Implications for treatment: GABAA receptors in aging, Down syndrome and Alzheimer's disease.

    PubMed

    Rissman, Robert A; Mobley, William C

    2011-05-01

    In addition to progressive dementia, Alzheimer's disease (AD) is characterized by increased incidence of seizure activity. Although originally discounted as a secondary process occurring as a result of neurodegeneration, more recent data suggest that alterations in excitatory-inhibitory (E/I) balance occur in AD and may be a primary mechanism contributing AD cognitive decline. In this study, we discuss relevant research and reports on the GABA(A) receptor in developmental disorders, such as Down syndrome, in healthy aging, and highlight documented aberrations in the GABAergic system in AD. Stressing the importance of understanding the subunit composition of individual GABA(A) receptors, investigations demonstrate alterations of particular GABA(A) receptor subunits in AD, but overall sparing of the GABAergic system. In this study, we review experimental data on the GABAergic system in the pathobiology of AD and discuss relevant therapeutic implications. When developing AD therapeutics that modulate GABA it is important to consider how E/I balance impacts AD pathogenesis and the relationship between seizure activity and cognitive decline.

  20. Allopregnanolone microinjected into the lateral septum or dorsal hippocampus reduces immobility in the forced swim test: participation of the GABAA receptor.

    PubMed

    Rodríguez-Landa, Juan Francisco; Contreras, Carlos M; García-Ríos, Rosa Isela

    2009-10-01

    Allopregnanolone is a 5α-reduced metabolite of progesterone with actions on γ-aminobutyric acid-A (GABAA) receptors that produce antidepressant-like effects. However, little is known about the target brain regions that mediate its antidepressant-like effects. In this study, allopregnanolone (2.0 μg/0.3 μl/rat) or its vehicle (35% cyclodextrin solution) were microinjected into the lateral septum, septofimbrial, or dorsal hippocampus of male Wistar rats that had previously received intraperitoneal injections of either saline or the GABAA antagonist bicuculline (1.0 mg/kg), and its effects were evaluated in the open field and forced swim tests. Allopregnanolone microinjected into the lateral septum or dorsal hippocampus, but not septofimbrial nucleus, induced a longer latency to the first immobility and a shorter total immobility time in the forced swim test compared with vehicle. Bicuculline pretreatment reversed the effect of allopregnanolone. None of the treatments produced significant changes in crossings in the open field test. In conclusion, allopregnanolone produces an antidepressant-like effect in rats submitted to the forced swim test through actions on GABAA receptors located in the lateral septum and dorsal hippocampus, which is consistent with the antistress effect of GABAA agonists in these particular brain structures.

  1. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25675485

  3. Neurosteroids promote phosphorylation and membrane insertion of extrasynaptic GABAA receptors

    PubMed Central

    Abramian, Armen M.; Comenencia-Ortiz, Eydith; Modgil, Amit; Vien, Thuy N.; Nakamura, Yasuko; Moore, Yvonne E.; Maguire, Jamie L.; Terunuma, Miho; Davies, Paul A.; Moss, Stephen J.

    2014-01-01

    Neurosteroids are synthesized within the brain and act as endogenous anxiolytic, anticonvulsant, hypnotic, and sedative agents, actions that are principally mediated via their ability to potentiate phasic and tonic inhibitory neurotransmission mediated by γ-aminobutyric acid type A receptors (GABAARs). Although neurosteroids are accepted allosteric modulators of GABAARs, here we reveal they exert sustained effects on GABAergic inhibition by selectively enhancing the trafficking of GABAARs that mediate tonic inhibition. We demonstrate that neurosteroids potentiate the protein kinase C-dependent phosphorylation of S443 within α4 subunits, a component of GABAAR subtypes that mediate tonic inhibition in many brain regions. This process enhances insertion of α4 subunit-containing GABAAR subtypes into the membrane, resulting in a selective and sustained elevation in the efficacy of tonic inhibition. Therefore, the ability of neurosteroids to modulate the phosphorylation and membrane insertion of α4 subunit-containing GABAARs may underlie the profound effects these endogenous signaling molecules have on neuronal excitability and behavior. PMID:24778259

  4. Neurosteroids promote phosphorylation and membrane insertion of extrasynaptic GABAA receptors.

    PubMed

    Abramian, Armen M; Comenencia-Ortiz, Eydith; Modgil, Amit; Vien, Thuy N; Nakamura, Yasuko; Moore, Yvonne E; Maguire, Jamie L; Terunuma, Miho; Davies, Paul A; Moss, Stephen J

    2014-05-13

    Neurosteroids are synthesized within the brain and act as endogenous anxiolytic, anticonvulsant, hypnotic, and sedative agents, actions that are principally mediated via their ability to potentiate phasic and tonic inhibitory neurotransmission mediated by γ-aminobutyric acid type A receptors (GABAARs). Although neurosteroids are accepted allosteric modulators of GABAARs, here we reveal they exert sustained effects on GABAergic inhibition by selectively enhancing the trafficking of GABAARs that mediate tonic inhibition. We demonstrate that neurosteroids potentiate the protein kinase C-dependent phosphorylation of S443 within α4 subunits, a component of GABAAR subtypes that mediate tonic inhibition in many brain regions. This process enhances insertion of α4 subunit-containing GABAAR subtypes into the membrane, resulting in a selective and sustained elevation in the efficacy of tonic inhibition. Therefore, the ability of neurosteroids to modulate the phosphorylation and membrane insertion of α4 subunit-containing GABAARs may underlie the profound effects these endogenous signaling molecules have on neuronal excitability and behavior. PMID:24778259

  5. Early continuous white noise exposure alters auditory spatial sensitivity and expression of GAD65 and GABAA receptor subunits in rat auditory cortex.

    PubMed

    Xu, Jinghong; Yu, Liping; Cai, Rui; Zhang, Jiping; Sun, Xinde

    2010-04-01

    Sensory experiences have important roles in the functional development of the mammalian auditory cortex. Here, we show how early continuous noise rearing influences spatial sensitivity in the rat primary auditory cortex (A1) and its underlying mechanisms. By rearing infant rat pups under conditions of continuous, moderate level white noise, we found that noise rearing markedly attenuated the spatial sensitivity of A1 neurons. Compared with rats reared under normal conditions, spike counts of A1 neurons were more poorly modulated by changes in stimulus location, and their preferred locations were distributed over a larger area. We further show that early continuous noise rearing induced significant decreases in glutamic acid decarboxylase 65 and gamma-aminobutyric acid (GABA)(A) receptor alpha1 subunit expression, and an increase in GABA(A) receptor alpha3 expression, which indicates a returned to the juvenile form of GABA(A) receptor, with no effect on the expression of N-methyl-D-aspartate receptors. These observations indicate that noise rearing has powerful adverse effects on the maturation of cortical GABAergic inhibition, which might be responsible for the reduced spatial sensitivity. PMID:19620619

  6. Luteolin mediates the antidepressant-like effects of Cirsium japonicum in mice, possibly through modulation of the GABAA receptor.

    PubMed

    de la Peña, June Bryan I; Kim, Chong Ah; Lee, Hye Lim; Yoon, Seo Young; Kim, Hee Jin; Hong, Eun Young; Kim, Gun Hee; Ryu, Jong Hoon; Lee, Yong Soo; Kim, Kyeong Man; Cheong, Jae Hoon

    2014-02-01

    Cirsium japonicum (CJ) has been shown to possess antidepressant-like properties. In the present study, we sought to identify which constituent of CJ might be responsible for its antidepressant effects and determine probable mechanism of action. The ethanol extract of CJ was administered to mice then behavioral changes were evaluated in the forced-swimming test (FST) and open-field test (OFT). In addition, its effects on norepinephrine (NE) reuptake and intracellular chloride (Cl(-)) flux were determined, in vitro. The effects of CJ's major constituents (linarin, pectolinarin, chlorogenic acid, luteolin) were also evaluated. CJ showed antidepressant-like effect by significantly reducing immobile behavior of mice in the FST, without increasing locomotor activity in the OFT. CJ had no effect on monoamine (NE) uptake, but it significantly promoted Cl(-) ion influx in human neuroblastoma cells. This CJ-induced Cl(-) influx was significantly blocked by co-administration of the competitive GABAA receptor antagonist, bicuculline. Among the major constituents of the CJ extract, only luteolin produced similar antidepressant-like effect, in vivo, and Cl(-) ion influx, in vitro. Altogether, the present results suggest that the antidepressant-like effect of CJ was most probably induced by its constituent luteolin, mediated through potentiation of the GABAA receptor-Cl(-) ion channel complex. PMID:23925560

  7. Stable expression of cloned rat GABAA receptor subunits in a human kidney cell line.

    PubMed

    Hamilton, B J; Lennon, D J; Im, H K; Im, W B; Seeburg, P H; Carter, D B

    1993-04-30

    A predominant form of the GABAA/benzodiazepine receptor-Cl- channel complex is believed to consist of three different 48-55 kDa subunits (alpha, beta, gamma) with unknown stoichiometry. Plasmids containing the rat GABAA receptor cDNAs coding for alpha 1, beta 2, and gamma 2 were co-transfected, along with a plasmid encoding G418 resistance, into human embryonic kidney cells previously transformed with Adenovirus 5 (HEK-293) [J. Gen. Virol., 36 (1977) 59-72]. Four percent of the G418 resistant colonies were found to express mRNA for all three of the GABAA subunits constitutively. A single cell clone derived from one of the alpha 1 beta 2 gamma 2 expressors has demonstrated stable electrophysiological characteristics over 25 passages. The GABA-activated Cl- current in this cell line is blocked by picrotoxin and bicuculline, and is modulated by a variety of agonist and inverse agonist ligands including diazepam, Ro 154513, zolpidem, and beta-CCE. The cell line has been used successfully over a 12-month period as a screen for novel drugs modulating GABA-mediated polarization of neuronal cells. PMID:7687050

  8. Imidazenil: a low efficacy agonist at alpha1- but high efficacy at alpha5-GABAA receptors fail to show anticonvulsant cross tolerance to diazepam or zolpidem.

    PubMed

    Auta, James; Impagnatiello, Francesco; Kadriu, Bashkim; Guidotti, Alessandro; Costa, Erminio

    2008-08-01

    Whereas advances in the molecular biology of GABA(A) receptor complex using knock-out and knock-in mice have been valuable in unveiling the structure, composition, receptor assembly, and several functions of different GABA(A) receptor subtypes, the mechanism(s) underlying benzodiazepine (BZ) tolerance and withdrawal remain poorly understood. Studies using specific GABA(A) receptor subunit knock-in mice suggest that tolerance to sedative action of diazepam requires long-term activation of alpha1 and alpha5 GABA(A) receptor subunits. We investigated the role of long-term activation of these GABA(A) receptor subunits during anticonvulsant tolerance using high affinity and high intrinsic efficacy ligands for GABA(A) receptors expressing the alpha5 subunit (imidazenil) or alpha1 subunit (zolpidem), and a non-selective BZ recognition site ligand (diazepam). We report here that long-term activation of GABA(A) receptors by zolpidem and diazepam but not by imidazenil elicits anticonvulsant tolerance. Although anticonvulsant cross-tolerance occurs between diazepam and zolpidem, there is no cross-tolerance between imidazenil and diazepam or zolpidem. Furthermore, diazepam or zolpidem long-term treatment decreased the expression of mRNA encoding the alpha1 GABA(A) receptor subunit in prefrontal cortex by 43% and 20% respectively. In addition, diazepam but not zolpidem long-term treatment produced a 30% increase in the expression of the alpha5 GABA(A) receptor subunit mRNA in prefrontal cortex. In contrast, imidazenil which is devoid of anticonvulsant tolerance does not elicit significant changes in the expression of alpha1 or alpha5 GABA(A) receptor subunit. These findings suggest that long-term activation of GABA(A) receptors containing the alpha1 or other subunits but not the alpha5 receptor subunit is essential for the induction of anticonvulsant tolerance.

  9. Imidazenil: A Low Efficacy Agonist at α1- but High Efficacy at α5-GABAA Receptors Fail to Show Anticonvulsant Cross Tolerance to Diazepam or Zolpidem

    PubMed Central

    Auta, James; Impagnatiello, Francesco; Kadriu, Bashkim; Guidotti, Alessandro; Costa, Erminio

    2008-01-01

    SUMMARY Whereas advances in the molecular biology of GABAA receptor complex using knock-out and knock-in mice have been valuable in unveiling the structure, composition, receptor assembly, and several functions of different GABAA receptor subtypes, the mechanism(s) underlying benzodiazepine (BZ) tolerance and withdrawal remain poorly understood. Studies using specific GABAA receptor subunit knock-in mice suggest that tolerance to sedative action of diazepam requires long-term activation of α1 and α5 GABAA receptor subunits. We investigated the role of long-term activation of these GABAA receptor subunits during anticonvulsant tolerance using high affinity and high intrinsic efficacy ligands for GABAA receptors expressing the α5 subunit (imidazenil) or α1 subunit (Zolpidem), and a non-selective BZ recognition site ligand (diazepam). We report here that long-term activation of GABAA receptors by zolpidem and diazepam but not by imidazenil elicits anticonvulsant tolerance. Although anticonvulsant cross-tolerance occurs between diazepam and zolpidem, there is no cross-tolerance between imidazenil and diazepam or zolpidem. Furthermore, diazepam or zolpidem long-term treatment decreased the expression of mRNA encoding the α1 GABAA receptor subunit in prefrontal cortex by 43% and 20% respectively. In addition, diazepam but not zolpidem long-term treatment produced a 30% increase in the expression of the α5 GABAA receptor subunit mRNA in prefrontal cortex. In contrast, imidazenil which is devoid of anticonvulsant tolerance does not elicit significant changes in the expression of α1 or α5 GABAA receptor subunit. These findings suggest that long-term activation of GABAA receptors containing the α1 or other subunits but not the α5 receptor subunit is essential for the induction of anticonvulsant tolerance. PMID:18555494

  10. Axon-to-Glia Interaction Regulates GABAA Receptor Expression in Oligodendrocytes.

    PubMed

    Arellano, Rogelio O; Sánchez-Gómez, María Victoria; Alberdi, Elena; Canedo-Antelo, Manuel; Chara, Juan Carlos; Palomino, Aitor; Pérez-Samartín, Alberto; Matute, Carlos

    2016-01-01

    Myelination requires oligodendrocyte-neuron communication, and both neurotransmitters and contact interactions are essential for this process. Oligodendrocytes are endowed with neurotransmitter receptors whose expression levels and properties may change during myelination. However, only scant information is available about the extent and timing of these changes or how they are regulated by oligodendrocyte-neuron interactions. Here, we used electrophysiology to study the expression of ionotropic GABA, glutamate, and ATP receptors in oligodendrocytes derived from the optic nerve and forebrain cultured either alone or in the presence of dorsal root ganglion neurons. We observed that oligodendrocytes from both regions responded to these transmitters at 1 day in culture. After the first day in culture, however, GABA sensitivity diminished drastically to less than 10%, while that of glutamate and ATP remained constant. In contrast, the GABA response amplitude was sustained and remained stable in oligodendrocytes cocultured with dorsal root ganglion neurons. Immunochemistry and pharmacological properties of the responses indicated that they were mediated by distinctive GABAA receptors and that in coculture with neurons, the oligodendrocytes bearing the receptors were those in direct contact with axons. These results reveal that GABAA receptor regulation in oligodendrocytes is driven by axonal cues and that GABA signaling may play a role in myelination and/or during axon-glia recognition.

  11. Pharmacological plasticity of GABAA receptors at dentate gyrus synapses in a rat model of temporal lobe epilepsy

    PubMed Central

    Leroy, Claire; Poisbeau, Pierrick; Keller, A Florence; Nehlig, Astrid

    2004-01-01

    In the lithium–pilocarpine model (Li-pilocarpine) of temporal lobe epilepsy, GABAA receptor-mediated inhibitory postsynaptic currents (GABAA IPSCs) were recorded in dentate gyrus granule cells (GCs) from adult rat hippocampal slices. The properties of GABAA IPSCs were compared before and after superfusion of modulators in control conditions (Li-saline rats) and in Li-pilocarpine rats 24–48 h and 3–5 months (epileptic rats) after status epilepticus (SE). The mean peak amplitude of GABAA IPSCs increased by about 40% over Li-saline values in GCs 24–48 h after SE and remained higher in epileptic rats. In Li-pilocarpine rats, studied at 24–48 h after SE, diazepam (1 μm) lost 65% of its effectiveness at increasing the half-decay time (T50%) of GABAA miniature IPSCs (mIPSCs). Diazepam had no effects on mIPSC T50% in epileptic rats. The benzodiazepine ligand flumazenil (1 μm), acting as an antagonist in Li-saline rats, exhibited a potent inverse agonistic effect on GABAA mIPSCs of GCs from Li-pilocarpine rats 24–48 h and 3–5 months after SE. The neurosteroid allopregnanolone (100 nm), which considerably prolonged GABAA mIPSCs in Li-saline rats, totally lost its effect in rats studied 24–48 h after SE. However, this decrease in effectiveness was transient and was totally restored in epileptic rats. In addition to the up-regulation in the number of receptors at individual GC synapses, we propose that these ‘epileptic’ GABAA receptors possess benzodiazepine binding sites with altered allosteric properties. The failure of benzodiazepine and neurosteroid to potentiate inhibition early after SE may be a critical factor in the development of epileptogenesis and occurrence of seizures. PMID:15034126

  12. GABAA receptor α4 subunits mediate extrasynaptic inhibition in thalamus and dentate gyrus and the action of gaboxadol

    PubMed Central

    Chandra, D.; Jia, F.; Liang, J.; Peng, Z.; Suryanarayanan, A.; Werner, D. F.; Spigelman, I.; Houser, C. R.; Olsen, R. W.; Harrison, N. L.; Homanics, G. E.

    2006-01-01

    The neurotransmitter GABA mediates the majority of rapid inhibition in the CNS. Inhibition can occur via the conventional mechanism, the transient activation of subsynaptic GABAA receptors (GABAA-Rs), or via continuous activation of high-affinity receptors by low concentrations of ambient GABA, leading to “tonic” inhibition that can control levels of excitability and network activity. The GABAA-R α4 subunit is expressed at high levels in the dentate gyrus and thalamus and is suspected to contribute to extrasynaptic GABAA-R-mediated tonic inhibition. Mice were engineered to lack the α4 subunit by targeted disruption of the Gabra4 gene. α4 Subunit knockout mice are viable, breed normally, and are superficially indistinguishable from WT mice. In electrophysiological recordings, these mice show a lack of tonic inhibition in dentate granule cells and thalamic relay neurons. Behaviorally, knockout mice are insensitive to the ataxic, sedative, and analgesic effects of the novel hypnotic drug, gaboxadol. These data demonstrate that tonic inhibition in dentate granule cells and thalamic relay neurons is mediated by extrasynaptic GABAA-Rs containing the α4 subunit and that gaboxadol achieves its effects via the activation of this GABAA-R subtype. PMID:17005728

  13. GABAA Receptor α1 Subunit Mutation A322D Associated with Autosomal Dominant Juvenile Myoclonic Epilepsy Reduces the Expression and Alters the Composition of Wild Type GABAA Receptors*

    PubMed Central

    Ding, Li; Feng, Hua-Jun; Macdonald, Robert L.; Botzolakis, Emanuel J.; Hu, Ningning; Gallagher, Martin J.

    2010-01-01

    A GABAA receptor (GABAAR) α1 subunit mutation, A322D (AD), causes an autosomal dominant form of juvenile myoclonic epilepsy (ADJME). Previous studies demonstrated that the mutation caused α1(AD) subunit misfolding and rapid degradation, reducing its total and surface expression substantially. Here, we determined the effects of the residual α1(AD) subunit expression on wild type GABAAR expression to determine whether the AD mutation conferred a dominant negative effect. We found that although the α1(AD) subunit did not substitute for wild type α1 subunits on the cell surface, it reduced the surface expression of α1β2γ2 and α3β2γ2 receptors by associating with the wild type subunits within the endoplasmic reticulum and preventing them from trafficking to the cell surface. The α1(AD) subunit reduced surface expression of α3β2γ2 receptors by a greater amount than α1β2γ2 receptors, thus altering cell surface GABAAR composition. When transfected into cultured cortical neurons, the α1(AD) subunit altered the time course of miniature inhibitory postsynaptic current kinetics and reduced miniature inhibitory postsynaptic current amplitudes. These findings demonstrated that, in addition to causing a heterozygous loss of function of α1(AD) subunits, this epilepsy mutation also elicited a modest dominant negative effect that likely shapes the epilepsy phenotype. PMID:20551311

  14. Reversal of neurosteroid effects at alpha4beta2delta GABAA receptors triggers anxiety at puberty.

    PubMed

    Shen, Hui; Gong, Qi Hua; Aoki, Chiye; Yuan, Maoli; Ruderman, Yevgeniy; Dattilo, Michael; Williams, Keith; Smith, Sheryl S

    2007-04-01

    Puberty is characterized by mood swings and anxiety, which are often produced by stress. Here we show that THP (allopregnanolone), a steroid that is released as a result of stress, increases anxiety in pubertal female mice, in contrast to its anxiety-reducing effect in adults. Anxiety is regulated by GABAergic inhibition in limbic circuits. Although this inhibition is increased by THP administration before puberty and in adults, during puberty THP reduces the tonic inhibition of pyramidal cells in hippocampal region CA1, leading to increased excitability. This paradoxical effect of THP results from inhibition of alpha4betadelta GABAA receptors. These receptors are normally expressed at very low levels, but at puberty, their expression is increased in hippocampal area CA1, where they generate outward currents. THP also decreases the outward current at recombinant alpha4beta2delta receptors, and this effect depends on arginine 353 in the alpha4 subunit, a putative site for modulation by Cl-. Therefore, inhibition of alpha4beta2delta GABAA receptors by THP provides a mechanism for the generation of anxiety at puberty.

  15. 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

  16. Pain-related deactivation of medial prefrontal cortical neurons involves mGluR1 and GABAA receptors

    PubMed Central

    Ji, Guangchen

    2011-01-01

    Pain-related hyperactivity in the amygdala leads to deactivation of the medial prefrontal cortex (mPFC) and decision-making deficits. The mechanisms of pain-related inhibition of the mPFC are not yet known. Here, we used extracellular single-unit recordings of prelimbic mPFC neurons to determine the role of GABAA receptors and metabotropic glutamate receptor (mGluR) subtypes, mGluR1 and mGluR5, in pain-related activity changes of mPFC neurons. Background and evoked activity of mPFC neurons decreased after arthritis induction. To determine pain-related changes, the same neuron was recorded continuously before and after induction of arthritis in one knee joint by intra-articular injection of kaolin/carrageenan. Stereotaxic administration of a GABAA receptor antagonist {[R-(R*,S*)]-5-(6,8-dihydro-8-oxofuro[3,4-e]-1,3-benzodioxol-6-yl)-5,6,7,8-tetrahydro-6,6-dimethyl-1,3-dioxolo[4,5-g]isoquinolinium iodide (bicuculline)} into the mPFC by microdialysis reversed pain-related inhibition, whereas offsite injections into the adjacent anterior cingulate cortex had no or opposite effects on prelimbic mPFC neurons. A selective mGluR1/5 agonist [(S)-3,5-dihydroxyphenylglycine (DHPG)] inhibited background and evoked activity under normal conditions through a GABAergic mechanism, because the inhibitory effect was blocked with bicuculline. In the arthritis pain state, DHPG, alone or in the presence of bicuculline, had no effect. Consistent with the involvement of mGluR1 in pain-related inhibition of the mPFC, a selective mGluR1 antagonist [(S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid] reversed the pain-related decrease of background and evoked activity of mPFC neurons in arthritis, whereas a selective mGluR5 antagonist [2-methyl-6-(phenylethynyl)pyridine hydrochloride] had no effect. The mGluR antagonists had no effect under normal conditions. We interpret our data to suggest that pain-related inhibition of mPFC neurons in the arthritis model depends on mGluR1-mediated

  17. Distinct activities of GABA agonists at synaptic- and extrasynaptic-type GABAA receptors.

    PubMed

    Mortensen, Martin; Ebert, Bjarke; Wafford, Keith; Smart, Trevor G

    2010-04-15

    The activation characteristics of synaptic and extrasynaptic GABA(A) receptors are important for shaping the profile of phasic and tonic inhibition in the central nervous system, which will critically impact on the activity of neuronal networks. Here, we study in isolation the activity of three agonists, GABA, muscimol and 4,5,6,7-tetrahydoisoxazolo[5,4-c]pyridin-3(2H)-one (THIP), to further understand the activation profiles of alpha 1 beta 3 gamma 2, alpha 4 beta 3 gamma 2 and alpha 4 beta 3 delta receptors that typify synaptic- and extrasynaptic-type receptors expressed in the hippocampus and thalamus. The agonists display an order of potency that is invariant between the three receptors, which is reliant mostly on the agonist dissociation constant. At delta subunit-containing extrasynaptic-type GABA(A) receptors, both THIP and muscimol additionally exhibited, to different degrees, superagonist behaviour. By comparing whole-cell and single channel currents induced by the agonists, we provide a molecular explanation for their different activation profiles. For THIP at high concentrations, the unusual superagonist behaviour on alpha 4 beta 3 delta receptors is a consequence of its ability to increase the duration of longer channel openings and their frequency, resulting in longer burst durations. By contrast, for muscimol, moderate superagonist behaviour was caused by reduced desensitisation of the extrasynaptic-type receptors. The ability to specifically increase the efficacy of receptor activation, by selected exogenous agonists over that obtained with the natural transmitter, may prove to be of therapeutic benefit under circumstances when synaptic inhibition is compromised or dysfunctional.

  18. In vitro blood-brain barrier permeability predictions for GABAA receptor modulating piperine analogs.

    PubMed

    Eigenmann, Daniela Elisabeth; Dürig, Carmen; Jähne, Evelyn Andrea; Smieško, Martin; Culot, Maxime; Gosselet, Fabien; Cecchelli, Romeo; Helms, Hans Christian Cederberg; Brodin, Birger; Wimmer, Laurin; Mihovilovic, Marko D; Hamburger, Matthias; Oufir, Mouhssin

    2016-06-01

    The alkaloid piperine from black pepper (Piper nigrum L.) and several synthetic piperine analogs were recently identified as positive allosteric modulators of γ-aminobutyric acid type A (GABAA) receptors. In order to reach their target sites of action, these compounds need to enter the brain by crossing the blood-brain barrier (BBB). We here evaluated piperine and five selected analogs (SCT-66, SCT-64, SCT-29, LAU397, and LAU399) regarding their BBB permeability. Data were obtained in three in vitro BBB models, namely a recently established human model with immortalized hBMEC cells, a human brain-like endothelial cells (BLEC) model, and a primary animal (bovine endothelial/rat astrocytes co-culture) model. For each compound, quantitative UHPLC-MS/MS methods in the range of 5.00-500ng/mL in the corresponding matrix were developed, and permeability coefficients in the three BBB models were determined. In vitro predictions from the two human BBB models were in good agreement, while permeability data from the animal model differed to some extent, possibly due to protein binding of the screened compounds. In all three BBB models, piperine and SCT-64 displayed the highest BBB permeation potential. This was corroborated by data from in silico prediction. For the other piperine analogs (SCT-66, SCT-29, LAU397, and LAU399), BBB permeability was low to moderate in the two human BBB models, and moderate to high in the animal BBB model. Efflux ratios (ER) calculated from bidirectional permeability experiments indicated that the compounds were likely not substrates of active efflux transporters. PMID:27018328

  19. Interaction between cyclodextrin and neuronal membrane results in modulation of GABAA receptor conformational transitions

    PubMed Central

    Pytel, Maria; Mercik, Katarzyna; Mozrzymas, Jerzy W

    2006-01-01

    Cyclodextrins (CDs) are nanostructures widely applied in biotechnology and chemistry. Owing to partially hydrophobic character, CDs interact with biological membranes. While the mechanisms of CDs interactions with lipids were widely studied, their effects on proteins are less understood. In the present study we investigated the effects of beta cyclodextrin (βCD) on GABAA receptor (GABAAR) gating. To reliably resolve the kinetics of conformational transitions, currents were elicited by ultrafast gamma-aminobutyric acid (GABA) applications to outside-out patches from rat cultured hippocampal neurons. βCD increased the amplitude of responses to saturating GABA concentration ([GABA]) in a dose-dependent manner and this effect was accompanied by profound alterations in the current kinetics. Current deactivation was slowed down by βCD but this effect was biphasic with a maximum at around 0.5 mM βCD. While the fast deactivation time constant was monotonically slowed down within considered βCD concentration range, the slow component first increased and then, at millimolar βCD concentration, decreased. The rate and extent of desensitization was decreased by βCD in a dose-dependent manner. The analysis of current responses to nonsaturating [GABA] indicated that βCD affected the GABAAR agonist binding site by slowing down the unbinding rate. Modulation of GABAAR desensitization and binding showed different concentration-dependence suggesting different modualtory sites with higher affinity of the latter one. All the βCD effects were fully reversible indicating that cholesterol uptake into βCD was not the primary mechanism. We conclude that βCD is a strong modulator of GABAAR conformational transitions. PMID:16702996

  20. Interaction between cyclodextrin and neuronal membrane results in modulation of GABA(A) receptor conformational transitions.

    PubMed

    Pytel, Maria; Mercik, Katarzyna; Mozrzymas, Jerzy W

    2006-06-01

    Cyclodextrins (CDs) are nanostructures widely applied in biotechnology and chemistry. Owing to partially hydrophobic character, CDs interact with biological membranes. While the mechanisms of CDs interactions with lipids were widely studied, their effects on proteins are less understood. In the present study we investigated the effects of beta cyclodextrin (betaCD) on GABA(A) receptor (GABA(A)R) gating. To reliably resolve the kinetics of conformational transitions, currents were elicited by ultrafast gamma-aminobutyric acid (GABA) applications to outside-out patches from rat cultured hippocampal neurons. betaCD increased the amplitude of responses to saturating GABA concentration ([GABA]) in a dose-dependent manner and this effect was accompanied by profound alterations in the current kinetics. Current deactivation was slowed down by betaCD but this effect was biphasic with a maximum at around 0.5 mM betaCD. While the fast deactivation time constant was monotonically slowed down within considered betaCD concentration range, the slow component first increased and then, at millimolar betaCD concentration, decreased. The rate and extent of desensitization was decreased by betaCD in a dose-dependent manner. The analysis of current responses to nonsaturating [GABA] indicated that betaCD affected the GABA(A)R agonist binding site by slowing down the unbinding rate. Modulation of GABA(A)R desensitization and binding showed different concentration-dependence suggesting different modualtory sites with higher affinity of the latter one. All the betaCD effects were fully reversible indicating that cholesterol uptake into betaCD was not the primary mechanism. We conclude that betaCD is a strong modulator of GABA(A)R conformational transitions.

  1. Inactivation of GABAA receptor is related to heat shock stress response in organism model Caenorhabditis elegans.

    PubMed

    Camargo, Gabriela; Elizalde, Alejandro; Trujillo, Xochitl; Montoya-Pérez, Rocío; Mendoza-Magaña, María Luisa; Hernandez-Chavez, Abel; Hernandez, Leonardo

    2016-09-01

    The mechanisms underlying oxidative stress (OS) resistance are not completely clear. Caenorhabditis elegans (C. elegans) is a good organism model to study OS because it displays stress responses similar to those in mammals. Among these mechanisms, the insulin/IGF-1 signaling (IIS) pathway is thought to affect GABAergic neurotransmission. The aim of this study was to determine the influence of heat shock stress (HS) on GABAergic activity in C. elegans. For this purpose, we tested the effect of exposure to picrotoxin (PTX), gamma-aminobutyric acid (GABA), hydrogen peroxide, and HS on the occurrence of a shrinking response (SR) after nose touch stimulus in N2 (WT) worms. Moreover, the effect of HS on the expression of UNC-49 (GABAA receptor ortholog) in the EG1653 strain and the effect of GABA and PTX exposure on HSP-16.2 expression in the TJ375 strain were analyzed. PTX 1 mM- or H2O2 0.7 mM-exposed worms displayed a SR in about 80 % of trials. GABA exposure did not cause a SR. HS prompted the occurrence of a SR as did PTX 1 mM or H2O2 0.7 mM exposure. In addition, HS increased UNC-49 expression, and PTX augmented HSP-16.2 expression. Thus, the results of the present study suggest that oxidative stress, through either H2O2 exposure or application of heat shock, inactivates the GABAergic system, which subsequently would affect the oxidative stress response, perhaps by enhancing the activity of transcription factors DAF-16 and HSF-1, both regulated by the IIS pathway and related to hsp-16.2 expression.

  2. Inactivation of GABAA receptor is related to heat shock stress response in organism model Caenorhabditis elegans.

    PubMed

    Camargo, Gabriela; Elizalde, Alejandro; Trujillo, Xochitl; Montoya-Pérez, Rocío; Mendoza-Magaña, María Luisa; Hernandez-Chavez, Abel; Hernandez, Leonardo

    2016-09-01

    The mechanisms underlying oxidative stress (OS) resistance are not completely clear. Caenorhabditis elegans (C. elegans) is a good organism model to study OS because it displays stress responses similar to those in mammals. Among these mechanisms, the insulin/IGF-1 signaling (IIS) pathway is thought to affect GABAergic neurotransmission. The aim of this study was to determine the influence of heat shock stress (HS) on GABAergic activity in C. elegans. For this purpose, we tested the effect of exposure to picrotoxin (PTX), gamma-aminobutyric acid (GABA), hydrogen peroxide, and HS on the occurrence of a shrinking response (SR) after nose touch stimulus in N2 (WT) worms. Moreover, the effect of HS on the expression of UNC-49 (GABAA receptor ortholog) in the EG1653 strain and the effect of GABA and PTX exposure on HSP-16.2 expression in the TJ375 strain were analyzed. PTX 1 mM- or H2O2 0.7 mM-exposed worms displayed a SR in about 80 % of trials. GABA exposure did not cause a SR. HS prompted the occurrence of a SR as did PTX 1 mM or H2O2 0.7 mM exposure. In addition, HS increased UNC-49 expression, and PTX augmented HSP-16.2 expression. Thus, the results of the present study suggest that oxidative stress, through either H2O2 exposure or application of heat shock, inactivates the GABAergic system, which subsequently would affect the oxidative stress response, perhaps by enhancing the activity of transcription factors DAF-16 and HSF-1, both regulated by the IIS pathway and related to hsp-16.2 expression. PMID:27230213

  3. Structure of excitatory synapses and GABAA receptor localization at inhibitory synapses are regulated by neuroplastin-65.

    PubMed

    Herrera-Molina, Rodrigo; Sarto-Jackson, Isabella; Montenegro-Venegas, Carolina; Heine, Martin; Smalla, Karl-Heinz; Seidenbecher, Constanze I; Beesley, Philip W; Gundelfinger, Eckart D; Montag, Dirk

    2014-03-28

    Formation, maintenance, and activity of excitatory and inhibitory synapses are essential for neuronal network function. Cell adhesion molecules (CAMs) are crucially involved in these processes. The CAM neuroplastin-65 (Np65) highly expressed during periods of synapse formation and stabilization is present at the pre- and postsynaptic membranes. Np65 can translocate into synapses in response to electrical stimulation and it interacts with subtypes of GABAA receptors in inhibitory synapses. Here, we report that in the murine hippocampus and in hippocampal primary culture, neurons of the CA1 region and the dentate gyrus (DG) express high Np65 levels, whereas expression in CA3 neurons is lower. In neuroplastin-deficient (Np(-/-)) mice the number of excitatory synapses in CA1 and DG, but not CA3 regions is reduced. Notably this picture is mirrored in mature Np(-/-) hippocampal cultures or in mature CA1 and DG wild-type (Np(+/+)) neurons treated with a function-blocking recombinant Np65-Fc extracellular fragment. Although the number of GABAergic synapses was unchanged in Np(-/-) neurons or in mature Np65-Fc-treated Np(+/+) neurons, the ratio of excitatory to inhibitory synapses was significantly lower in Np(-/-) cultures. Furthermore, GABAA receptor composition was altered at inhibitory synapses in Np(-/-) neurons as the α1 to α2 GABAA receptor subunit ratio was increased. Changes of excitatory and inhibitory synaptic function in Np(-/-) neurons were confirmed evaluating the presynaptic release function and using patch clamp recording. These data demonstrate that Np65 is an important regulator of the number and function of synapses in the hippocampus.

  4. Development of GABAA Receptor Subtype-Selective Imidazobenzodiazepines as Novel Asthma Treatments.

    PubMed

    Forkuo, Gloria S; Guthrie, Margaret L; Yuan, Nina Y; Nieman, Amanda N; Kodali, Revathi; Jahan, Rajwana; Stephen, Michael R; Yocum, Gene T; Treven, Marco; Poe, Michael M; Li, Guanguan; Yu, Olivia B; Hartzler, Benjamin D; Zahn, Nicolas M; Ernst, Margot; Emala, Charles W; Stafford, Douglas C; Cook, James M; Arnold, Leggy A

    2016-06-01

    Recent studies have demonstrated that subtype-selective GABAA receptor modulators are able to relax precontracted human airway smooth muscle ex vivo and reduce airway hyper-responsiveness in mice upon aerosol administration. Our goal in this study was to investigate systemic administration of subtype-selective GABAA receptor modulators to alleviate bronchoconstriction in a mouse model of asthma. Expression of GABAA receptor subunits was identified in mouse lungs, and the effects of α4-subunit-selective GABAAR modulators, XHE-III-74EE and its metabolite XHE-III-74A, were investigated in a murine model of asthma (ovalbumin sensitized and challenged BALB/c mice). We observed that chronic treatment with XHE-III-74EE significantly reduced airway hyper-responsiveness. In addition, acute treatment with XHE-III-74A but not XHE-III-74EE decreased airway eosinophilia. Immune suppressive activity was also shown in activated human T-cells with a reduction in IL-2 expression and intracellular calcium concentrations [Ca(2+)]i in the presence of GABA or XHE-III-74A, whereas XHE-III-74EE showed only partial reduction of [Ca(2+)]i and no inhibition of IL-2 secretion. However, both compounds significantly relaxed precontracted tracheal rings ex vivo. Overall, we conclude that the systemic delivery of a α4-subunit-selective GABAAR modulator shows good potential for a novel asthma therapy; however, the pharmacokinetic properties of this class of drug candidates have to be improved to enable better beneficial systemic pharmacodynamic effects.

  5. Studying Cerebellar Circuits by Remote Control of Selected Neuronal Types with GABAA Receptors

    PubMed Central

    Wisden, William; Murray, Andrew J.; McClure, Christina; Wulff, Peer

    2009-01-01

    Although GABAA receptor-mediated inhibition of cerebellar Purkinje cells by molecular layer interneurons (MLIs) has been studied intensely at the cellular level, it has remained unclear how this inhibition regulates cerebellum-dependent behaviour. We have implemented two complementary approaches to investigate the function of the MLI-Purkinje cell synapse on the behavioural level. In the first approach we permanently disrupted inhibitory fast synaptic transmission at the synapse by genetically removing the postsynaptic GABAA receptors from Purkinje cells (PC-Δγ2 mice). We found that chronic disruption of the MLI-Purkinje cell synapse strongly impaired cerebellar learning of the vestibular occular reflex (VOR), presumably by disrupting the temporal patterns of Purkinje cell activity. However, in PC-Δγ2 mice the baseline VOR reflex was only mildly affected; indeed PC-Δγ2 mice show no ataxia or gait abnormalities, suggesting that MLI control of Purkinje cell activity is either not involved in ongoing motor tasks or that the system compensates for its loss. To investigate the latter possibility we developed an alternative genetic technique; we made the MLI-Purkinje cell synapse selectively sensitive to rapid manipulation with the GABAA receptor modulator zolpidem (PC-γ2-swap mice). Minutes after intraperitoneal zolpidem injection, these PC-γ2-swap mice developed severe motor abnormalities, revealing a substantial contribution of the MLI-Purkinje cell synapses to real time motor control. The cell-type selective permanent knockout of synaptic GABAergic input and the fast reversible modulation of GABAergic input at the same synapse illustrate how pursuing both strategies gives a fuller view. PMID:20076763

  6. Local oxytocin tempers anxiety by activating GABAA receptors in the hypothalamic paraventricular nucleus.

    PubMed

    Smith, Adam S; Tabbaa, Manal; Lei, Kelly; Eastham, Patrick; Butler, Michael J; Linton, Latanya; Altshuler, Randy; Liu, Yan; Wang, Zuoxin

    2016-01-01

    Oxytocin (Oxt) is released in various hypothalamic and extrahypothalamic brain areas in response to anxiogenic stimuli to regulate aspects of emotionality and stress coping. We examined the anxiolytic action of Oxt in the hypothalamic paraventricular nucleus (PVN) while appraising if Oxt recruits GABA neurons to inhibit the behavioral, hormonal, and neuronal response to stress in female prairie voles (Microtus ochrogaster). Voles received an injection of Oxt in the PVN either before or after an elevated platform stress to determine a time-course for the effects of Oxt on the hormonal stress response. Subsequently, we evaluated if ante-stress injections of Oxt affected anxiety-like behaviors as well as neuronal activity in the PVN, using real-time in-vivo retrodialysis and immunohistochemistry with c-Fos expression as a biomarker of neural activity. In addition, we exposed voles to Oxt and a GABAA receptor antagonist, concurrently, to evaluate the impact of pharmacological blockade of GABAA receptors on the anxiolytic effects of Oxt. Elevated platform stress amplified anxiety-like behaviors and hypothalamic-pituitary-adrenal (HPA) axis activity-catalyzing corticotrophin-releasing hormone (CRH) neuronal activity and augmenting corticosterone release in circulation. Ante-stress Oxt injections in the PVN blocked these stress effects while promoting PVN GABA activity and release. Post-stress Oxt treatments were ineffective. The anxiolytic effects of Oxt were hindered by concurrent pharmacological blockade of GABAA receptors. Together, our data demonstrate ante-stress treatments of Oxt in the PVN inhibit stress activation of the HPA axis through recruitment of GABAergic neurons, providing insights to the local circuitry and potential therapeutically-relevant mechanisms.

  7. Development of GABAA Receptor Subtype-Selective Imidazobenzodiazepines as Novel Asthma Treatments.

    PubMed

    Forkuo, Gloria S; Guthrie, Margaret L; Yuan, Nina Y; Nieman, Amanda N; Kodali, Revathi; Jahan, Rajwana; Stephen, Michael R; Yocum, Gene T; Treven, Marco; Poe, Michael M; Li, Guanguan; Yu, Olivia B; Hartzler, Benjamin D; Zahn, Nicolas M; Ernst, Margot; Emala, Charles W; Stafford, Douglas C; Cook, James M; Arnold, Leggy A

    2016-06-01

    Recent studies have demonstrated that subtype-selective GABAA receptor modulators are able to relax precontracted human airway smooth muscle ex vivo and reduce airway hyper-responsiveness in mice upon aerosol administration. Our goal in this study was to investigate systemic administration of subtype-selective GABAA receptor modulators to alleviate bronchoconstriction in a mouse model of asthma. Expression of GABAA receptor subunits was identified in mouse lungs, and the effects of α4-subunit-selective GABAAR modulators, XHE-III-74EE and its metabolite XHE-III-74A, were investigated in a murine model of asthma (ovalbumin sensitized and challenged BALB/c mice). We observed that chronic treatment with XHE-III-74EE significantly reduced airway hyper-responsiveness. In addition, acute treatment with XHE-III-74A but not XHE-III-74EE decreased airway eosinophilia. Immune suppressive activity was also shown in activated human T-cells with a reduction in IL-2 expression and intracellular calcium concentrations [Ca(2+)]i in the presence of GABA or XHE-III-74A, whereas XHE-III-74EE showed only partial reduction of [Ca(2+)]i and no inhibition of IL-2 secretion. However, both compounds significantly relaxed precontracted tracheal rings ex vivo. Overall, we conclude that the systemic delivery of a α4-subunit-selective GABAAR modulator shows good potential for a novel asthma therapy; however, the pharmacokinetic properties of this class of drug candidates have to be improved to enable better beneficial systemic pharmacodynamic effects. PMID:27120014

  8. The effect of GABA stimulation on GABAA receptor subunit protein and mRNA expression in rat cultured cerebellar granule cells.

    PubMed Central

    Platt, K. P.; Zwartjes, R. E.; Bristow, D. R.

    1996-01-01

    1. After 8 days in vitro, rat cerebellar granule cells were exposed to 1 mM gamma-aminobutyric acid (GABA) for periods of 1, 2, 4, 6, 8 and 10 days. The effect of the GABA exposure on GABAA receptor alpha 1, alpha 6 and beta 2,3 subunit protein expression and alpha 1 and alpha 6 subunit steady-state mRNA levels, was examined using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. 2. GABA exposure for 2 days decreased alpha 1 (35 +/- 10%, mean +/- s.e.mean), beta 2,3 (21 +/- 9%) and alpha 6 (28 +/- 10%) subunit protein expression compared to control levels. The GABA-mediated reduction in alpha 1 subunit expression after 2 days treatment was abolished in the presence of the GABAA receptor antagonist, Ru 5135 (10 microM). 3. GABA exposure for 8 days increased alpha 1 (26 +/- 10%, mean +/- s.e.mean) and beta 2,3 (56 +/- 23%) subunit protein expression over control levels, whereas alpha 6 subunit protein expression remained below control levels (by 38 +/- 10%). However, after 10 days GABA exposure, alpha 6 subunit protein expression was also increased over control levels by 65 +/- 29% (mean +/- s.e.mean). 4. GABA exposure did not change the alpha 1 or alpha 6 subunit steady-state mRNA levels over and 8 day period, nor did it alter the expression of cyclophilin mRNA over 1-8 days. 5. These results suggest that chronic GABA exposure of rat cerebellar granule cells has a bi-phasic effect on GABAA receptor subunit expression that is independent of changes to mRNA levels. Therefore, the regulation of the GABAA receptor expression by chronic agonist treatment appears to involve post-transcriptional and/or post-translational processes. Images Figure 1 Figure 3 Figure 4 PMID:8968548

  9. Variations in maternal care alter GABA(A) receptor subunit expression in brain regions associated with fear.

    PubMed

    Caldji, Christian; Diorio, Josie; Meaney, Michael J

    2003-11-01

    Maternal care influences the development of stress reactivity in the offspring. These effects are accompanied by changes in corticotropin-releasing factor (CRF) expression in brain regions that regulate responses to stress. However, such effects appear secondary to those involving systems that normally serve to inhibit CRF expression and release. Thus, maternal care over the first week of life alters GABA(A) (gamma-aminobutyric acid)(A) receptor mRNA subunit expression. The adult offspring of mothers that exhibit increased levels of pup licking/grooming and arched back-nursing (high LG-ABN mothers) show increased alpha1 mRNA levels in the medial prefrontal cortex, the hippocampus as well as the basolateral and central regions, of the amygdala and increased gamma2 mRNA in the amygdala. Western blot analyses confirm these effects at the level of protein. In contrast, the offspring of low LG-ABN mothers showed increased levels of alpha3 and alpha4 subunit mRNAs. The results of an adoption study showed that the biological offspring of low LG-ABN mothers fostered shortly after birth to high LG-ABN dams showed the increased levels of both alpha1 and gamma2 mRNA expression in the amygdala in comparison to peers fostered to other low LG-ABN mothers (the reverse was true for the biological offspring of high LG-ABN mothers). These findings are consistent with earlier reports of the effects of maternal care on GABA(A)/benzodiazepine receptor binding and suggest that maternal care can permanently alter the subunit composition of the GABA(A) receptor complex in brain regions that regulate responses to stress.

  10. Neurosteroids shift partial agonist activation of GABA(A) receptor channels from low- to high-efficacy gating patterns.

    PubMed

    Bianchi, Matt T; Macdonald, Robert L

    2003-11-26

    Although GABA activates synaptic (alphabetagamma) GABA(A) receptors with high efficacy, partial agonist activation of alphabetagamma isoforms and GABA activation of the primary extrasynaptic (alphabetadelta) GABA(A) receptors are limited to low-efficacy activity, characterized by minimal desensitization and brief openings. The unusual sensitivity of alphabetadelta receptor channels to neurosteroid modulation prompted investigation of whether this high sensitivity was dependent on the delta subunit or the low-efficacy channel function that it confers. We show that the isoform specificity (alphabetadelta > alphabetagamma) of neurosteroid modulation could be reversed by conditions that reversed isoform-specific activity modes, including the use of beta-alanine to achieve increased efficacy with alphabetadelta receptors and taurine to render alphabetagamma receptors low efficacy. We suggest that neurosteroids preferentially enhance low-efficacy GABA(A) receptor activity independent of subunit composition. Allosteric conversion of partial to full agonism may be a general mechanism for reversibly scaling the efficacy of GABA(A) receptors to endogenous partial agonists.

  11. Systemic availability of guanidinoacetate affects GABAA receptor function and seizure threshold in GAMT deficient mice.

    PubMed

    Schulze, A; Tran, C; Levandovskiy, V; Patel, V; Cortez, M A

    2016-08-01

    Deficiency of guanidinoacetate methyltransferase (GAMT) causes creatine depletion and guanidinoacetate accumulation in brain with the latter deemed to be responsible for the severe seizure disorder seen in affected patients. We studied electrical brain activity and GABAA mediated mechanisms of B6J.Cg-Gamt(tm1Isb) mice. Electrocorticographic (ECoG) monitoring of pharmacological treatments with ornithine (5 % in drinking water for 5-18 days) and/or Picrotoxin (PTX) (a GABAA receptor antagonist) (1.5 mg/kg, I.P.) in Gamt(MUT) and Gamt(WT) groups [n = 3, mean age (SEM) = 6.9 (0.2) weeks]. Mice were fitted with two frontal and two parietal epidural electrodes under ketamine/xylazine anesthesia. Baseline and test recordings were performed for determination of seizure activity over a 2 h period. The ECoG baseline of Gamt(MUT) exhibited an abnormal monotonous cortical rhythm (7-8 Hz) with little variability during awake and sleep states compared to wild type recordings. Ornithine treatment and also PTX administration led to a relative normalization of the Gamt(MUT) ECoG phenotype. Gamt(WT) on PTX exhibited electro-behavioral seizures, whereas the Gamt(MUT) did not have PTX induced seizures at the same PTX dose. Gamt(MUT) treated with both ornithine and PTX did not show electro-behavioral seizures while ornithine elevated the PTX seizure threshold of Gamt(MUT) mice even further. These data demonstrate: (1) that there is expression of electrical seizure activity in this Gamt-deficient transgenic mouse strain, and (2) that the systemic availability of guanidinoacetate affects GABAA receptor function and seizure thresholds. These findings are directly and clinically relevant for patients with a creatine-deficiency syndrome due to genetic defects in GAMT and provide a rational basis for a combined ornithine/picrotoxin therapeutic intervention.

  12. The GABAA receptor complex in hepatic encephalopathy. Autoradiographic evidence for the presence of elevated levels of a benzodiazepine receptor ligand

    SciTech Connect

    Basile, A.S.; Ostrowski, N.L.; Gammal, S.H.; Jones, E.A.; Skolnick, P. )

    1990-02-01

    Autoradiographic analysis was used to examine radioligand binding to benzodiazepine (BZ) and GABAA receptors in the brains of rabbits with hepatic encephalopathy (HE). Thin sections of whole brain from normal rabbits and rabbits with HE were mounted on slides and subdivided into two groups. One group was washed before incubation with radioligand, while the second group was not prewashed. (3H)Flunitrazepam binding to BZ receptors was decreased by 22% to 42% (p less than 0.05) in the cerebral cortex, superior and inferior colliculi, and cerebellum of unwashed sections from rabbits with HE compared to all other groups. The binding of (3H)Ro 15-1788 to unwashed sections from rabbits with HE was reduced by a similar degree (18% to 37%, p less than 0.05) in the cerebral cortex, hippocampus, superior colliculus, and cerebellar cortex. Incubation of sections with the GABA-mimetic muscimol and NaCl produced an additional decrease in (3H)flunitrazepam binding to the cortex and hippocampus (25% to 31%, p less than 0.05) in unwashed HE rabbit brain, but increased radioligand binding (27% to 71%, p less than 0.05) to several regions in control rabbits. No changes in radioligand binding to either GABAA or peripheral benzodiazepine receptors was observed between HE and control rabbit sections. These findings are consistent with previous electrophysiologic and neurochemical observations indicating no significant changes in either the function or density of GABAA or BZ receptors in this model of HE. Further, they indicate that a reversible BZ receptor ligand with agonist properties is present in the brain in HE. This substance may contribute to the enhancement of GABAergic tone observed in this syndrome.

  13. GABAA receptor antagonism ameliorates behavioral and synaptic impairments associated with MeCP2 overexpression.

    PubMed

    Na, Elisa S; Morris, Michael J; Nelson, Erika D; Monteggia, Lisa M

    2014-07-01

    Methyl-CpG-binding protein 2 (MeCP2) is a ubiquitously expressed transcriptional regulator with functional importance in the central nervous system. Loss-of-function mutations in MECP2 results in the neurodevelopmental disorder, Rett syndrome, whereas increased expression levels are associated with the neurological disorder, MECP2 duplication syndrome. Previous characterization of a mouse line overexpressing Mecp2 demonstrated that this model recapitulated key behavioral features of MECP2 duplication syndrome with specific deficits in synaptic plasticity and neurotransmission. Alterations in excitation/inhibition balance have been suggested to underlie neurodevelopmental disorders with recent data suggesting that picrotoxin (PTX), a GABAA receptor antagonist, rescues certain behavioral and synaptic phenotypes in a mouse model of Down syndrome. We therefore examined whether a similar treatment regimen would impact the behavioral and synaptic phenotypes in a mouse model of MECP2 duplication syndrome. We report that chronic treatment with low doses of PTX ameliorates specific behavioral phenotypes, including motor coordination, episodic memory impairments, and synaptic plasticity deficits. These findings suggest that GABAA receptor antagonists may offer a possible therapeutic target for the treatment of MECP2 duplication syndrome.

  14. 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

  15. Alcohol use disorders and current pharmacological therapies: the role of GABA(A) receptors.

    PubMed

    Liang, Jing; Olsen, Richard W

    2014-08-01

    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.

  16. Alcohol use disorders and current pharmacological therapies: the role of GABA(A) receptors.

    PubMed

    Liang, Jing; Olsen, Richard W

    2014-08-01

    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

  17. Molecular mechanisms of benzodiazepine-induced down-regulation of GABAA receptor alpha 1 subunit protein in rat cerebellar granule cells.

    PubMed Central

    Brown, M. J.; Bristow, D. R.

    1996-01-01

    1. Chronic benzodiazepine treatment of rat cerebellar granule cells induced a transient down-regulation of the gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit protein, that was dose-dependent (1 nM-1 microM) and prevented by the benzodiazepine antagonist flumazenil (1 microM). After 2 days of treatment with 1 microM flunitrazepam the alpha 1 subunit protein was reduced by 41% compared to untreated cells, which returned to, and remained at, control cell levels from 4-12 days of treatment. Chronic flunitrazepam treatment did not significantly alter the GABAA receptor alpha 6 subunit protein over the 2-12 day period. 2. GABA treatment for 2 days down-regulates the alpha 1 subunit protein in a dose-dependent (10 microM-1 mM) manner that was prevented by the selective GABAA receptor antagonist bicuculline (10 microM). At 10 microM and 1 mM GABA the reduction in alpha 1 subunit expression compared to controls was 31% and 66%, respectively. 3. The flunitrazepam-induced decrease in alpha 1 subunit protein is independent of GABA, which suggests that it involves a mechanism distinct from the GABA-dependent action of benzodiazepines on GABAA receptor channel activity. 4. Simultaneous treatment with flunitrazepam and GABA did not produce an additive down-regulation of alpha 1 subunit protein, but produced an effect of the same magnitude as that of flunitrazepam alone. This down-regulation induced by the combination of flunitrazepam and GABA was inhibited by flumazenil (78%), but unaffected by bicuculline. 5. The flunitrazepam-induced down-regulation of alpha 1 subunit protein at 2 days was completely reversed by the protein kinase inhibitor staurosporine (0.3 microM). 6. This study has shown that both flunitrazepam and GABA treatment, via their respective binding sites, caused a reduction in the expression of the GABAA receptor alpha 1 subunit protein; an effect mediated through the same neurochemical mechanism. The results also imply that the benzodiazepine effect

  18. The role of GABAA receptors in the acute and chronic effects of ethanol: a decade of progress

    PubMed Central

    Kumar, Sandeep; Porcu, Patrizia; Werner, David F.; Matthews, Douglas B.; Diaz-Granados, Jaime L.; Helfand, Rebecca S.

    2010-01-01

    The past decade has brought many advances in our understanding of GABAA receptor-mediated ethanol action in the central nervous system. We now know that specific GABAA receptor subtypes are sensitive to ethanol at doses attained during social drinking while other subtypes respond to ethanol at doses attained by severe intoxication. Furthermore, ethanol increases GABAergic neurotransmission through indirect effects, including the elevation of endogenous GABAergic neuroactive steroids, presynaptic release of GABA, and dephosphorylation of GABAA receptors promoting increases in GABA sensitivity. Ethanol’s effects on intracellular signaling also influence GABAergic transmission in multiple ways that vary across brain regions and cell types. The effects of chronic ethanol administration are influenced by adaptations in GABAA receptor function, expression, trafficking, and subcellular localization that contribute to ethanol tolerance, dependence, and withdrawal hyperexcitability. Adolescents exhibit altered sensitivity to ethanol actions, the tendency for higher drinking and longer lasting GABAergic adaptations to chronic ethanol administration. The elucidation of the mechanisms that underlie adaptations to ethanol exposure are leading to a better understanding of the regulation of inhibitory transmission and new targets for therapies to support recovery from ethanol withdrawal and alcoholism. PMID:19455309

  19. The diversity of GABA(A) receptor subunit distribution in the normal and Huntington's disease human brain.

    PubMed

    Waldvogel, H J; Faull, R L M

    2015-01-01

    GABA(A) receptors are assembled into pentameric receptor complexes from a total of 19 different subunits derived from a variety of different subunit classes (α1-6, β1-3, γ1-3, δ, ɛ, θ, and π) which surround a central chloride ion channel. GABA(A) receptor complexes are distributed heterogeneously throughout the brain and spinal cord and are activated by the extensive GABAergic inhibitory system. In this chapter, we describe the heterogeneous distribution of six of the most widely distributed subunits (α1, α2, α3, β2,3, and γ2) throughout the human basal ganglia. This review describes the studies we have carried out on the normal and Huntington's disease human basal ganglia using autoradiographic labeling and immunohistochemistry in the human basal ganglia. GABA(A) receptors are known to react to changing conditions in the brain in neurological disorders, especially in Huntington's disease and display a high degree of plasticity which is thought to compensate for loss of function caused by disease. In Huntington's disease, the variable loss of GABAergic medium spiny striatopallidal projection neurons is associated with a loss of GABA(A) receptor subunits in the striosome and/or the matrix compartments of the striatum. By contrast in the globus pallidus, a loss of the GABAergic striatal projection neurons results in a dramatic upregulation of subunits on the large postsynaptic pallidal neurons; this is thought to be a compensatory plastic mechanism resulting from the loss of striatal GABAergic input. Most interestingly, our studies have revealed that the subventricular zone overlying the caudate nucleus contains a variety of proliferating progenitor stem cells that possess a heterogeneity of GABA(A) receptor subunits which may play a role in human brain repair mechanisms.

  20. Zolpidem and eszopiclone prime α1β2γ2 GABAA receptors for longer duration of activity

    PubMed Central

    Dixon, Christine L; Harrison, Neil L; Lynch, Joseph W; Keramidas, Angelo

    2015-01-01

    Background and Purpose GABAA receptors mediate neuronal inhibition in the brain. They are the primary targets for benzodiazepines, which are widely used to treat neurological disorders including anxiety, epilepsy and insomnia. The mechanism by which benzodiazepines enhance GABAA receptor activity has been extensively studied, but there is little mechanistic information on how non-benzodiazepine drugs that bind to the same site exert their effects. Eszopiclone and zolpidem are two non-benzodiazepine drugs for which no mechanism of action has yet been proposed, despite their clinical importance as sleeping aids. Here we investigate how both drugs enhance the activity of α1β2γ2 GABAA receptors. Experimental Approach We used rapid ligand application onto macropatches and single-channel kinetic analysis to assess rates of current deactivation. We also studied synaptic currents in primary neuronal cultures and in heterosynapses, whereby native GABAergic nerve terminals form synapses with HEK293 cells expressing α1β2γ2 GABAA receptors. Drug binding and modulation was quantified with the aid of an activation mechanism. Key Results At the single-channel level, the drugs prolonged the duration of receptor activation, with similar KD values of ∼80 nM. Channel activation was prolonged primarily by increasing the equilibrium constant between two connected shut states that precede channel opening. Conclusions and Implications As the derived mechanism successfully simulated the effects of eszopiclone and zolpidem on ensemble currents, we propose it as the definitive mechanism accounting for the effects of both drugs. Importantly, eszopiclone and zolpidem enhanced GABAA receptor currents via a mechanism that differs from that proposed for benzodiazepines. PMID:25817320

  1. Functional sites involved in modulation of the GABAA receptor channel by the intravenous anesthetics propofol, etomidate and pentobarbital.

    PubMed

    Maldifassi, Maria C; Baur, Roland; Sigel, Erwin

    2016-06-01

    GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs. Among the many modulatory compounds are also the intravenous anesthetics propofol and etomidate, and barbiturates. The mechanism of receptor modulation by these compounds is of mayor relevance. The site of action of these compounds has been located to subunit interfaces in the intra-membrane region of the receptor. In α1β2γ2 GABAA receptors there are five such interfaces, two β+/α- and one each of α+/β-, α+/γ- and γ+/β- subunit interfaces. We have used reporter mutations located in the second trans-membrane region in different subunits to probe the effects of changes at these subunit interfaces on modulation by propofol, etomidate and pentobarbital. We provide evidence for the fact that each of these compounds either modulates through a different set of subunit interfaces or through the same set of subunit interfaces to a different degree. As a GABAA receptor pentamer harbors two β+/α- subunit interfaces, we used concatenated receptors to dissect the contribution of individual interfaces and show that only one of these interfaces is important for receptor modulation by etomidate. PMID:26767954

  2. 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

  3. Protons inhibit Cl- conductance by direct or allosteric interaction with the GABA-binding site in the rat recombinant alpha1beta2gamma2L and alpha1beta2 GABAA receptor.

    PubMed

    Wang, Ming-De; Rahman, Mozibur; Zhu, Di

    2005-12-28

    Functional roles of external pH on the Cl- conductance were examined on Xenopus oocytes expressing rat recombinant alpha1beta2gamma2L and alpha1beta2 GABAA receptors. Acidic pH inhibited GABA-response in a reversible and concentration-dependent manner, significantly increasing the EC50 without appreciably changing the slope or maximal currents induced by GABA in the alpha1beta2gamma2L and alpha1beta2 receptors. In contrast, protonation did not influence the pentobarbital-gated currents in the alpha1beta2gamma2L receptors, suggesting that protons do not modulate channel activity by directly affecting the channel gating process. Protons competitively inhibited the bicuculline-induced antagonism on GABA in the alpha1beta2gamma2L receptors. The data support the hypothesis that protons inhibit GABAA receptor function by direct or allosteric interaction with the GABA-binding site.

  4. The neuroplastic index p-FADD/FADD and phosphoprotein PEA-15, interacting at GABAA receptor, are upregulated in brain cortex during midazolam-induced hypnosis in mice.

    PubMed

    Álvaro-Bartolomé, María; García-Sevilla, Jesús A

    2015-11-01

    Fas-associated death domain (FADD) adaptor is involved in the signaling of metabotropic G protein-coupled receptors, whose agonists stimulate its phosphoryaltion (p) increasing p-FADD/FADD ratio in brain. Whether FADD might also participate in the activation of dissimilar receptors such as the ligand-gated ion channels is not known. This study investigated the role of FADD and phosphoprotein-enriched in astrocytes of 15 kDa (PEA-15, a FADD partner) in the activation of γ-aminobutyric acid-A (GABAA) receptor, which mediates the hypnotic effect of midazolam. The main findings revealed that during the time course of midazolam (60 mg/kg)-induced hypnosis in mice (about 2 h) p-FADD (and p-FADD/FADD ratio) as well as p-PEA (and its phosphorylating Akt1 kinase) were markedly increased (36-80%) in brain cortex, and these effects were partially (only p-FADD) or fully prevented by flumazenil (a neutral allosteric ligand) and FG 7142 (a partial negative allosteric ligand) acting at GABAA receptors. The upregulation of cortical p-FADD/FADD was exclusively observed in the nucleus (up to 2.8-fold), where the transciption factor NF-κB was also increased (up to 46%), and that of p-PEA/p-Akt1 only in the cytosol (up to 53%), suggesting that p-FADD/p-PEA/p-Akt1 are involved in sleep-induced neuroplasticity. Repeated treatment with midazolam (60 mg/kg, 4 days) induced behavioral (prolonged sleep latency and reduced sleeping time) and neurochemical (reduced p-FADD/p-PEA contents) tolerance. These findings indicated that p-FADD/p-PEA are novel molecules in GABAA receptor signaling and that cortical p-PEA and p-FADD, working in tandem, are involved in the complex molecular processes leading to the hypnotic effect of midazolam in mice.

  5. The neuroplastic index p-FADD/FADD and phosphoprotein PEA-15, interacting at GABAA receptor, are upregulated in brain cortex during midazolam-induced hypnosis in mice.

    PubMed

    Álvaro-Bartolomé, María; García-Sevilla, Jesús A

    2015-11-01

    Fas-associated death domain (FADD) adaptor is involved in the signaling of metabotropic G protein-coupled receptors, whose agonists stimulate its phosphoryaltion (p) increasing p-FADD/FADD ratio in brain. Whether FADD might also participate in the activation of dissimilar receptors such as the ligand-gated ion channels is not known. This study investigated the role of FADD and phosphoprotein-enriched in astrocytes of 15 kDa (PEA-15, a FADD partner) in the activation of γ-aminobutyric acid-A (GABAA) receptor, which mediates the hypnotic effect of midazolam. The main findings revealed that during the time course of midazolam (60 mg/kg)-induced hypnosis in mice (about 2 h) p-FADD (and p-FADD/FADD ratio) as well as p-PEA (and its phosphorylating Akt1 kinase) were markedly increased (36-80%) in brain cortex, and these effects were partially (only p-FADD) or fully prevented by flumazenil (a neutral allosteric ligand) and FG 7142 (a partial negative allosteric ligand) acting at GABAA receptors. The upregulation of cortical p-FADD/FADD was exclusively observed in the nucleus (up to 2.8-fold), where the transciption factor NF-κB was also increased (up to 46%), and that of p-PEA/p-Akt1 only in the cytosol (up to 53%), suggesting that p-FADD/p-PEA/p-Akt1 are involved in sleep-induced neuroplasticity. Repeated treatment with midazolam (60 mg/kg, 4 days) induced behavioral (prolonged sleep latency and reduced sleeping time) and neurochemical (reduced p-FADD/p-PEA contents) tolerance. These findings indicated that p-FADD/p-PEA are novel molecules in GABAA receptor signaling and that cortical p-PEA and p-FADD, working in tandem, are involved in the complex molecular processes leading to the hypnotic effect of midazolam in mice. PMID:26282360

  6. Would right atrial stretch inhibit sodium intake following GABAA receptor activation in the lateral parabrachial nucleus?

    PubMed

    Shimoura, Caroline Gusson; Barbosa, Silas Pereira; Menani, Jose Vanderlei; De Gobbi, Juliana Irani Fratucci

    2013-10-11

    The knowledge of the mechanisms underlying circulating volume control may be achieved by stretching a balloon placed at the junction of the superior vena cava-right atrial junction (SVC-RAJ). We investigated whether the inflation of a balloon at the SVC-RAJ inhibits the intake of 0.3M NaCl induced by GABAA receptor activation in the lateral parabrachial nucleus (LPBN) in euhydrated and satiated rats. Male Wistar rats (280-300 g) with bilateral stainless steel LPBN cannulae and balloons implanted at the SVC-RAJ were used. Bilateral injections of the GABAA receptor agonist muscimol (0.5 ηmol/0.2l) in the LPBN with deflated balloons increased intake of 0.3M NaCl (30.1 ± 3.9 vs. saline: 2.2 ± 0.7)ml/210 min, n=8) and water (17.7 ± 1.9 vs. saline: 2.9 ± 0.5 ml/210 min). Conversely, 0.3M NaCl (27.8 ± 2.1 ml/210 min) and water (22.8 ± 2.3 ml/210 min) intake were not affected in rats with inflated balloons at the SVC-RAJ. The results show that sodium and water intake induced by muscimol injected into the LPBN was not affected by balloon inflation at the SVC-RAJ. We suggest that the blockade of LPBN neuronal activity with muscimol injections impairs inhibitory mechanisms activated by signals from cardiopulmonary volume receptors determined by balloon inflation.

  7. Exploring the molecular basis of neurosteroid binding to the β3 homopentameric GABAA receptor.

    PubMed

    Alvarez, Lautaro D; Estrin, Darío A

    2015-11-01

    Neurosteroids are the principal endogenous modulators of GABA(A) receptors (GABA(A)Rs), which are pentameric membrane-bound proteins that regulate the passage of chloride ions from the extracellular to the intracellular compartment. As consequence of their ability to modify inhibitory functions in the brain, neurosteroids have high physiological and clinical importance and may act as anesthetic, anticonvulsant and anxiolytic drugs. Despite their relevance, essential issues regarding neurosteroid action on GABA(A)Rs are still unsettled. In particular, residues taking part of the steroid recognition are not definitely identified. Taking as starting point the first reported crystal structure of a human GABAA receptor (a β3 homopentamer), we have explored through a combination of computational methods (a cavity-detection algorithm, docking and molecular dynamics simulations) the binding mode of two structurally different representative neurosteroids, pregnanolone and allopregnanolone. We have identified a neurosteroid binding site between the TM3 of one subunit and TM1 and TM4 of the adjacent subunit that is consistent with the set of experimental data reported for the action of neurosteroids on β3 homopentamers. These sites are able to properly accommodate both overall torsioned and flat steroidal structures and they specifically recognize the 3-OH group, explaining the requirement of a 3α-configuration for the activity. We believe that this work provides for first time convincing information about the molecular interaction between neurosteroids and a GABA(A)R. This information largely increases our understanding of this fundamental ligand-receptor system. PMID:26223009

  8. Ethanol Regulation of Synaptic GABAA α4 Receptors Is Prevented by Protein Kinase A Activation.

    PubMed

    Carlson, Stephen L; Bohnsack, John Peyton; Morrow, A Leslie

    2016-04-01

    Ethanol alters GABAA receptor trafficking and function through activation of protein kinases, and these changes may underlie ethanol dependence and withdrawal. In this study, we used subsynaptic fraction techniques and patch-clamp electrophysiology to investigate the biochemical and functional effects of protein kinase A (PKA) and protein kinase C (PKC) activation by ethanol on synaptic GABAA α4 receptors, a key target of ethanol-induced changes. Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or kinase modulators for 4 hours, a paradigm that recapitulates GABAergic changes found after chronic ethanol exposure in vivo. PKA activation by forskolin or rolipram during ethanol exposure prevented increases in P2 fraction α4 subunit abundance, whereas inhibiting PKA had no effect. Similarly, in the synaptic fraction, activation of PKA by rolipram in the presence of ethanol prevented the increase in synaptic α4 subunit abundance, whereas inhibiting PKA in the presence of ethanol was ineffective. Conversely, PKC inhibition in the presence of ethanol prevented the ethanol-induced increases in synaptic α4 subunit abundance. Finally, we found that either activating PKA or inhibiting PKC in the presence of ethanol prevented the ethanol-induced decrease in GABA miniature inhibitory postsynaptic current decay τ1, whereas inhibiting PKA had no effect. We conclude that PKA and PKC have opposing effects in the regulation of synaptic α4 receptors, with PKA activation negatively modulating, and PKC activation positively modulating, synaptic α4 subunit abundance and function. These results suggest potential targets for restoring normal GABAergic functioning in the treatment of alcohol use disorders.

  9. GABAA Receptor Binding Assays of Standardized Leonurus cardiaca and Leonurus japonicus Extracts as Well as Their Isolated Constituents.

    PubMed

    Rauwald, Hans Wilhelm; Savtschenko, Alex; Merten, Alexander; Rusch, Christian; Appel, Kurt; Kuchta, Kenny

    2015-08-01

    A main traditional use of European Leonurus cardiaca and East Asian Leonurus japonicus is in the treatment of neurological disorders such as anxiety, depression, nervousness, and as a sedative for insomnia. However, their mechanism of action is still under discussion. As anxiety and depressive disorders are increasingly being recognized as connected to dysfunctions of the gamma-aminobutyric acid system, the in vitro effects of standardized L. cardiaca and L japonicus extracts as well as five of their isolated constituents, namely, the labdane-type isoleosibirin, the novel iridoid 7R-chloro-6-desoxy-harpagide, the phenylethanoid lavandulifolioside, and the N-containing compounds stachydrine and leonurine, on this type of neuronal receptor were investigated for the first time. Extracts of L. cardiaca and L. japonicus, characterized by reversed-phase high-performance liquid chromatography determination, as well as their above named isolated, possible active constituents of different chemical nature were tested in several receptor binding assays at rat GABAA receptors using [(3)H]-SR95 531 and [(3)H]-Ro-15-1788 (flumazenil)/diazepam control. The L. cardiaca and L. japonicus extracts as well as leonurine inhibited the concentration-dependent binding of [(3)H]-SR95 531 to the gamma-aminobutyric acid site of the gamma-aminobutyric acid type A receptor with a high binding affinity: IC50s 21 µg/ml, 46 µg/ml, and 15 µg/ml, respectively. In contrast, binding to the benzodiazepine site of the rat gamma-aminobutyric acid type A receptor had a 15 to 30 times lower binding affinity than to the gamma-aminobutyric acid site. The presented experiments provide hints that the neurological mechanism of action of L. cardiaca and L. japonicus may essentially be based on their interaction to the gamma-aminobutyric acid site of the gamma-aminobutyric acid type A receptor, while the benzodiazepine site most probably does not contribute to this effect. In the case of L

  10. Developmental expression of cerebellar GABAA-receptor subunit mRNAs. Nature versus nurture.

    PubMed

    Siegel, R E

    1998-01-01

    Recent studies have demonstrated that many of the mRNAs encoding GABAA-receptor subunits in the cerebellum exhibit distinct temporal profiles of expression. The levels of six of these subunit transcripts increase severalfold in the second week of postnatal ontogeny. Findings from a variety of experimental systems suggest that the onset and increases in subunit mRNA expression are mediated by the interaction of genetic and epigenetic programs. The initiation of subunit mRNA expression occurs relatively early in cellular maturation and may be directed by intrinsic mechanisms. However, the levels of expression attained in adult animals may be controlled by extrinsic signals received by neurons during the postnatal maturation process. PMID:9777637

  11. GABAA receptor cysteinyl mutants and the ginkgo terpenoid lactones bilobalide and ginkgolides.

    PubMed

    Ng, Chiu Chin; Duke, Rujee K; Hinton, Tina; Johnston, Graham A R

    2016-04-15

    The terpenoid lactones from Ginkgo biloba, bilobalide and ginkgolides, have been shown to act as negative modulators at α1β2γ2L GABAA receptors. They have structural features similar to those of the chloride channel blocker picrotoxinin. Unlike picrotoxinin, however they are not known to produce convulsant effects. Using two-electrode voltage clamp electrophysiology, this study compared the effect of mutation of 2', 6' and 15' pore facing M2 domain residues to cysteine on the action of picrotoxinin, bilobalide and ginkgolides at α1β2γ2L GABAA receptors expressed in Xenopus oocytes. Picrotoxinin was affected by mutation differently from the ginkgo terpenoid lactones. Although some of these compounds were affected by the mutation at same position and/or subunit, the changes in their potency were found to be dissimilar. The results suggest that the intracellular pore binding site for picrotoxinin, bilobalide, ginkgolide A, ginkgolide B and ginkgolide C is comprised of 2'β-6'β6'γ, 2'α2'β-6'α6'β, 2'α2'β2'γ-6'β6'γ, 2'α, 2'β2'γ-6'β and 2'α2'β, respectively. Unlike bilobalide and ginkgolides, the inhibitory action of picrotoxinin was not affected by mutations at 15' position. It is proposed that 15'α15'β, 15'β, 15'α15'β and 15'α15'β15'γ forms an extracellular pore binding site for bilobalide, ginkgolide A, ginkgolide B and ginkgolide C, respectively. The lack of convulsant effects of bilobalide, and ginkgolide A and B may be associated in part with their different binding locations within the chloride channel. PMID:26953225

  12. Membrane voltage modulates the GABA(A) receptor gating in cultured rat hippocampal neurons.

    PubMed

    Pytel, Maria; Mercik, Katarzyna; Mozrzymas, Jerzy W

    2006-02-01

    The kinetics of GABAergic currents in neurons is known to be modulated by the membrane voltage but the underlying mechanisms have not been fully explored. In particular, the impact of membrane potential on the GABA(A) receptor gating has not been elucidated. In the present study, the effect of membrane voltage on current responses elicited by ultrafast GABA applications was studied in cultured hippocampal neurons. The current to voltage relationship (I-V) for responses to saturating [GABA] (10 mM) showed an inward rectification (slope conductance at positive voltages was 0.62 +/- 0.05 of that at negative potentials). On the contrary, I-V for currents evoked by low [GABA] (1 microM) showed an outward rectification. The onset of currents elicited by saturating [GABA] was significantly accelerated at positive potentials. Analysis of currents evoked by prolonged applications of saturating [GABA] revealed that positive voltages significantly increased the rate and extent of desensitization. The onsets of current responses to non-saturating [GABA] were significantly accelerated at positive voltages indicating an enhancement of the binding rate. However, at low [GABA] at which the onset rate is expected to approach an asymptote set by opening/closing and unbinding rates, no significant modification of current onset by voltage was observed. Quantitative analysis based on model simulations indicated that the major effect of membrane depolarization was to increase the rates of binding, desensitization and of opening as well as to slightly reduce the rate of exit from desensitization. In conclusion, we provide evidence that membrane voltage affects the GABA(A) receptor microscopic gating.

  13. Characterization of Inhibitory GABA-A Receptor Activation during Spreading Depolarization in Brain Slice

    PubMed Central

    Aiba, Isamu; Shuttleworth, C. William

    2014-01-01

    Spreading depolarization (SD) is a slowly propagating wave of near complete depolarizations of neurons and glia. Previous studies have reported large GABA releases during SD, but there is limited understanding of how GABA release and receptor activation are regulated and influence the propagating SD wavefront, as well as an excitatory phase immediately following the passage of SD. The present study characterized GABA-A type receptor (GABAAR) currents during SD generated by KCl microinjection in acute hippocampal slices from adult mice. Spontaneous GABAAR-mediated currents (sIPSCs) were initially enhanced, and were followed by a large outward current at the wavefront. sIPSC were then transiently supressed during the late SD phase, resulting in a significant reduction of the sIPSC/sEPSC ratio. The large outward current generated during SD was eliminated by the GABAAR antagonist gabazine, but the channel potentiator/agonist propofol failed to potentiate the current, likely because of a ceiling effect. Extracellular Cl− decreases recorded during SD were reduced by the antagonist but were not increased by the potentiator. Together with effects of GABAAR modulators on SD propagation rate, these results demonstrate a significant inhibitory role of the initial GABAAR activation and suggest that intracellular Cl− loading is insufficient to generate excitatory GABAAR responses during SD propagation. These results provide a mechanistic explanation for facilitating effects of GABAAR antagonists, and the lack of inhibitory effect of GABAAR potentiators on SD propagation. In addition, selective suppression of GABA transmission in the late SD period and the lack of effect of GABAA modulators on the duration of SD suggests that GABA modulation may not be effective approach to protect neurons during the vulnerable phase of SD. PMID:25338191

  14. Involvement of AMPA/kainate and GABAA receptors in topiramate neuroprotective effects against methylphenidate abuse sequels involving oxidative stress and inflammation in rat isolated hippocampus.

    PubMed

    Motaghinejad, Majid; Motevalian, Manijeh

    2016-08-01

    Abuses of methylphenidate (MPH) as psychostimulant cause neural damage of brain cells. Neuroprotective properties of topiramate (TPM) have been indicated in several studies but its exact mechanism of action remains unclear. The current study evaluates protective role of various doses of TPM and its mechanism of action in MPH induced oxidative stress and inflammation. The neuroprotective effects of various doses of TPM against MPH induced oxidative stress and inflammation were evaluated and then the action of TPM was studied in presence of domoic acid (DOM), as AMPA/kainate receptor agonist and bicuculline (BIC) as GABAA receptor antagonist, in isolated rat hippocampus. Open Field Test (OFT) was used to investigate motor activity changes. Oxidative, antioxidant and inflammatory factors were measured in isolated hippocampus. TPM (70 and 100mg/kg) decreased MPH induced motor activity disturbances and inhibit MPH induced oxidative stress and inflammation. On the other hand pretreatment of animals with DOM or BIC, inhibit this effect of TPM and potentiate MPH induced motor activity disturbances and increased lipid peroxidation, mitochondrial oxidized form of glutathione (GSSG) level, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in isolated hippocampal cells and decreased reduced form of glutathione (GSH) level, superoxide dismutase, glutathione peroxidase and glutathione reductase activity. It seems that TPM can protect cells of hippocampus from oxidative stress and neuroinflammation and it could be partly by activation of GABAA receptor and inhibition of AMPA/kainite receptor.

  15. Involvement of AMPA/kainate and GABAA receptors in topiramate neuroprotective effects against methylphenidate abuse sequels involving oxidative stress and inflammation in rat isolated hippocampus.

    PubMed

    Motaghinejad, Majid; Motevalian, Manijeh

    2016-08-01

    Abuses of methylphenidate (MPH) as psychostimulant cause neural damage of brain cells. Neuroprotective properties of topiramate (TPM) have been indicated in several studies but its exact mechanism of action remains unclear. The current study evaluates protective role of various doses of TPM and its mechanism of action in MPH induced oxidative stress and inflammation. The neuroprotective effects of various doses of TPM against MPH induced oxidative stress and inflammation were evaluated and then the action of TPM was studied in presence of domoic acid (DOM), as AMPA/kainate receptor agonist and bicuculline (BIC) as GABAA receptor antagonist, in isolated rat hippocampus. Open Field Test (OFT) was used to investigate motor activity changes. Oxidative, antioxidant and inflammatory factors were measured in isolated hippocampus. TPM (70 and 100mg/kg) decreased MPH induced motor activity disturbances and inhibit MPH induced oxidative stress and inflammation. On the other hand pretreatment of animals with DOM or BIC, inhibit this effect of TPM and potentiate MPH induced motor activity disturbances and increased lipid peroxidation, mitochondrial oxidized form of glutathione (GSSG) level, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in isolated hippocampal cells and decreased reduced form of glutathione (GSH) level, superoxide dismutase, glutathione peroxidase and glutathione reductase activity. It seems that TPM can protect cells of hippocampus from oxidative stress and neuroinflammation and it could be partly by activation of GABAA receptor and inhibition of AMPA/kainite receptor. PMID:27105819

  16. Cytoplasmic domain of δ subunit is important for the extra-synaptic targeting of GABAA receptor subtypes.

    PubMed

    Arslan, Ayla; von Engelhardt, Jakob; Wisden, William

    2014-12-01

    GABA(A) receptors (GABA(A)Rs) are hetero-pentameric chloride channels and the primary sites for fast synaptic inhibition. We have expressed recombinant γ2 and δ subunits of GABA(A)Rs in cultured hippocampal neurons to analyze the membrane targeting of synaptic and extra-synaptic GABA(A)Rs, a phenomenon not well understood. Our data demonstrate that the synaptic targeting of γ2-containing GABA(A)Rs (γ2-GABA(A)Rs) does not depend on the cytoplasmic loop of γ2 subunit, in parallel with previous findings, showing that the synaptic localization of γ2-GABA(A)Rs requires the TM4 domain of γ2 rather than the large cytoplasmic loop. On the other hand, we showed here that the extrasynaptic targeting of the δ-containing GABA(A)Rs (δ-GABA(A)Rs) depends on the cytoplasmic loop of δ subunit via an active or a passive mechanism. We also show that the amino acid sequences of δ loop is highly conserved across the whole span of vertebrate evolution suggesting an active role of δ loop in extra-synaptic targeting of corresponding receptor subtypes. PMID:25233879

  17. Cytoplasmic domain of δ subunit is important for the extra-synaptic targeting of GABAA receptor subtypes.

    PubMed

    Arslan, Ayla; von Engelhardt, Jakob; Wisden, William

    2014-12-01

    GABA(A) receptors (GABA(A)Rs) are hetero-pentameric chloride channels and the primary sites for fast synaptic inhibition. We have expressed recombinant γ2 and δ subunits of GABA(A)Rs in cultured hippocampal neurons to analyze the membrane targeting of synaptic and extra-synaptic GABA(A)Rs, a phenomenon not well understood. Our data demonstrate that the synaptic targeting of γ2-containing GABA(A)Rs (γ2-GABA(A)Rs) does not depend on the cytoplasmic loop of γ2 subunit, in parallel with previous findings, showing that the synaptic localization of γ2-GABA(A)Rs requires the TM4 domain of γ2 rather than the large cytoplasmic loop. On the other hand, we showed here that the extrasynaptic targeting of the δ-containing GABA(A)Rs (δ-GABA(A)Rs) depends on the cytoplasmic loop of δ subunit via an active or a passive mechanism. We also show that the amino acid sequences of δ loop is highly conserved across the whole span of vertebrate evolution suggesting an active role of δ loop in extra-synaptic targeting of corresponding receptor subtypes.

  18. Effects of the chlorotriazine herbicide, cyanazine, on GABA(A) receptors in cortical tissue from rat brain.

    PubMed

    Shafer, T J; Ward, T R; Meacham, C A; Cooper, R L

    1999-12-20

    Chlorotriazine herbicides disrupt luteinizing hormone (LH) release in female rats following in vivo exposure. Although the mechanism of action is unknown, significant evidence suggests that inhibition of LH release by chlorotriazines may be mediated by effects in the central nervous system. GABA(A) receptors are important for neuronal regulation of gonadotropin releasing hormone and LH release. The ability of chlorotriazine herbicides to interact with GABA(A) receptors was examined by measuring their effects on [3H]muscimol, [3H]Ro15-4513 and [35S]tert-butylbicyclophosphorothionate (TBPS) binding to rat cortical membranes. Cyanazine (1-400 microM) inhibited [3H]Ro15-4513 binding with an IC50 of approximately 105 microM (n=4). Atrazine (1-400 microM) also inhibited [3H]Ro15-4513 binding, but was less potent than cyanazine (IC50 = 305 microM). However, the chlorotriazine metabolites diaminochlorotriazine, 2-amino-4-chloro-6-ethylamino-s-triazine and 2-amino-4-chloro-6-isopropylamino-s-triazine were without significant effect on [3H]Ro15-4513 binding. Cyanazine and the other chlorotriazines were without effect on [3H]muscimol or [35S]TBPS binding. To examine whether cyanazine altered GABA(A) receptor function, GABA-stimulated 36Cl- flux into synaptoneurosomes was examined. Cyanazine (50-100 microM) alone did not significantly decrease GABA-stimulated 36Cl- flux. Diazepam (10 microM) and pentobarbital (100 microM) potentiated GABA-stimulated 36Cl- flux to 126 and 166% of control, respectively. At concentrations of 50 and 100 microM, cyanazine decreased potentiation by diazepam to 112 and 97% of control, respectively, and decreased potentiation by pentobarbital to 158 and 137% of control (n = 6). Interestingly, at lower concentrations (5 microM), cyanazine shifted the EC50 for GABA-stimulated 36Cl- flux into synaptoneurosomes from 28.9 to 19.4 microM, respectively (n = 5). These results suggest that cyanazine modulates benzodiazepine, but not the muscimol (GABA receptor

  19. Regulation of dopamine D2 receptor-mediated extracellular signal-regulated kinase signaling and spine formation by GABAA receptors in hippocampal neurons.

    PubMed

    Yoon, Dong-Hoon; Yoon, Sehyoun; Kim, Donghoon; Kim, Hyun; Baik, Ja-Hyun

    2015-01-23

    Dopamine (DA) signaling via DA receptors is known to control hippocampal activity that contributes to learning, memory, and synaptic plasticity. In primary hippocampal neuronal culture, we observed that dopamine D2 receptors (D2R) co-localized with certain subtypes of GABAA receptors, namely α1, β3, and γ2 subunits, as revealed by double immunofluorocytochemical analysis. Treatment with the D2R agonist, quinpirole, was shown to elicit an increase in phosphorylation of extracellular signal-regulated kinase (ERK) in hippocampal neurons. This phosphorylation was inhibited by pretreatment with the GABAA receptor agonist, muscimol. Furthermore, treatment of hippocampal neurons with quinpirole increased the dendritic spine density and this regulation was totally blocked by pretreatment with a MAP kinase kinase (MEK) inhibitor (PD98059), D2R antagonist (haloperidol), or by the GABAA receptor agonist, muscimol. These results suggest that D2R-mediated ERK phosphorylation can control spine formation and that the GABAA receptor negatively regulates the D2R-induced spine formation through ERK signaling in hippocampal neurons, thus indicating a potential role of D2R in the control of hippocampal neuronal excitability. PMID:25483619

  20. The effect of BLA GABA(A) receptors in anxiolytic-like effect and aversive memory deficit induced by ACPA

    PubMed Central

    Kangarlu-Haghighi, Katayoon; Oryan, Shahrbanoo; Nasehi, Mohammad; Zarrindast, Mohammad-Reza

    2015-01-01

    The roles of GABAergic receptors of the Basolateral amygdala (BLA) in the cannabinoid CB1 receptor agonist (arachydonilcyclopropylamide; ACPA)-induced anxiolytic-like effect and aversive memory deficit in adult male mice were examined in elevated plus-maze task. Results showed that pre-test intra-peritoneal injection of ACPA induced anxiolytic-like effect (at dose of 0.05 mg/kg) and aversive memory deficit (at doses of 0.025 and 0.05 mg/kg). The results revealed that Pre-test intra-BLA infusion of muscimol (GABAA receptor agonist; at doses of 0.1 and 0.2 µg/mouse) or bicuculline (GABAA receptor antagonist; at all doses) impaired and did not alter aversive memory, respectively. All previous GABA agents did not have any effects on anxiety-like behaviors. Interestingly, pretreatment with a sub-threshold dose of muscimol (0.025 µg/mouse) and bicuculline (0.025 µg/mouse) did not alter anxiolytic-like behaviors induced by ACPA, while both drugs restored ACPA-induced amnesia. Moreover, muscimol or bicuculline increased and decreased ACPA-induced locomotor activity, respectively. Finally the data may indicate that BLA GABAA receptors have critical and different roles in anxiolytic-like effect, aversive memory deficit and locomotor activity induced by ACPA. PMID:26648818

  1. Propofol depresses cerebellar Purkinje cell activity via activation of GABA(A) and glycine receptors in vivo in mice.

    PubMed

    Jin, Ri; Liu, Heng; Jin, Wen-Zhe; Shi, Jin-Di; Jin, Qing-Hua; Chu, Chun-Ping; Qiu, De-Lai

    2015-10-01

    Propofol is an intravenous sedative-hypnotic agen, which causes rapid and reliable loss of consciousness. Under in vitro conditions, propofol activates GABAA and glycine receptors in spinal cord, hippocampus and hypothalamus neurons. However, the effects of propofol on the cerebellar neuronal activity under in vivo conditions are currently unclear. In the present study, we examined the effects of propofol on the spontaneous activity of Purkinje cells (PCs) in urethane-anesthetized mice by cell-attached recording and pharmacological methods. Our results showed that cerebellar surface perfusion of propofol (10-1000 μM) induced depression of the PC simple spike (SS) firing rate in a dose-dependent manner, but without significantly changing the properties of complex spikes (CS). The IC50 of propofol for inhibiting SS firing of PCs was 144.5 μM. Application of GABAA receptor antagonist, SR95531 (40 μM) or GABAB receptor antagonist, saclofen (20 μM), as well as glycine receptor antagonist, strychnine (10 μM) alone failed to prevent the propofol-induced inhibition of PCs spontaneous activity. However, application the mixture of SR95531 (40 μM) and strychnine (10 μM) completely blocked the propofol-induced inhibition of PC SS firing. These data indicated that cerebellar surface application of propofol depressed PC SS firing rate via facilitation of GABAA and functional glycine receptors activity in adult cerebellar PCs under in vivo conditions. Our present results provide a new insight of the anesthetic action of propofol in cerebellar cortex, suggesting that propofol depresses the SS outputs of cerebellar PCs which is involved in both GABAA and glycine receptors activity.

  2. 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

  3. Profound desensitization by ambient GABA limits activation of δ-containing GABAA receptors during spillover.

    PubMed

    Bright, Damian P; Renzi, Massimiliano; Bartram, Julian; McGee, Thomas P; MacKenzie, Georgina; Hosie, Alastair M; Farrant, Mark; Brickley, Stephen G

    2011-01-12

    High-affinity extrasynaptic GABA(A) receptors (GABA(A)Rs) are a prominent feature of cerebellar granule neurons and thalamic relay neurons. In both cell types, the presence of synaptic glomeruli would be expected to promote activation of these GABA(A)Rs, contributing to phasic spillover-mediated currents and tonic inhibition. However, the precise role of different receptor subtypes in these two phenomena is unclear. To address this question, we made recordings from neurons in acute brain slices from mice, and from tsA201 cells expressing recombinant GABA(A)Rs. We found that δ subunit-containing GABA(A)Rs of both cerebellar granule neurons and thalamic relay neurons of the lateral geniculate nucleus contributed to tonic conductance caused by ambient GABA but not to spillover-mediated currents. In the presence of a low "ambient" GABA concentration, recombinant "extrasynaptic" δ subunit-containing GABA(A)Rs exhibited profound desensitization, rendering them insensitive to brief synaptic- or spillover-like GABA transients. Together, our results demonstrate that phasic spillover and tonic inhibition reflect the activation of distinct receptor populations.

  4. GABAA receptor partial agonists and antagonists: structure, binding mode, and pharmacology.

    PubMed

    Krall, Jacob; Balle, Thomas; Krogsgaard-Larsen, Niels; Sørensen, Troels E; Krogsgaard-Larsen, Povl; Kristiansen, Uffe; Frølund, Bente

    2015-01-01

    A high degree of structural heterogeneity of the GABAA receptors (GABAARs) has been revealed and is reflected in multiple receptor subtypes. The subunit composition of GABAAR subtypes is believed to determine their localization relative to the synapses and adapt their functional properties to the local temporal pattern of GABA impact, enabling phasic or tonic inhibition. Specific GABAAR antagonists are essential tools for physiological and pharmacological elucidation of the different type of GABAAR inhibition. However, distinct selectivity among the receptor subtypes (populations) has been shown for only a few orthosteric ligands. Still, these examples show that it is indeed possible to obtain orthosteric subtype selectivity and they serve as models for further development in the orthosteric GABAAR ligand area. This review presents the very few existing structural classes of orthosteric GABAAR antagonists and describes the development of potent antagonists from partial agonists originally derived from the potent GABAAR agonist muscimol. In this process, several heterocyclic aromatic systems have been used in combination with structural models in order to map the orthosteric binding site and to reveal structural details to be used for obtaining potency and subtype selectivity. The challenges connected to functional characterization of orthosteric GABAAR partial agonists and antagonists, especially with regard to GABAAR stoichiometry and alternative binding sites are discussed. GABAAR antagonists have been essential in defining the tonic current but both remaining issues concerning the GABAARs involved and the therapeutic possibilities of modulating tonic inhibition underline the need for GABAAR antagonists with improved selectivity.

  5. Generation of Functional Inhibitory Synapses Incorporating Defined Combinations of GABA(A) or Glycine Receptor Subunits

    PubMed Central

    Dixon, Christine L.; Zhang, Yan; Lynch, Joseph W.

    2015-01-01

    Fast inhibitory neurotransmission in the brain is mediated by wide range of GABAA receptor (GABAAR) and glycine receptor (GlyR) isoforms, each with different physiological and pharmacological properties. Because multiple isoforms are expressed simultaneously in most neurons, it is difficult to define the properties of individual isoforms under synaptic stimulation conditions in vivo. Although recombinant expression systems permit the expression of individual isoforms in isolation, they require exogenous agonist application which cannot mimic the dynamic neurotransmitter profile characteristic of native synapses. We describe a neuron-HEK293 cell co-culture technique for generating inhibitory synapses incorporating defined combinations of GABAAR or GlyR subunits. Primary neuronal cultures, prepared from embryonic rat cerebral cortex or spinal cord, are used to provide presynaptic GABAergic and glycinergic terminals, respectively. When the cultures are mature, HEK293 cells expressing the subunits of interest plus neuroligin 2A are plated onto the neurons, which rapidly form synapses onto HEK293 cells. Patch clamp electrophysiology is then used to analyze the physiological and pharmacological properties of the inhibitory postsynaptic currents mediated by the recombinant receptors. The method is suitable for investigating the kinetic properties or the effects of drugs on inhibitory postsynaptic currents mediated by defined GABAAR or GlyR isoforms of interest, the effects of hereditary disease mutations on the formation and function of both types of synapses, and synaptogenesis and synaptic clustering mechanisms. The entire cell preparation procedure takes 2–5 weeks. PMID:26778954

  6. Regulation of GABAA Receptor Dynamics by Interaction with Purinergic P2X2 Receptors*

    PubMed Central

    Shrivastava, Amulya Nidhi; Triller, Antoine; Sieghart, Werner; Sarto-Jackson, Isabella

    2011-01-01

    γ-Aminobutyric acid type A receptors (GABAARs) in the spinal cord are evolving as an important target for drug development against pain. Purinergic P2X2 receptors (P2X2Rs) are also expressed in spinal cord neurons and are known to cross-talk with GABAARs. Here, we investigated a possible “dynamic” interaction between GABAARs and P2X2Rs using co-immunoprecipitation and fluorescence resonance energy transfer (FRET) studies in human embryonic kidney (HEK) 293 cells along with co-localization and single particle tracking studies in spinal cord neurons. Our results suggest that a significant proportion of P2X2Rs forms a transient complex with GABAARs inside the cell, thus stabilizing these receptors and using them for co-trafficking to the cell surface, where P2X2Rs and GABAARs are primarily located extra-synaptically. Furthermore, agonist-induced activation of P2X2Rs results in a Ca2+-dependent as well as an apparently Ca2+-independent increase in the mobility and an enhanced degradation of GABAARs, whereas P2X2Rs are stabilized and form larger clusters. Antagonist-induced blocking of P2XRs results in co-stabilization of this receptor complex at the cell surface. These results suggest a novel mechanism where association of P2X2Rs and GABAARs could be used for specific targeting to neuronal membranes, thus providing an extrasynaptic receptor reserve that could regulate the excitability of neurons. We further conclude that blocking the excitatory activity of excessively released ATP under diseased state by P2XR antagonists could simultaneously enhance synaptic inhibition mediated by GABAARs. PMID:21343285

  7. GABAA receptor antagonism at the hypoglossal motor nucleus increases genioglossus muscle activity in NREM but not REM sleep.

    PubMed

    Morrison, Janna L; Sood, Sandeep; Liu, Hattie; Park, Eileen; Nolan, Philip; Horner, Richard L

    2003-04-15

    The pharyngeal muscles, such as the genioglossus (GG) muscle of the tongue, are important for effective lung ventilation since they maintain an open airspace. Rapid-eye-movement (REM) sleep, however, recruits powerful neural mechanisms that can abolish GG activity, even during strong reflex respiratory stimulation by elevated CO2. In vitro studies have demonstrated the presence of GABAA receptors on hypoglossal motoneurons, and these and other data have led to the speculation that GABAA mechanisms may contribute to the suppression of hypoglossal motor outflow to the GG muscle in REM sleep. We have developed an animal model that allows us to chronically manipulate neurotransmission at the hypoglossal motor nucleus using microdialysis across natural sleep-wake states in rats. The present study tests the hypothesis that microdialysis perfusion of the GABAA receptor antagonist bicuculline into the hypoglossal motor nucleus will prevent the suppression of GG muscle activity in REM sleep during both room-air and CO2-stimulated breathing. Ten rats were implanted with electroencephalogram and neck muscle electrodes to record sleep-wake states, and GG and diaphragm electrodes for respiratory muscle recording. Microdialysis probes were implanted into the hypoglossal motor nucleus for perfusion of artificial cerebrospinal fluid (ACSF) or 100 microM bicuculline during room-air and CO2-stimulated breathing (7 % inspired CO2). GABAA receptor antagonism at the hypoglossal motor nucleus increased respiratory-related GG activity during both room-air (P = 0.01) and CO2-stimulated breathing (P = 0.007), indicating a background inhibitory GABA tone. However, the effects of bicuculline on GG activity depended on the prevailing sleep-wake state (P < 0.005), with bicuculline increasing GG activity in non-REM (NREM) sleep and wakefulness both in room air and hypercapnia (P < 0.01), but GG activity was effectively abolished in those REM periods without phasic twitches in the GG muscle

  8. Hypothermic activity of acetaminophen; involvement of GABAA receptor, theoretical and experimental studies

    PubMed Central

    Ahangar, Nematollah; Esam, Zohreh; Bekhradnia, Ahmadreza; Ebrahimzadeh, Mohammad Ali

    2016-01-01

    Objective(s): The mechanism of hypothermia action of acetaminophen (APAP) remains unclear even 125 years after its synthesis. Acetaminophen produces hypothermia. The mechanism of this reduction in core body temperature is not clear but evidence shows that it is not dependent on opioid and cannabinoid receptors. Because of strong documents about the roles of GABA and benzodiazepine receptors in hypothemic activity of some drugs such as diazepam, we determined if these receptors also contributes to the hypothermic effect of APAP. Materials and Methods: Diazepam (5 mg/kg, IP) was used for induction of hypothermia. Flumazenil (10 mg/kg, IP) or picrotoxin (2 mg/kg, IP) used for reversal of this effect. Rats injected with APAP (100, 200 or 300 mg/kg, IP). Baseline temperature measurements were taken with a digital thermometer via rectum. To evaluate the structural correlation between APAP and benzodiazepine receptor ligands, numerous models are selected and studied at HF/6-31G* level of theory. Relative energies, enthalpies and Gibbs free energies were calculated for all selected drugs. Results Diazepam induced hypothermia was reversed by flumazenil or picrotoxin. Rats injected with APAP displayed dose- and time-related hypothermia. For combined administration, the hypothermic effect of APAP (200 mg/kg) was strongly reduced by pretreatment with picrotoxin or flumazenil P<0.0001and P<0.01, respectively. Selective structural data, bond length, dihedral angles, and related distance in pharmacophore of APAP and BZDR models were the same. Some significant structural analogues were obtained between these drugs. Conclusion: Results suggest hypothermic action of acetaminophen may be mediate by its effect at GABAA benzodiazepine receptor. PMID:27403252

  9. RNA editing of the GABA(A) receptor alpha3 subunit alters the functional properties of recombinant receptors.

    PubMed

    Nimmich, Mitchell L; Heidelberg, Laura S; Fisher, Janet L

    2009-04-01

    RNA editing provides a post-transcriptional mechanism to increase structural heterogeneity of gene products. Recently, the alpha3 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 alpha3 subunit along with beta3 and gamma2L. 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 alpha1 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 alpha3 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 alpha3 subunit.

  10. Astrocytes regulate inhibitory synapse formation via Trk-mediated modulation of postsynaptic GABAA receptors.

    PubMed

    Elmariah, Sarina B; Oh, Eun Joo; Hughes, Ethan G; Balice-Gordon, Rita J

    2005-04-01

    Astrocytes promote the formation and function of excitatory synapses in the CNS. However, whether and how astrocytes modulate inhibitory synaptogenesis are essentially unknown. We asked whether astrocytes regulate the formation of inhibitory synapses between hippocampal neurons during maturation in vitro. Neuronal coculture with astrocytes or treatment with astrocyte-conditioned medium (ACM) increased the number of inhibitory presynaptic terminals, the frequency of miniature IPSCs, and the number and synaptic localization of GABA(A) receptor (GABA(A)R) clusters during the first 10 d in vitro. We asked whether neurotrophins, which are potent modulators of inhibitory synaptic structure and function, mediate the effects of astrocytes on inhibitory synapses. ACM from BDNF- or tyrosine receptor kinase B (TrkB)-deficient astrocytes increased inhibitory presynaptic terminals and postsynaptic GABA(A)R clusters in wild-type neurons, suggesting that BDNF and TrkB expression in astrocytes is not required for these effects. In contrast, although the increase in the number of inhibitory presynaptic terminals persisted, no increase was observed in postsynaptic GABA(A)R clusters after ACM treatment of hippocampal neurons lacking BDNF or TrkB. These results suggest that neurons, not astrocytes, are the relevant source of BDNF and are the site of TrkB activation required for postsynaptic GABA(A)R modulation. These data also suggest that astrocytes may modulate postsynaptic development indirectly by stimulating Trk signaling between neurons. Together, these data show that astrocytes modulate inhibitory synapse formation via distinct presynaptic and postsynaptic mechanisms.

  11. Side chain flexibility and the pore dimensions in the GABAA receptor.

    PubMed

    Rossokhin, Alexey V; Zhorov, Boris S

    2016-07-01

    Permeation of ions through open channels and their accessibility to pore-targeting drugs depend on the pore cross-sectional dimensions, which are known only for static X-ray and cryo-EM structures. Here, we have built homology models of the closed, open and desensitized α1β2γ2 GABAA receptor (GABAAR). The models are based, respectively, on the X-ray structure of α3 glycine receptor (α3 GlyR), cryo-EM structure of α1 GlyR and X-ray structure of β3 GABAAR. We employed Monte Carlo energy minimizations to explore how the pore lumen may increase due to repulsions of flexible side chains from a variable-diameter electroneutral atom (an expanding sphere) pulled through the pore. The expanding sphere computations predicted that the pore diameter averaged along the permeation pathway is larger by approximately 3 Å than that computed for the models with fixed sidechains. Our models predict three major pore constrictions located at the levels of -2', 9' and 20' residues. Residues around the -2' and 9' rings are known to form the desensitization and activation gates of GABAAR. Our computations predict that the 20' ring may also serve as GABAAR gate whose physiological role is unclear. The side chain flexibility of residues -2', 9' and 20' and hence the dimensions of the constrictions depend on the GABAAR functional state. PMID:27460059

  12. Oxytocin regulates neurosteroid modulation of GABA(A) receptors in supraoptic nucleus around parturition.

    PubMed

    Koksma, Jan-Jurjen; van Kesteren, Ronald E; Rosahl, Thomas W; Zwart, Ruud; Smit, August B; Lüddens, Hartmut; Brussaard, Arjen B

    2003-02-01

    In this study, we investigate how neurosteroid sensitivity of GABA(A) receptors (GABA(A)Rs) is regulated. We examined this issue in neurons of the supraoptic nucleus (SON) of the rat and found that, during parturition, the GABA(A)Rs become insensitive to the neurosteroid allopregnanolone attributable to a shift in the balance between the activities of endogenous Ser/Thr phosphatase and PKC. In particular, a constitutive endogenous tone of oxytocin within the SON after parturition suppressed neurosteroid sensitivity of GABA(A)Rs via activation of PKC. Vice versa before parturition, during late pregnancy, application of exogenous oxytocin brings the GABA(A)Rs from a neurosteroid-sensitive mode toward a condition in which the receptors are not sensitive. This indicates that there may be an inverse causal relationship between the extent to which the GABA(A)R or one of its interacting proteins is phosphorylated and the neurosteroid sensitivity of the GABA(A)R. Neurosteroid sensitivity was not affected by changes in subunit composition of GABA(A)Rs known to occur concurrently in these cells.

  13. Side chain flexibility and the pore dimensions in the GABAA receptor.

    PubMed

    Rossokhin, Alexey V; Zhorov, Boris S

    2016-07-01

    Permeation of ions through open channels and their accessibility to pore-targeting drugs depend on the pore cross-sectional dimensions, which are known only for static X-ray and cryo-EM structures. Here, we have built homology models of the closed, open and desensitized α1β2γ2 GABAA receptor (GABAAR). The models are based, respectively, on the X-ray structure of α3 glycine receptor (α3 GlyR), cryo-EM structure of α1 GlyR and X-ray structure of β3 GABAAR. We employed Monte Carlo energy minimizations to explore how the pore lumen may increase due to repulsions of flexible side chains from a variable-diameter electroneutral atom (an expanding sphere) pulled through the pore. The expanding sphere computations predicted that the pore diameter averaged along the permeation pathway is larger by approximately 3 Å than that computed for the models with fixed sidechains. Our models predict three major pore constrictions located at the levels of -2', 9' and 20' residues. Residues around the -2' and 9' rings are known to form the desensitization and activation gates of GABAAR. Our computations predict that the 20' ring may also serve as GABAAR gate whose physiological role is unclear. The side chain flexibility of residues -2', 9' and 20' and hence the dimensions of the constrictions depend on the GABAAR functional state.

  14. Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β- Interface.

    PubMed

    Jayakar, Selwyn S; Zhou, Xiaojuan; Savechenkov, Pavel Y; Chiara, David C; Desai, Rooma; Bruzik, Karol S; Miller, Keith W; Cohen, Jonathan B

    2015-09-18

    In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABAARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABAAR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343-19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABAAR responses. In the α1β3γ2 GABAAR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ(+)-β(-) subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β(+)-α(-) subunit interfaces. GABA inhibits S-[(3)H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2-15') in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[(3)H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ(+)-β(-) site, based upon the distance in GABAAR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABAAR function and provide a first demonstration that an intersubunit-binding site in the GABAAR transmembrane domain binds negative and positive allosteric modulators.

  15. Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β− Interface*

    PubMed Central

    Jayakar, Selwyn S.; Zhou, Xiaojuan; Savechenkov, Pavel Y.; Chiara, David C.; Desai, Rooma; Bruzik, Karol S.; Miller, Keith W.; Cohen, Jonathan B.

    2015-01-01

    In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABAARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABAAR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343–19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABAAR responses. In the α1β3γ2 GABAAR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ+-β− subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β+-α− subunit interfaces. GABA inhibits S-[3H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2–15′) in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[3H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ+-β− site, based upon the distance in GABAAR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABAAR function and provide a first demonstration that an intersubunit-binding site in the GABAAR transmembrane domain binds negative and positive allosteric modulators. PMID:26229099

  16. Competitive antagonists facilitate the recovery from desensitization of α1β2γ2 GABAA receptors expressed in Xenopus oocytes

    PubMed Central

    Xu, Xiao-jun; Roberts, Diane; Zhu, Guo-nian; Chang, Yong-chang

    2016-01-01

    Aim: The continuous presence of an agonist drives its receptor into a refractory state, termed desensitization. In this study, we tested the hypothesis that a competitive antagonist, SR95531, could facilitate the recovery of α1β2γ2 GABAA receptor from functional desensitization. Methods: α1β2γ2 GABAA receptors were expressed in Xenopus oocytes. GABA-evoked currents were recorded using two-electrode voltage-clamp technique. Drugs were applied through perfusion. Results: Long application of GABA (100 μmol/L) evoked a large peak current followed by a small amplitude steady-state current (desensitization). Co-application of SR95531 during the desensitization caused a larger rebound of GABA current after removal of SR95531. Furthermore, application of SR95531 after removal of GABA increased the rate of receptor recovery from desensitization, and the recovery time constant was decreased from 59±3.2 s to 33±1.6 s. SR95531-facilitated receptor recovery from desensitization was dependent on the perfusion duration of SR95531. It was also dependent on the concentration of SR95531, and the curve fitting with Hill equation revealed two potency components, which were similar to the two potency components in inhibition of the steady-state current by SR95531. Bicuculline caused similar facilitation of desensitization recovery. Conclusion: SR95531 facilitates α1β2γ2 GABAA receptor recovery from desensitization, possibly through two mechanisms: binding to the desensitized receptor and converting it to the non-desensitized state, and binding to the resting state receptor and preventing re-desensitization. PMID:27374488

  17. Amygdala-specific reduction of α1-GABAA receptors disrupts the anticonvulsant, locomotor, and sedative, but not anxiolytic effects of benzodiazepines in mice

    PubMed Central

    Heldt, Scott A.; Ressler, Kerry J.

    2010-01-01

    The heterogeneity and distribution of GABAA receptor subunits mediates differential roles in behavior. It is thought that particular behavioral responses to benzodiazepine (BZ) ligands might be associated with an action at a regionally defined receptor subtype. However, the role of specific GABAA receptor subtypes in particular brain regions is less clear. Such detailed knowledge of regional α1-GABAA receptor function will advance our understanding of the neural circuitry underlying the role of GABAA receptors and the effects of GABAA-modulating drugs on behavior. By combining inducible, site-specific α1 subunit deletion, using a lentivirus expressing Cre-recombinase in mice with the α1 subunit gene flanked by loxP sites, we examine baseline and pharmacological effects of deletion of amygdala α1-GABAA receptors. We find that amygdala-specific reduction of α1 receptor subunits does not affect mRNA or protein levels of amygdala α2 or α3 subunit receptors. Nor does this inducible reduction affect baseline locomotion or measures of anxiety. However, we also find that this inducible, site-specific deletion does disrupt the normal sedative-locomotor inhibition as well as the anticonvulsive effects, of two distinct benzodiazepine-site ligands, diazepam and zolpidem, which is relatively α1-subunit selective. These data, utilizing inducible, region and subunit-specific deletion, combined with pharmacogenetic approaches, demonstrate that amygdala expression of the α1-GABAA receptor subunit is required for normal benzodiazepine effects on sedation, locomotion, and seizure inhibition, but not for anxiolysis. PMID:20505082

  18. Adolescent Alcohol Exposure Alters GABAA Receptor Subunit Expression in Adult Hippocampus

    PubMed Central

    Centanni, Samuel W.; Teppen, Tara; Risher, Mary-Louise; Fleming, Rebekah L.; Moss, Julia L.; Acheson, Shawn K.; Mulholland, Patrick J.; Pandey, Subhash C.; Chandler, L. Judson; Swartzwelder, H. Scott

    2014-01-01

    Background The long-term consequences of adolescent alcohol abuse that persist into adulthood are poorly understood and have not been widely investigated. We have shown that intermittent exposure to alcohol during adolescence decreased the amplitude of GABAA receptor-mediated tonic currents in hippocampal dentate granule cells in adulthood. The aim of the present study was to investigate the enduring effects of chronic intermittent alcohol exposure during adolescence or adulthood on the expression of hippocampal GABAA receptors (GABAARs). Methods We used a previously characterized tissue fractionation method to isolate detergent resistant membranes and soluble fractions, followed by western blots to measure GABAAR protein expression. We also measured mRNA levels of GABAAR subunits using quantitative real-time PCR. Results Although the protein levels of α1-, α4- and δ-GABAAR subunits remained stable between postnatal day (PD) 30 (early adolescence) and PD71 (adulthood), the α5-GABAAR subunit was reduced across that period. In rats that were subjected to adolescent intermittent ethanol (AIE) exposure between PD30–46, there was a significant reduction in the protein levels of the δ-GABAAR, in the absence of any changes in mRNA levels, at 48 hours and 26 days after the last ethanol exposure. Protein levels of the α4-GABAAR subunit were significantly reduced, but mRNA levels were increased, 26 days (but not 48 hours) after the last AIE exposure. Protein levels of α5-GABAAR were not changed by AIE, but mRNA levels were reduced at 48hrs but normalized 26 days after AIE. In contrast to the effects of AIE, chronic intermittent exposure to ethanol during adulthood (CIE) had no effect on expression of any of the GABAAR subunits examined. Conclusions AIE produced both short- and long-term alterations of GABAAR subunits mRNA and protein expression in the hippocampus, whereas CIE produced no long lasting effects on those measures. The observed reduction of protein

  19. NMDA and GABAA receptors in the rat Kölliker-Fuse area control cardiorespiratory responses evoked by trigeminal ethmoidal nerve stimulation

    PubMed Central

    Dutschmann, Mathias; Herbert, Horst

    1998-01-01

    Electrical stimulation (10 s) of the ethmoidal nerve (EN5) evokes the nasotrigeminal reflex responses, including apnoea, bradycardia and rise in arterial blood pressure. In the present study, we examined the involvement of N-methyl-D-aspartate (NMDA), AMPA/kainate, (γ-aminobutyric acidA (GABAA) and glycine receptors in the Kölliker-Fuse (KF) nucleus in the mediation of the nasotrigeminal reflex responses.Unilateral injections (n = 6) of 50-100 nl of the NMDA receptor antagonist AP5 into the KF area led to a significant blockade of the EN5-evoked respiratory depression and bradycardia. Injections placed into the midlevel of the KF area were most effective (80-90% blockade). The rise in arterial blood pressure remained unaffected.Unilateral injections (n = 6) of the AMPA/kainate receptor antagonist CNQX into the KF area failed to block EN5-evoked autonomic responses significantly.Unilateral injections (n = 5) of the GABAA receptor antagonist bicuculline enhanced the EN5-evoked respiratory depression and bradycardia. The effect persisted for up to 30 s after stimulation. Bicuculline injections into the midlevel of the KF area were most effective. The increase in arterial blood pressure remained unaffected.Unilateral injections (n = 5) of the glycine receptor antagonist strychnine into the KF area did not produce any significant effects on EN5-evoked autonomic responses.Our results suggest that the KF area represents a mandatory relay for the nasotrigeminally induced apnoea and bradycardia which are predominantly mediated by NMDA receptors in the KF. Furthermore, it appears that KF neurons are under a potent GABAergic inhibitory control. The EN5-evoked rise in arterial blood pressure was not altered by any of the drugs and, therefore, appears not to be mediated via the KF. PMID:9660894

  20. Autoradiographic analysis of GABAA receptor binding in the neural anxiety network of postpartum and non-postpartum laboratory rats

    PubMed Central

    Miller, Stephanie M.; Lonstein, Joseph S.

    2011-01-01

    Postpartum female rats exhibit a suppression of anxiety-related behaviors when compared to diestrous virgin females, pregnant females, and males. This blunted anxiety promotes optimal maternal care and involves elevated GABA neurotransmission, possibly including greater density of GABAA and benzodiazepine receptors in the postpartum brain. We here examined autoradiographic binding of [3H]muscimol to measure the total population of GABAA receptors and [3H]flunitrazepam to assess density of benzodiazepine sites in the medial prefrontal cortex, bed nucleus of the stria terminalis, amygdala, hippocampus, and periaqueductal gray of female rats sacrificed on day 7 postpartum, day 10 of pregnancy, or as diestrous virgins. A group of sexually naïve male rats was also included. We found that [3H]muscimol binding did not differ among groups in any site but that diestrous virgin females had greater [3H]flunitrazepam binding in the CA1 and dentate gyrus of the hippocampus compared to mid-pregnant females and males. Notably, postpartum and diestrous virgin females did not significantly differ in binding of either ligand in any site examined. This is the first study to evaluate the densities of GABAA and benzodiazepine binding sites simultaneously across three female reproductive states and sex with a focus on brain sites influencing anxiety-related behaviors. The results suggest that changes other GABAA receptor characteristics, such as subunit composition or increased presynaptic GABA release during interactions with offspring, must instead play a greater role in the postpartum suppression of anxiety in laboratory rats. PMID:21664440

  1. Enhanced behavioral sensitivity to the competitive GABA agonist, gaboxadol, in transgenic mice over-expressing hippocampal extrasynaptic alpha6beta GABA(A) receptors.

    PubMed

    Saarelainen, Kati S; Ranna, Martin; Rabe, Holger; Sinkkonen, Saku T; Möykkynen, Tommi; Uusi-Oukari, Mikko; Linden, Anni-Maija; Lüddens, Hartmut; Korpi, Esa R

    2008-04-01

    The behavioral and functional significance of the extrasynaptic inhibitory GABA(A) receptors in the brain is still poorly known. We used a transgenic mouse line expressing the GABA(A) receptor alpha6 subunit gene in the forebrain under the Thy-1.2 promoter (Thy1alpha6) mice ectopically expressing alpha6 subunits especially in the hippocampus to study how extrasynaptically enriched alphabeta(gamma2)-type receptors alter animal behavior and receptor responses. In these mice extrasynaptic alpha6beta receptors make up about 10% of the hippocampal GABA(A) receptors resulting in imbalance between synaptic and extrasynaptic inhibition. The synthetic GABA-site competitive agonist gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; 3 mg/kg) induced remarkable anxiolytic-like response in the light : dark exploration and elevated plus-maze tests in Thy1alpha6 mice, while being almost inactive in wild-type mice. The transgenic mice also lost quicker and for longer time their righting reflex after 25 mg/kg gaboxadol than wild-type mice. In hippocampal sections of Thy1alpha6 mice, the alpha6beta receptors could be visualized autoradiographically by interactions between gaboxadol and GABA via [(35)S]TBPS binding to the GABA(A) receptor ionophore. Gaboxadol inhibition of the binding could be partially prevented by GABA. Electrophysiology of recombinant GABA(A) receptors revealed that GABA was a partial agonist at alpha6beta3 and alpha6beta3delta receptors, but a full agonist at alpha6beta3gamma2 receptors when compared with gaboxadol. The results suggest strong behavioral effects via selective pharmacological activation of enriched extrasynaptic alphabeta GABA(A) receptors, and the mouse model represents an example of the functional consequences of altered balance between extrasynaptic and synaptic inhibition.

  2. Protein kinase C regulates tonic GABAA receptor-mediated inhibition in the hippocampus and thalamus

    PubMed Central

    Bright, Damian P; Smart, Trevor G

    2013-01-01

    Tonic inhibition mediated by extrasynaptic GABAA receptors (GABAARs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABAARs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole-cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC-mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABAAR-mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABAAR activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABAARs, which represent a key extrasynaptic GABAAR isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABAARs. The inhibitory effects of PKC activation on α4β2δ GABAAR activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC-mediated phosphorylation can be an important physiological regulator of tonic GABAAR-mediated inhibition. PMID:24102973

  3. Thyroid hormones modulate GABA(A) receptor-mediated currents in hippocampal neurons.

    PubMed

    Puia, G; Losi, G

    2011-06-01

    Thyroid hormones (THs) play a crucial role in the maturation and functioning of mammalian central nervous system. Thyroxine (T4) and 3, 3', 5-L-triiodothyronine (T3) are well known for their genomic effects, but recently attention has been focused on their non genomic actions as modulators of neuronal activity. In the present study we report that T4 and T3 reduce, in a non competitive manner, GABA-evoked currents in rat hippocampal cultures with IC₅₀s of 13±4μM and 12±3μM, respectively. The genomically inactive compound rev-T3 was also able to inhibit the currents elicited by GABA. Blocking PKC or PKA activity, chelating intracellular calcium, or antagonizing the integrin receptor αVβ3 with TETRAC did not affect THs modulation of GABA-evoked currents. THs affect also synaptic activity in hippocampal and cortical cultured neurons. T3 and T4 reduced to approximately 50% the amplitude and frequency of spontaneous inhibitory synaptic currents (sIPSCs), without altering their decay kinetic. Tonic currents evoked by low GABA concentrations were also reduced by T3 (40±5%, n=14), but not by T4. Similarly, T3 decreased currents elicited by low concentrations of THIP, a low affinity GABAA receptor agonist that preferentially activates extrasynaptic receptors, whereas T4 was ineffective. Thus, our data demonstrate that T3 and T4 selectively affect GABAergic phasic and tonic neurotransmission. Since THs concentrations can be regulated at the level of the synapses these data suggest that the network activity of the whole brain could be differently modulated depending on the relative amount of these two hormones. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'. PMID:21215272

  4. GABAA receptors expression pattern in rat brain following low pressure distension of the stomach.

    PubMed

    Sabbatini, M; Molinari, C; Grossini, E; Piffanelli, V; Mary, D A S G; Vacca, G; Cannas, M

    2008-03-18

    It is known that gastric mechanoreceptor stimuli are widely integrated into neuronal circuits that involve visceral nuclei of hindbrain as well as several central brain areas. GABAergic neurons are widely represented in hindbrain nuclei controlling gastric motor functions, but limited information is available specifically about GABA(A)-responding neurons in brain visceral areas. The present investigation was designed to determine the central sensory neuronal pathways and their GABA(A)-alpha1 and -alpha3 receptor presenting neurons that respond to gastric mechanoreceptor stimulation within the entire rat brain. Low pressure gastric distension was used to deliver physiological mechanical stimuli in anesthetized rats, and different protocols of gastric distension were performed to mimic different stimulation patterns with and without sectioning vagal and/or splanchnic afferent nerves. Mapping of activated neurons was investigated using double colorimetric immunohistochemistry for GABA(A)-alpha1 or -alpha3 subunits and c-Fos. Following stomach distension, neurons expressing GABA(A) receptors with alpha1 or alpha3 subunits were detected. Low frequency gastric distension induced c-Fos expression in nucleus tractus solitarii (NTS) only, whereas in the high frequency gastric distension c-Fos positive nuclei were found in lateral reticular nucleus and in NTS in addition to some forebrain areas. In contrast, during the tonic-rapid gastric distension the neuronal activation was found in hindbrain, midbrain and forebrain areas. Moreover different protocols of gastric stimulation activated diverse patterns of neurons presenting GABA(A)-alpha1 or -alpha3 receptors within responding brain nuclei, which may indicate a probable functional significance of differential expression of GABA(A)-responding neurons. The same protocol of gastric distension performed in vagotomized rats has confirmed the primary role of the vagus in the response of activation of gastric brain areas, whereas

  5. Discovery of anxiolytic 2-ferrocenyl-1,3-thiazolidin-4-ones exerting GABAA receptor interaction via the benzodiazepine-binding site.

    PubMed

    Pejović, Anka; Denić, Marija S; Stevanović, Dragana; Damljanović, Ivan; Vukićević, Mirjana; Kostova, Kalina; Tavlinova-Kirilova, Maya; Randjelović, Pavle; Stojanović, Nikola M; Bogdanović, Goran A; Blagojević, Polina; D'hooghe, Matthias; Radulović, Niko S; Vukićević, Rastko D

    2014-08-18

    Herein, we report on the synthesis, spectral, crystallographic and electrochemical properties of a small library of N-substituted 2-ferrocenyl-1,3-thiazolidin-4-ones, designed as novel GABAA benzodiazepine-binding site ligands. The anxiolytic properties of the title compounds were evaluated in several different in vivo models, whereas the involvement of the GABAA receptor complex in the activity of the most potent compound, 2-ferrocenyl-3-(4-methoxyphenylethyl)-1,3-thiazolidin-4-one, was inferred from experiments with known GABAA-targeting agents. Ligand docking experiments revealed that the high, dose-dependent, anxiolytic activity of the new compounds might be due to their favorable interactions with the benzodiazepine-binding site of the GABAA receptor complex. The incorporation of the ferrocene core and fine tuning of the distance between the thiazolidinone core and an additional aromatic ring were judged to be crucial structural requirements for the observed anxiolytic effect. PMID:24950490

  6. Effect of (+)-dehydrofukinone on GABAA receptors and stress response in fish model

    PubMed Central

    Garlet, Q.I.; Pires, L.C.; Silva, D.T.; Spall, S.; Gressler, L.T.; Bürger, M.E.; Baldisserotto, B.; Heinzmann, B.M.

    2015-01-01

    (+)-Dehydrofukinone (DHF) is a major component of the essential oil of Nectandra grandiflora (Lauraceae), and exerts a depressant effect on the central nervous system of fish. However, the neuronal mechanism underlying DHF action remains unknown. This study aimed to investigate the action of DHF on GABAA receptors using a silver catfish (Rhamdia quelen) model. Additionally, we investigated the effect of DHF exposure on stress-induced cortisol modulation. Chemical identification was performed using gas chromatography-mass spectrometry and purity was evaluated using gas chromatography with a flame ionization detector. To an aquarium, we applied between 2.5 and 50 mg/L DHF diluted in ethanol, in combination with 42.7 mg/L diazepam. DHF within the range of 10-20 mg/L acted collaboratively in combination with diazepam, but the sedative action of DHF was reversed by 3 mg/L flumazenil. Additionally, fish exposed for 24 h to 2.5-20 mg/L DHF showed no side effects and there was sustained sedation during the first 12 h of drug exposure with 10-20 mg/L DHF. DHF pretreatment did not increase plasma cortisol levels in fish subjected to a stress protocol. Moreover, the stress-induced cortisol peak was absent following pretreatment with 20 mg/L DHF. DHF proved to be a relatively safe sedative or anesthetic, which interacts with GABAergic and cortisol pathways in fish. PMID:26628396

  7. Structural plasticity of GABAergic axons is regulated by network activity and GABAA receptor activation

    PubMed Central

    Schuemann, Anne; Klawiter, Agnieszka; Bonhoeffer, Tobias; Wierenga, Corette J.

    2013-01-01

    Coordinated changes at excitatory and inhibitory synapses are essential for normal brain development and function. It is well established that excitatory neurons undergo structural changes, but our knowledge about inhibitory structural plasticity is rather scarce. Here we present a quantitative analysis of the dynamics of GABAergic boutons in the dendritic region of the hippocampal CA1 area using time-lapse two-photon imaging in organotypic hippocampal cultures from GAD65-GFP mice. We show that ~20% of inhibitory boutons are not stable. They are appearing, disappearing and reappearing at specific locations along the inhibitory axon and reflect immature or incomplete synapses. Furthermore, we observed that persistent boutons show large volume fluctuations over several hours, suggesting that presynaptic content of inhibitory synapses is not constant. Our data show that inhibitory boutons are highly dynamic structures and suggest that inhibitory axons are continuously probing potential locations for inhibitory synapse formation by redistributing presynaptic material along the axon. In addition, we found that neuronal activity affects the exploratory dynamics of inhibitory axons. Blocking network activity rapidly reduces the number of transient boutons, whereas enhancing activity reduces the number of persistent inhibitory boutons, possibly reflecting enhanced competition between boutons along the axon. The latter effect requires signaling through GABAA receptors. We propose that activity-dependent regulation of bouton dynamics contributes to inhibitory synaptic plasticity. PMID:23805077

  8. Spatial and Temporal Dynamics in the Ionic Driving Force for GABAA Receptors

    PubMed Central

    Wright, R.; Raimondo, J. V.; Akerman, C. J.

    2011-01-01

    It is becoming increasingly apparent that the strength of GABAergic synaptic transmission is dynamic. One parameter that can establish differences in the actions of GABAergic synapses is the ionic driving force for the chloride-permeable GABAA receptor (GABAAR). Here we review some of the sophisticated ways in which this ionic driving force can vary within neuronal circuits. This driving force for GABAARs is subject to tight spatial control, with the distribution of Cl− transporter proteins and channels generating regional variation in the strength of GABAAR signalling across a single neuron. GABAAR dynamics can result from short-term changes in their driving force, which involve the temporary accumulation or depletion of intracellular Cl−. In addition, activity-dependent changes in the expression and function of Cl− regulating proteins can result in long-term shifts in the driving force for GABAARs. The multifaceted regulation of the ionic driving force for GABAARs has wide ranging implications for mature brain function, neural circuit development, and disease. PMID:21766044

  9. Similar GABAA receptor subunit composition in somatic and axon initial segment synapses of hippocampal pyramidal cells

    PubMed Central

    Kerti-Szigeti, Katalin; Nusser, Zoltan

    2016-01-01

    Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in α1 and α2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the α1, α2, β1, β2, β3 and γ2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the α1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The α2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content. DOI: http://dx.doi.org/10.7554/eLife.18426.001 PMID:27537197

  10. Postnatal development of the alpha1 containing GABAA receptor subunit in rat hippocampus.

    PubMed

    Lopez-Tellez, Juan Felix; Vela, Jose; del Rio, Juan Carlos; Ramos, Blanca; Baglietto-Vargas, David; Santa-Maria, Consuelo; Ruano, Diego; Gutierrez, Antonia; Vitorica, Javier

    2004-01-31

    Here we have studied the developmental expression of alpha1 subunit of the GABAA receptor in comparison with the expression of alpha2 subunit and several GABAergic markers (parvalbumin (PV), calretinin (CR), somatostatin (SOM), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP)). The alpha1 expression (mRNA and protein) was low at birth and increased progressively until the adulthood. This expression pattern was similar to that observed for PV, opposite to that of CR (high at birth and decreased continuously until the adulthood) and differed from that observed for the alpha2 and neuropeptides (SOM, NPY and VIP) (in all cases, a clear peak in expression was observed at P10). We further investigated the expression of alpha1, PV and CR by immunohistochemistry. As expected, the alpha1 and the PV expression were low at birth and increased progressively until the adulthood. Both alpha1 and PV were co-expressed by the same interneuronal population, however, the maturation of the alpha1 subunit preceded to that of PV. Finally, we observed a gradient of maturation between the different fields of the hippocampus proper (CA2-3 preceded to CA1 and DG). This gradient could be related to the high expression of CR positive cells and fibers during the first 10 postnatal days, located principally in the stratum lacunosum moleculare of the CA2-3 layers.

  11. Effect of (+)-dehydrofukinone on GABAA receptors and stress response in fish model.

    PubMed

    Garlet, Q I; Pires, L C; Silva, D T; Spall, S; Gressler, L T; Bürger, M E; Baldisserotto, B; Heinzmann, B M

    2016-01-01

    (+)-Dehydrofukinone (DHF) is a major component of the essential oil of Nectandra grandiflora (Lauraceae), and exerts a depressant effect on the central nervous system of fish. However, the neuronal mechanism underlying DHF action remains unknown. This study aimed to investigate the action of DHF on GABAA receptors using a silver catfish (Rhamdia quelen) model. Additionally, we investigated the effect of DHF exposure on stress-induced cortisol modulation. Chemical identification was performed using gas chromatography-mass spectrometry and purity was evaluated using gas chromatography with a flame ionization detector. To an aquarium, we applied between 2.5 and 50 mg/L DHF diluted in ethanol, in combination with 42.7 mg/L diazepam. DHF within the range of 10-20 mg/L acted collaboratively in combination with diazepam, but the sedative action of DHF was reversed by 3 mg/L flumazenil. Additionally, fish exposed for 24 h to 2.5-20 mg/L DHF showed no side effects and there was sustained sedation during the first 12 h of drug exposure with 10-20 mg/L DHF. DHF pretreatment did not increase plasma cortisol levels in fish subjected to a stress protocol. Moreover, the stress-induced cortisol peak was absent following pretreatment with 20 mg/L DHF. DHF proved to be a relatively safe sedative or anesthetic, which interacts with GABAergic and cortisol pathways in fish.

  12. GABAA receptor-acting neurosteroids: A role in the development and regulation of the stress response

    PubMed Central

    Gunn, Benjamin G.; Cunningham, Linda; Mitchell, Scott G.; Swinny, Jerome D.; Lambert, Jeremy J.; Belelli, Delia

    2015-01-01

    Regulation of hypothalamic–pituitary–adrenocortical (HPA) axis activity by stress is a fundamental survival mechanism and HPA-dysfunction is implicated in psychiatric disorders. Adverse early life experiences, e.g. poor maternal care, negatively influence brain development and programs an abnormal stress response by encoding long-lasting molecular changes, which may extend to the next generation. How HPA-dysfunction leads to the development of affective disorders is complex, but may involve GABAA receptors (GABAARs), as they curtail stress-induced HPA axis activation. Of particular interest are endogenous neurosteroids that potently modulate the function of GABAARs and exhibit stress-protective properties. Importantly, neurosteroid levels rise rapidly during acute stress, are perturbed in chronic stress and are implicated in the behavioural changes associated with early-life adversity. We will appraise how GABAAR-active neurosteroids may impact on HPA axis development and the orchestration of the stress-evoked response. The significance of these actions will be discussed in the context of stress-associated mood disorders. PMID:24929099

  13. Effect of (+)-dehydrofukinone on GABAA receptors and stress response in fish model.

    PubMed

    Garlet, Q I; Pires, L C; Silva, D T; Spall, S; Gressler, L T; Bürger, M E; Baldisserotto, B; Heinzmann, B M

    2016-01-01

    (+)-Dehydrofukinone (DHF) is a major component of the essential oil of Nectandra grandiflora (Lauraceae), and exerts a depressant effect on the central nervous system of fish. However, the neuronal mechanism underlying DHF action remains unknown. This study aimed to investigate the action of DHF on GABAA receptors using a silver catfish (Rhamdia quelen) model. Additionally, we investigated the effect of DHF exposure on stress-induced cortisol modulation. Chemical identification was performed using gas chromatography-mass spectrometry and purity was evaluated using gas chromatography with a flame ionization detector. To an aquarium, we applied between 2.5 and 50 mg/L DHF diluted in ethanol, in combination with 42.7 mg/L diazepam. DHF within the range of 10-20 mg/L acted collaboratively in combination with diazepam, but the sedative action of DHF was reversed by 3 mg/L flumazenil. Additionally, fish exposed for 24 h to 2.5-20 mg/L DHF showed no side effects and there was sustained sedation during the first 12 h of drug exposure with 10-20 mg/L DHF. DHF pretreatment did not increase plasma cortisol levels in fish subjected to a stress protocol. Moreover, the stress-induced cortisol peak was absent following pretreatment with 20 mg/L DHF. DHF proved to be a relatively safe sedative or anesthetic, which interacts with GABAergic and cortisol pathways in fish. PMID:26628396

  14. Phasic, Nonsynaptic GABA-A Receptor-Mediated Inhibition Entrains Thalamocortical Oscillations

    PubMed Central

    Rovó, Zita; Mátyás, Ferenc; Barthó, Péter; Slézia, Andrea; Lecci, Sandro; Pellegrini, Chiara; Astori, Simone; Dávid, Csaba; Hangya, Balázs

    2014-01-01

    GABA-A receptors (GABA-ARs) are typically expressed at synaptic or nonsynaptic sites mediating phasic and tonic inhibition, respectively. These two forms of inhibition conjointly control various network oscillations. To disentangle their roles in thalamocortical rhythms, we focally deleted synaptic, γ2 subunit-containing GABA-ARs in the thalamus using viral intervention in mice. After successful removal of γ2 subunit clusters, spontaneous and evoked GABAergic synaptic currents disappeared in thalamocortical cells when the presynaptic, reticular thalamic (nRT) neurons fired in tonic mode. However, when nRT cells fired in burst mode, slow phasic GABA-AR-mediated events persisted, indicating a dynamic, burst-specific recruitment of nonsynaptic GABA-ARs. In vivo, removal of synaptic GABA-ARs reduced the firing of individual thalamocortical cells but did not abolish slow oscillations or sleep spindles. We conclude that nonsynaptic GABA-ARs are recruited in a phasic manner specifically during burst firing of nRT cells and provide sufficient GABA-AR activation to control major thalamocortical oscillations. PMID:24849349

  15. Early postnatal GABAA receptor modulation reverses deficits in neuronal maturation in a conditional neurodevelopmental mouse model of DISC1

    PubMed Central

    Saito, Atsushi; Taniguchi, Yu; Rannals, Matthew D.; Merfeld, Emily B.; Ballinger, Michael D.; Koga, Minori; Ohtani, Yoshikazu; Gurley, David A.; Sedlak, Thomas W.; Cross, Alan; Moss, Stephen J.; Brandon, Nicholas J.; Maher, Brady J.; Kamiya, Atsushi

    2015-01-01

    Exploring drug targets based on disease-associated molecular mechanisms during development is crucial for the generation of novel prevention and treatment strategies for neurodevelopmental psychiatric conditions. We report that prefrontal cortex-specific postnatal knockdown of DISC1 via in utero electroporation combined with an inducible knockdown expression system drives deficits in synaptic GABAA function and dendritic development in pyramidal neurons, as well as abnormalities in sensorimotor gating, albeit without profound memory deficits. We show for the first time that DISC1 is specifically involved in regulating cell surface expression of α2 subunit-containing GABAA receptors in immature developing neurons, but not after full maturation. Notably, pharmacological intervention with α2/3 subtype-selective GABAA receptor positive allosteric modulators during the early postnatal period ameliorates dendritic deficits and behavioral abnormalities induced by knockdown of DISC1. These findings highlight a critical role of DISC1-mediated disruption of postnatal GABA signaling in aberrant prefrontal cortex maturation and function. PMID:26728564

  16. Flumazenil decreases surface expression of α4β2δ GABAA receptors by increasing the rate of receptor internalization.

    PubMed

    Kuver, Aarti; Smith, Sheryl S

    2016-01-01

    Increases in expression of α4βδ GABAA receptors (GABARs), triggered by fluctuations in the neurosteroid THP (3α-OH-5α[β]-pregnan-20-one), are associated with changes in mood and cognition. We tested whether α4βδ trafficking and surface expression would be altered by in vitro exposure to flumazenil, a benzodiazepine ligand which reduces α4βδ expression in vivo. We first determined that flumazenil (100 nM-100 μM, IC50=∼1 μM) acted as a negative modulator, reducing GABA (10 μM)-gated current in the presence of 100 nM THP (to increase receptor efficacy), assessed with whole cell patch clamp recordings of recombinant α4β2δ expressed in HEK-293 cells. Surface expression of recombinant α4β2δ receptors was detected using a 3XFLAG reporter at the C-terminus of α4 (α4F) using confocal immunocytochemical techniques following 48 h exposure of cells to GABA (10 μM)+THP (100 nM). Flumazenil (10 μM) decreased surface expression of α4F by ∼60%, while increasing its intracellular accumulation, after 48 h. Reduced surface expression of α4β2δ after flumazenil treatment was confirmed by decreases in the current responses to 100 nM of the GABA agonist gaboxadol. Flumazenil-induced decreases in surface expression of α4β2δ were prevented by the dynamin blocker, dynasore, and by leupeptin, which blocks lysosomal enzymes, suggesting that flumazenil is acting to increase endocytosis and lysosomal degradation of the receptor. Flumazenil increased the rate of receptor removal from the cell surface by 2-fold, assessed using botulinum toxin B to block insertion of new receptors. These findings may suggest new therapeutic strategies for regulation of α4β2δ expression using flumazenil.

  17. Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors.

    PubMed

    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

  18. 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

  19. Significance of GABA(A) receptor heterogeneity: clues from developing neurons.

    PubMed

    Fritschy, Jean-Marc

    2015-01-01

    Briefly after the landmark discovery by Hanns Möhler that GABA(A) receptors (GABA(A)R) are the site of action of benzodiazepine site ligands, their distribution in the rodent CNS during development was mapped by autoradiography, demonstrating early and widespread expression of GABA(A)R in the brain and spinal cord. Ten years later, the first studies using subunit-specific antibodies revealed unsuspected heterogeneity in the subunit composition of GABA(A)R in developing brain, with striking regional specificity and rapid changes in expression and subcellular localization correlating with the maturation of neuronal circuits. These data contributed to the wealth of evidence that GABAergic neurotransmission, acting both synaptically and extrasynaptically, modulates major steps of neuronal development (proliferation, migration, differentiation, and circuit formation). In immature neurons, GABA(A)R activation leads to neuronal depolarization and activation of Ca(2+) signals, which mediate many of the developmental effects of GABA. Therefore, GABA(A)R heterogeneity might be essential to fine-tune GABA actions in the dynamics of CNS maturation. Furthermore, since a disturbance of GABAergic function during ontogeny can potentially affect many aspects of CNS maturation and contribute to the etiology of major brain disorders, GABA(A)R heterogeneity provides a substrate for the development of a tailored pharmacology targeting specific receptor subtypes. This chapter provides a brief overview of these issues with a special focus on the seminal contributions of Hanns Möhler to the emergence of these concepts of fundamental relevance in today's neuroscience research and pharmacological developments. PMID:25637436

  20. GODZ-mediated palmitoylation of GABA(A) receptors is required for normal assembly and function of GABAergic inhibitory synapses.

    PubMed

    Fang, Cheng; Deng, Lunbin; Keller, Cheryl A; Fukata, Masaki; Fukata, Yuko; Chen, Gong; Lüscher, Bernhard

    2006-12-01

    Golgi-specific DHHC (Asp-His-His-Cys) zinc finger protein (GODZ) is a DHHC family palmitoyl acyltransferase that is implicated in palmitoylation and regulated trafficking of diverse substrates that function either at inhibitory or excitatory synapses. Of particular interest is the gamma2 subunit of GABA(A) receptors, which is required for targeting these receptors to inhibitory synapses. Here, we report that GODZ and, to a lesser extent, its close paralog sertoli cell gene with a zinc finger domain-beta (SERZ-beta) are the main members of the DHHC family of enzymes that are able to palmitoylate the gamma2 subunit in heterologous cells. Yeast two-hybrid and colocalization assays in human embryonic kidney 293T (HEK293T) cells indicate that GODZ and SERZ-beta show indistinguishable palmitoylation-dependent interaction with the gamma2 subunit. After coexpression in HEK293T cells, they form homomultimers and heteromultimers, as shown by coimmunoprecipitation and in vivo cross-linking experiments. Analyses in neurons transfected with dominant-negative GODZ (GODZ(C157S)) or plasmid-based GODZ-specific RNAi indicate that GODZ is required for normal accumulation of GABA(A) receptors at synapses, for normal whole-cell and synaptic GABAergic inhibitory function and, indirectly, for GABAergic innervation. Unexpectedly, GODZ was found to be dispensable for normal postsynaptic AMPA receptor-mediated glutamatergic transmission. We conclude that GODZ-mediated palmitoylation of GABA(A) receptors and possibly other substrates contributes selectively to the formation and normal function of GABAergic inhibitory synapses.

  1. SSRI augmentation of antipsychotic alters expression of GABA(A) receptor and related genes in PMC of schizophrenia patients.

    PubMed

    Silver, Henry; Susser, Ehud; Danovich, Lena; Bilker, Warren; Youdim, Moussa; Goldin, Vladimir; Weinreb, Orly

    2011-06-01

    Clinical studies have shown that negative symptoms of schizophrenia unresponsive to antipsychotic given alone can improve after augmentation with SSRI antidepressant. Laboratory investigations into the mechanism of this synergism showed that co-administration of SSRI and antipsychotic produces changes in GABA(A) receptor and related systems, which differ from the effects of each drug alone. To examine the clinical relevance of these findings, the current study examined the effects of SSRI augmentation treatment on GABA(A) receptor and related systems in schizophrenia patients. Schizophrenia patients with high levels of negative symptoms unresponsive to antipsychotic treatment received add-on fluvoxamine (100 mg/d). Blood was taken before and 1, 3 and 6 wk after adding fluvoxamine and peripheral mononuclear cells (PMC) isolated. RNA encoding for GABA(A)β3, 5-HT2A, and 5-HT7 receptors, PKCβ2, and brain-derived neurotrophic factor (BDNF) was assayed with real-time RT-PCR. Plasma BDNF protein was assayed using ELISA. Clinical symptoms were assessed with validated rating scales. We found significant increase in mRNA encoding for GABA(A)β3 and 5-HT2A, 5-HT7 receptors and BDNF and a reduction in PKCβ2 mRNA. Plasma BDNF protein concentrations were increased. There were significant correlations among the genes. Clinical symptoms improved significantly. mRNA expression of PKCβ2, 5-HT2A and 5-HT7 showed significant associations with clinical symptoms. Combined SSRI+antipsychotic treatment is associated with changes in GABA(A) receptor and in related signalling systems in patients. These changes may be part of the mechanism of clinically effective drug action and may prove to be biomarkers of pharmacological response.

  2. Structure of alpha6 beta3 delta GABA(A) receptors and their lack of ethanol sensitivity.

    PubMed

    Baur, Roland; Kaur, Kuldeep H; Sigel, Erwin

    2009-12-01

    Delta (delta) subunit containing GABA(A) receptors are expressed extra-synaptically and mediate tonic inhibition. In cerebellar granule cells, they often form a receptor together with alpha(6) subunits. We were interested to determine the architecture of these receptors. We predefined the subunit arrangement of 24 different GABA(A) receptor pentamers by subunit concatenation. These receptors (composed of alpha(6), beta(3) and delta subunits) were expressed in Xenopus oocytes and their electrophysiological properties analyzed. Currents elicited in response to GABA were determined in presence and absence of 3alpha, 21-dihydroxy-5alpha-pregnan-20-one and to 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol. alpha(6)-beta(3)-alpha(6)/delta receptors showed a substantial response to GABA alone. Three receptors, beta(3)-alpha(6)-delta/alpha(6)-beta(3), alpha(6)-beta(3)-alpha(6)/beta(3)-delta and beta(3)-delta-beta(3)/alpha(6)-beta(3), were only uncovered in the combined presence of the neurosteroid 3alpha, 21-dihydroxy-5alpha-pregnan-20-one with GABA. All four receptors were activated by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol. None of the functional receptors was modulated by physiological concentrations (up to 30 mM) of ethanol. GABA concentration response curves indicated that the delta subunit can contribute to the formation of an agonist site. We conclude from the investigated receptors that the delta subunit can assume multiple positions in a receptor pentamer composed of alpha(6), beta(3) and delta subunits.

  3. Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.

    PubMed

    Middendorp, Simon J; Maldifassi, Maria C; Baur, Roland; Sigel, Erwin

    2015-08-01

    GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ- subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ- site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.

  4. 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

  5. The blockade of GABAA receptors attenuates the inhibitory effect of orexin type 1 receptors antagonist on morphine withdrawal syndrome in rats.

    PubMed

    Davoudi, Mahnaz; Azizi, Hossein; Mirnajafi-Zadeh, Javad; Semnanian, Saeed

    2016-03-23

    The aim of present study was to investigate the involvement of orexin-A neuropeptide in naloxone-induced morphine withdrawal syndrome via modulating neurons bearing GABAA receptors. The locus coeruleus (LC) is a sensitive site for expression of the somatic aspects of morphine withdrawal. Intra-LC microinjection of GABAA receptor agonist attenuates morphine withdrawal signs in rats. Here we studied the influence of LC orexin type 1 receptors blockade by SB-334867 in presence of bicuculline, a GABAA receptor antagonist, on naloxone-induced morphine withdrawal syndrome. Adult male Wistar rats, weighing 250-300 g, were rendered dependent on morphine by subcutaneous (s.c.) injection of increasing morphine doses (6, 16, 26, 36, 46, 56 and 66 mg/kg, 2 ml/kg) at set intervals of 24 h for 7 days. On 8th day, naloxone (3 mg/kg, s.c.) was injected and the somatic signs of morphine withdrawal were evaluated. Intra-LC microinjections (0.2 μl) of either bicuculline (15 μM) or SB-334867 (3 mM) or a combination of both chemicals were done immediately before naloxone injection. Intra-LC microinjection of bicuculline (15 μM) had no significant effect on morphine withdrawal signs, whereas intra-LC microinjection of SB-334867 considerably attenuated morphine withdrawal signs. However, the effect of SB-334867 in attenuating naloxone-induced morphine withdrawal signs was blocked in presence of bicuculline. This finding, for the first time, indicated that orexin-A may participate in expression of naloxone-induced morphine withdrawal syndrome partly through decreasing the activity of neurons bearing GABAA receptors.

  6. Electrophysiological study of SR 42641, a novel aminopyridazine derivative of GABA: antagonist properties and receptor selectivity of GABAA versus GABAB responses.

    PubMed Central

    Desarmenien, M.; Desaulles, E.; Feltz, P.; Hamann, M.

    1987-01-01

    A new arylamino-pyridazine gamma-aminobutyric acid (GABA) derivative, SR 42641, has been tested for its ability to antagonize the actions of GABA on mammalian sensory neurones. SR 42641 and bicuculline reversibly decreased GABAA-induced depolarizations and currents recorded intracellularly from dorsal root ganglion neurons (DRG). Dose-response curves were shifted to the right in a parallel fashion. KB values (determined under voltage clamp conditions) were respectively 0.12 +/- 0.05 and 0.38 +/- 0.08 microM. Similar values were obtained with current clamp recording conditions. The study of the GABA-induced Cl- current under voltage-clamp conditions did not show any voltage-dependency of the antagonist effect of SR 42641. In nodose ganglion neurones, SR 42641 (0.4-4.5 microM) did not alter the (-)-baclofen-induced shortening of the calcium component of action potentials. At concentrations higher than 10 microM, SR 42641 itself prolonged calcium-dependent action potentials. Patch-clamp recordings from DRG cultured neurones indicated that SR 42641 did not affect the calcium current responsible for sustained calcium entry into cells. We conclude that SR 42641 is a potent competitive GABA antagonist, specific for the GABAA receptor. It does not act at the level of the chloride ionophore. PMID:2435350

  7. 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

  8. Activity-dependent bidirectional regulation of GABAA receptor channels by the 5-HT4 receptor-mediated signalling in rat prefrontal cortical pyramidal neurons

    PubMed Central

    Cai, Xiang; Flores-Hernandez, Jorge; Feng, Jian; Yan, Zhen

    2002-01-01

    Emerging evidence has implicated a potential role for 5-HT4 receptors in cognition and anxiolysis. One of the main target structures of 5-HT4 receptors on ‘cognitive and emotional’ pathways is the prefrontal cortex (PFC). As GABAergic signalling plays a key role in regulating PFC functions, we examined the effect of 5-HT4 receptors on GABAA receptor channels in PFC pyramidal neurons. Application of 5-HT4 receptor agonists produced either an enhancement or a reduction of GABA-evoked currents in PFC neurons, which are both mediated by anchored protein kinase A (PKA). Although PKA phosphorylation of GABAA receptor β3 or β1 subunits leads to current enhancement or reduction respectively in heterologous expression systems, we found that β3 and β1 subunits are co-expressed in PFC pyramidal neurons. Interestingly, altering PKA activation levels can change the direction of the dual effect, switching enhancement to reduction and vice versa. In addition, increased neuronal activity in PFC slices elevated the PKA activation level, changing the enhancing effect of 5-HT4 receptors on the amplitude of GABAergic inhibitory postsynaptic currents (IPSCs) to a reduction. These results suggest that 5-HT4 receptors can modulate GABAergic signalling bidirectionally, depending on the basal PKA activation levels that are determined by neuronal activity. This modulation provides a unique and flexible mechanism for 5-HT4 receptors to dynamically regulate synaptic transmission and neuronal excitability in the PFC network. PMID:11986365

  9. Early postnatal switch in GABAA receptor α-subunits in the reticular thalamic nucleus.

    PubMed

    Pangratz-Fuehrer, Susanne; Sieghart, Werner; Rudolph, Uwe; Parada, Isabel; Huguenard, John R

    2016-03-01

    The GABAergic neurons of the thalamic reticular nucleus (nRt) provide the primary source of inhibition within the thalamus. Using physiology, pharmacology, and immunohistochemistry in mice, we characterized postsynaptic developmental changes in these inhibitory projection neurons. First, at postnatal days 3-5 (P3-5), inhibitory postsynaptic currents (IPSCs) decayed very slowly, followed by a biphasic developmental progression, becoming faster at P6-8 and then slower again at P9-11 before stabilizing in a mature form around P12. Second, the pharmacological profile of GABA(A) receptor (GABA(A)R)-mediated IPSCs differed between neonatal and mature nRt neurons, and this was accompanied by reciprocal changes in α3 (late) and α5 (early) subunit expression in nRt. Zolpidem, selective for α1- and α3-containing GABA(A)Rs, augmented only mature IPSCs, whereas clonazepam enhanced IPSCs at all stages. This effect was blocked by the α5-specific inverse agonist L-655,708, but only in immature neurons. In α3(H126R) mice, in which α3-subunits were mutated to become benzodiazepine insensitive, IPSCs were enhanced compared with those in wild-type animals in early development. Third, tonic GABA(A)R activation in nRt is age dependent and more prominent in immature neurons, which correlates with early expression of α5-containing GABA(A)Rs. Thus neonatal nRt neurons show relatively high expression of α5-subunits, which contributes to both slow synaptic and tonic extrasynaptic inhibition. The postnatal switch in GABA(A)R subunits from α5 to α3 could facilitate spontaneous network activity in nRt that occurs at this developmental time point and which is proposed to play a role in early circuit development. PMID:26631150

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

    PubMed

    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 GABA(A) receptor (GABA(A)R), including some benzodiazepines. The ligands satisfy the Lipinski rules and contain a pharmacophore core that has been previously reported to be a GABA(A)R 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 GABA(A)R 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 GABA(A)R-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 GABA(A)R-D1, and GABA(A)R-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.

  11. GABAA receptor activity shapes the formation of inhibitory synapses between developing medium spiny neurons

    PubMed Central

    Arama, Jessica; Abitbol, Karine; Goffin, Darren; Fuchs, Celine; Sihra, Talvinder S.; Thomson, Alex M.; Jovanovic, Jasmina N.

    2015-01-01

    Basal ganglia play an essential role in motor coordination and cognitive functions. The GABAergic medium spiny neurons (MSNs) account for ~95% of all the neurons in this brain region. Central to the normal functioning of MSNs is integration of synaptic activity arriving from the glutamatergic corticostriatal and thalamostriatal afferents, with synaptic inhibition mediated by local interneurons and MSN axon collaterals. In this study we have investigated how the specific types of GABAergic synapses between the MSNs develop over time, and how the activity of GABAA receptors (GABAARs) influences this development. Isolated embryonic (E17) MSNs form a homogenous population in vitro and display spontaneous synaptic activity and functional properties similar to their in vivo counterparts. In dual whole-cell recordings of synaptically connected pairs of MSNs, action potential (AP)-activated synaptic events were detected between 7 and 14 days in vitro (DIV), which coincided with the shift in GABAAR operation from depolarization to hyperpolarization, as detected indirectly by intracellular calcium imaging. In parallel, the predominant subtypes of inhibitory synapses, which innervate dendrites of MSNs and contain GABAAR α1 or α2 subunits, underwent distinct changes in the size of postsynaptic clusters, with α1 becoming smaller and α2 larger over time, while both the percentage and the size of mixed α1/α2-postsynaptic clusters were increased. When activity of GABAARs was under chronic blockade between 4–7 DIV, the structural properties of these synapses remained unchanged. In contrast, chronic inhibition of GABAARs between 7–14 DIV led to reduction in size of α1- and α1/α2-postsynaptic clusters and a concomitant increase in number and size of α2-postsynaptic clusters. Thus, the main subtypes of GABAergic synapses formed by MSNs are regulated by GABAAR activity, but in opposite directions, and thus appear to be driven by different molecular mechanisms. PMID

  12. Targeted disruption of layer 4 during development increases GABAA receptor neurotransmission in the neocortex

    PubMed Central

    Abbah, J.; Braga, Maria F. M.

    2013-01-01

    Cortical dysplasia (CD) associates with clinical pathologies, including epilepsy and mental retardation. CD results from impaired migration of immature neurons to their cortical targets, leading to clustering of neural cells and changes in cortical properties. We developed a CD model by administering methylazoxymethanol (MAM), an anti-mitotic, to pregnant ferrets on embryonic day 33; this leads to reduction in cortical thickness in addition to redistribution and increased expression of GABAA receptors (GABAAR). We evaluated the impact of MAM treatment on GABAAR-mediated synaptic transmission in postnatal day 0–1 neurons, leaving the ganglionic eminence (GE) and in layer 2/3 pyramidal cells of postnatal day 28–38 ferrets. Embryonic day 33 MAM treatment significantly increases the amplitude and frequency of spontaneous GABAAR-mediated inhibitory postsynaptic currents (IPSCs) in the cells leaving the GE. In older MAM-treated animals, the amplitude and frequency of GABAAR-mediated spontaneous IPSCs in layer 2/3 pyramidal cells is increased, as are the amplitude and frequency of miniature IPSCs. The kinetics of GABAAR opening also altered following treatment with MAM. Western blot analysis shows that the expression of the GABAAα3R and GABAAγ2R subunits amplified in our model animals. We did not observe any significant change in the passive properties of either the layer 2/3 pyramidal cells or cells leaving the GE after MAM treatment. These observations reinforce the idea that synaptic neurotransmission through GABAAR enhances following treatment with MAM and coincides with our finding of increased GABAAαR expression within the upper cortical layers. Overall, we demonstrate that small amounts of toxins delivered during corticogenesis can result in long-lasting changes in ambient expression of GABAAR that influence intrinsic neuronal properties. PMID:24155012

  13. 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

  14. Artemisia capillaris Thunberg Produces Sedative-Hypnotic Effects in Mice, Which are Probably Mediated Through Potentiation of the GABAA Receptor.

    PubMed

    dela Peña, Irene Joy I; Hong, Eunyoung; Kim, Hee Jin; de la Peña, June Bryan; Woo, Tae Sun; Lee, Yong Soo; Cheong, Jae Hoon

    2015-01-01

    The Artemisia group of plants has long been used as a traditional remedy for various conditions. The present study assessed the sleep-promoting (sedative-hypnotic) effects of Artemisia capillaris Thunberg (A. capillaris), and elucidated a possible mechanism behind its effect. ICR mice were given A. capillaris extract (oral) at different dosages (50, 100, 200, 300, or 400 mg/kg), distilled water (oral; control), or diazepam (intraperitoneal; reference drug). One hour after administration, locomotion (open-field test) and motor coordination (rota-rod test) were assessed. The extract's effect on pentobarbital-induced sleep was also evaluated. Additionally, electroencephalographic (EEG) recordings were measured in rats. To evaluate a possible mechanism behind its effects, changes in chloride ( Cl (-)) ion influx were measured in human neuroblastoma cells. As compared to the control group, mice treated with A. capillaris demonstrated significantly decreased locomotor activity and impaired motor balance and coordination. The extract also shortened the onset and lengthened the duration of sleep induced by pentobarbital sodium. These effects were comparable to that induced by diazepam. Furthermore, A. capillaris-treated rats showed increased delta and decreased alpha EEG waves; an electroencephalographic pattern indicative of relaxation or sedation. In neuroblastoma cells, the extract dose-dependently increased Cl (-) ion influx, which was blocked by co-administration of bicuculline, a GABAA receptor competitive antagonist, suggesting that its effects are mediated through the GABAA receptor- Cl (-) ion channel complex. Altogether, the results of the present study demonstrate that A. capillaris possesses potent sedative-hypnotic effects, which are probably mediated through potentiation of the GABAA receptor- Cl (-) ion channel complex.

  15. Reinforcing effects of compounds lacking intrinsic efficacy at α1 subunit-containing GABAA receptor subtypes in midazolam- but not cocaine-experienced rhesus monkeys.

    PubMed

    Shinday, Nina M; Sawyer, Eileen K; Fischer, Bradford D; Platt, Donna M; Licata, Stephanie C; Atack, John R; Dawson, Gerard R; Reynolds, David S; Rowlett, James K

    2013-05-01

    Benzodiazepines are prescribed widely but their utility is limited by unwanted side effects, including abuse potential. The mechanisms underlying the abuse-related effects of benzodiazepines are not well understood, although α1 subunit-containing GABAA receptors have been proposed to have a critical role. Here, we examine the reinforcing effects of several compounds that vary with respect to intrinsic efficacy at α2, α3, and α5 subunit-containing GABAA receptors but lack efficacy at α1 subunit-containing GABAA receptors ('α1-sparing compounds'): MRK-623 (functional selectivity for α2/α3 subunit-containing receptors), TPA023B (functional selectivity for α2/α3/α5 subunit-containing receptors), and TP003 (functional selectivity for α3 subunit-containing receptors). The reinforcing effects of the α1-sparing compounds were compared with those of the non-selective benzodiazepine receptor partial agonist MRK-696, and non-selective benzodiazepine receptor full agonists, midazolam and lorazepam, in rhesus monkeys trained to self-administer midazolam or cocaine, under a progressive-ratio schedule of intravenous (i.v.) drug injection. The α1-sparing compounds were self-administered significantly above vehicle levels in monkeys maintained under a midazolam baseline, but not under a cocaine baseline over the dose ranges tested. Importantly, TP003 had significant reinforcing effects, albeit at lower levels of self-administration than non-selective benzodiazepine receptor agonists. Together, these results suggest that α1 subunit-containing GABAA receptors may have a role in the reinforcing effects of benzodiazepine-type compounds in monkeys with a history of stimulant self-administration, whereas α3 subunit-containing GABAA receptors may be important mediators of the reinforcing effects of benzodiazepine-type compounds in animals with a history of sedative-anxiolytic/benzodiazepine self-administration. PMID:23303046

  16. The non-anaesthetic propofol analogue 2,6-di-tert-butylphenol fails to modulate GABA(A) receptor function.

    PubMed

    Ahrens, Jörg; Leuwer, Martin; de la Roche, Jeanne; Foadi, Nilufar; Krampfl, Klaus; Haeseler, Gertrud

    2009-01-01

    Modulation of inhibitory synaptic transmission within the central nervous system contributes considerably to the anaesthetic effects of propofol and its analogues in vivo. We have studied the effects of the non-anaesthetic propofol analogue 2,6-di-tert-butylphenol on rat alpha(1)beta(2)gamma(2) GABA(A) receptors expressed in a mammalian expression system (HEK 293 cells) using the whole-cell patch clamp technique. Our experiments showed that 2,6-di-tert-butylphenol completely lacks co-activation and direct activation of the inhibitory GABA(A) receptor. Our results support the assumption that modulation of inhibitory GABA(A) receptor function is responsible for the anaesthetic effects of propofol in vivo.

  17. Gender and age differences in expression of GABAA receptor subunits in rat somatosensory thalamus and cortex in an absence epilepsy model

    PubMed Central

    Li, Huifang; Huguenard, John R.; Fisher, Robert S.

    2009-01-01

    Absence epilepsy is more prevalent in females, but reasons for this gender asymmetry are unknown. We reported previously that perinatal treatment of Long-Evans Hooded rats with the cholesterol synthesis inhibitor (CSI) AY9944 causes a life-long increase in EEG spike-wave discharges (SWDs), correlated with decreased expression of GABAA receptor subunit γ2 protein levels in thalamic reticular and ventrobasal nuclei (SS thalamus) (Li et al., 2006). In this study, we explored time course and gender different effects of perinatal AY9944 treatment on expression of GABAA receptor α1 and γ2 subunits in SS thalamus and SS cortex. Perinatal AY9944 treatment-induced decreases in GABAA γ2 receptor subunits in rat SS thalamus and increases in SS cortex are gender and age-specific. The findings suggest a mechanism for the higher prevalence of absence epilepsy in female patients. PMID:17208003

  18. Cyclin-Dependent Kinase 5 Is Involved in the Phosphorylation of Gephyrin and Clustering of GABAA Receptors at Inhibitory Synapses of Hippocampal Neurons

    PubMed Central

    Kalbouneh, Heba; Schlicksupp, Andrea; Kirsch, Joachim; Kuhse, Jochen

    2014-01-01

    CDK5 has been implicated in neural functions including growth, neuronal migration, synaptic transmission and plasticity of excitatory chemical synapses. Here we report robust effects of CDK5 on phosphorylation of the postsynaptic scaffold protein gephyrin and clustering of inhibitory GABAA receptors in hippocampal neurons. shRNA-mediated knockdown of CDK5 and pharmacological inhibition of cyclin-dependent kinases reduced phosphorylated gephyrin clusters and postsynaptic γ2-containing GABAA receptors. Phosphorylation of S270 is antagonized by PP1/PP2a phosphatase and site-directed mutagenesis and in vitro phosphorylation experiments indicate that S270 is a putative CDK5 phosphorylation site of gephyrin. Our data suggest that CDK5 plays an essential role for the stability of gephyrin-dependent GABAA receptor clusters in hippocampal neurons. PMID:25093719

  19. Pregnenolone sulfate block of GABA(A) receptors: mechanism and involvement of a residue in the M2 region of the alpha subunit.

    PubMed

    Akk, G; Bracamontes, J; Steinbach, J H

    2001-05-01

    Neurosteroids are produced in the brain, and can have rapid actions on membrane channels of neurons. Pregnenolone sulfate (PS) is a sulfated neurosteroid which reduces the responses of the [gamma]-aminobutyric acid A (GABA(A)) receptor. We analysed the actions of PS on single-channel currents from recombinant GABA(A) receptors formed from [alpha]1, [beta]2 and [gamma]2L subunits. Currents were elicited by a concentration of GABA eliciting a half-maximal response (50 microM) and a saturating concentration (1 mM). PS reduced the duration of clusters of single-channel activity at either concentration of GABA. PS had no discernable effect on rapid processes: no effects were apparent on channel opening and closing, nor on GABA affinity, and a rapidly recovering desensitised state was not affected. Instead, PS produced a slowly developing block which occurred at a similar rate for receptors with open or closed channels and with one or two bound GABA molecules. The rate of block was independent of membrane potential, implying that the charged sulfate moiety does not move through the membrane field. Change in a specific residue near the intracellular end of the channel lining portion of the [alpha]1 subunit had a major effect on the rate of block. Mutation of the residue [alpha]1 V256S reduced the rate of block by 30-fold. A mutation at the homologous position of the [beta]2 subunit ([beta]2 A252S) had no effect, nor did a complementary mutation in the [gamma]2L subunit ([gamma]2L S266A). It seems likely that this residue is involved in a conformational change underlying block by PS, instead of forming part of the binding site for PS.

  20. Biphasic GABA-A receptor-mediated effect on the spontaneous activity of the circular layer in cat terminal ileum.

    PubMed

    Pencheva, N; Radomirov, R

    1993-07-01

    1. The GABA and GABA-A receptor agonist muscimol changed the spontaneous mechanical activity of a circular layer isolated from cat terminal ileum, while the selective GABA-B receptor agonist (+/-)baclofen had no effect. 2. GABA at doses ranging from 1 microM to 2 mM elicited concentration-dependent biphasic responses which consisted of a relaxation followed by contraction, with a tonic and a phasic component. The EC50 values, calculated at 95% confidence limits (CL), were 94.9 microM (83.5-109.8 microM) and 66.0 microM (51.2-75.5 microM) for the relaxation and contractile phases, respectively. 3. The GABA-induced biphasic responses were sensitive to bicuculline and picrotoxinin and were entirely mimicked by muscimol. Bicuculline competitively antagonized the effects of GABA and gave closely similar pA2 values for both phases of these responses--inhibitory and stimulatory. Cross-desensitization occurred only between GABA and muscimol and not between (+/-)baclofen and GABA, or (+/-)baclofen and muscimol. 4. Both bicuculline-sensitive phases evoked by GABA and muscimol were abolished by tetrodotoxin or atropine, but were unaffected by guanethidine or naloxone. 5. The present results suggested that the biphasic GABA effect on the mechanical activity of the circular layer in cat terminal ileum was mediated by prejunctional GABA-A receptors, most probably through an action on the cholinergic pathway.

  1. 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

  2. NPY Y1 receptors differentially modulate GABAA and NMDA receptors via divergent signal-transduction pathways to reduce excitability of amygdala neurons.

    PubMed

    Molosh, Andrei I; Sajdyk, Tammy J; Truitt, William A; Zhu, Weiguo; Oxford, Gerry S; Shekhar, Anantha

    2013-06-01

    Neuropeptide Y (NPY) administration into the basolateral amygdala (BLA) decreases anxiety-like behavior, mediated in part through the Y1 receptor (Y1R) isoform. Activation of Y1Rs results in G-protein-mediated reduction of cAMP levels, which results in reduced excitability of amygdala projection neurons. Understanding the mechanisms linking decreased cAMP levels to reduced excitability in amygdala neurons is important for identifying novel anxiolytic targets. We studied the intracellular mechanisms of activation of Y1Rs on synaptic transmission in the BLA. Activating Y1Rs by [Leu(31),Pro(34)]-NPY (L-P NPY) reduced the amplitude of evoked NMDA-mediated excitatory postsynaptic currents (eEPSCs), without affecting AMPA-mediated eEPSCs, but conversely increased the amplitude of GABAA-mediated evoked inhibitory postsynaptic currents (eIPSCs). Both effects were abolished by the Y1R antagonist, PD160170. Intracellular GDP-β-S, or pre-treatment with either forskolin or 8Br-cAMP, eliminated the effects of L-P NPY on both NMDA- and GABAA-mediated currents. Thus, both the NMDA and GABAA effects of Y1R activation in the BLA are G-protein-mediated and cAMP-dependent. Pipette inclusion of protein kinase A (PKA) catalytic subunit blocked the effect of L-P NPY on GABAA-mediated eIPSCs, but not on NMDA-mediated eEPSCs. Conversely, activating the exchange protein activated by cAMP (Epac) with 8CPT-2Me-cAMP blocked the effect of L-P NPY on NMDA-mediated eEPSCs, but not on GABAA-mediated eIPSCs. Thus, NPY regulates amygdala excitability via two signal-transduction events, with reduced PKA activity enhancing GABAA-mediated eIPSCs and Epac deactivation reducing NMDA-mediated eEPSCs. This multipathway regulation of NMDA- and GABAA-mediated currents may be important for NPY plasticity and stress resilience in the amygdala.

  3. The GABA(A) receptor alpha1 subtype in the ventral pallidum regulates alcohol-seeking behaviors.

    PubMed

    Harvey, Scott C; Foster, Katrina L; McKay, Pete F; Carroll, Michelle R; Seyoum, Regat; Woods, James E; Grey, Collette; Jones, Cecily M; McCane, Shannan; Cummings, Rancia; Mason, Dynesha; Ma, Chunrong; Cook, James M; June, Harry L

    2002-05-01

    We investigated the potential role of the alpha1-containing GABA(A) receptor in regulating the reinforcing properties of alcohol. To accomplish this, we developed 3-propoxy-beta-carboline hydrochloride (3-PBC), a mixed agonist-antagonist benzodiazepine site ligand with binding selectivity at the alpha1 receptor. We then tested the capacity of 3-PBC to block alcohol-maintained responding in the ventral pallidum (VP), a novel alcohol reward substrate, which primarily expresses the alpha1-receptor isoform. Our results demonstrated that bilateral microinfusion of 3-PBC (0.5-40 microg) in the anterior and medial VP produced marked reductions in alcohol-maintained responding in a genetically selected rodent model of alcohol drinking. The VP infusions showed both neuroanatomical and reinforcer specificity because no effects were seen in sites dorsal to the VP (e.g., nucleus accumbens, caudate putamen). The saccharin-maintained responding was reduced only with the highest dose (40 microg). Parenteral injections of 3-PBC (1-20 mg/kg) also showed a similar selectivity on alcohol-maintained responding. Complementary in vitro studies revealed that 3-PBC exhibited a low partial agonist efficacy profile at recombinant diazepam-sensitive receptors (e.g., alpha1beta3gamma2, alpha2beta3gamma, and alpha3beta3gamma2). The selective suppression of 3-PBC on alcohol-maintained responding after central and parenteral administrations, together with its low-efficacy agonist profile, suggest that the reduction in alcohol-maintained behaviors was not attributable to a general suppression on consummatory behaviors. These results demonstrate that the alpha1-containing GABA(A) receptors in both the anterior and medial VP are important in regulating the reinforcing properties of alcohol. These receptors represent novel targets in the design and development of pharmacotherapies for alcohol-dependent subjects. PMID:11978852

  4. Benzodiazepine-insensitive mice generated by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid type A receptors.

    PubMed Central

    Günther, U; Benson, J; Benke, D; Fritschy, J M; Reyes, G; Knoflach, F; Crestani, F; Aguzzi, A; Arigoni, M; Lang, Y

    1995-01-01

    Vigilance, anxiety, epileptic activity, and muscle tone can be modulated by drugs acting at the benzodiazepine (BZ) site of gamma-aminobutyric acid type A (GABAA) receptors. In vivo, BZ sites are potential targets for endogenous ligands regulating the corresponding central nervous system states. To assess the physiological relevance of BZ sites, mice were generated containing GABAA receptors devoid of BZ sites. Following targeted disruption of the gamma 2 subunit gene, 94% of the BZ sites were absent in brain of neonatal mice, while the number of GABA sites was only slightly reduced. Except for the gamma 2 subunit, the level of expression and the regional and cellular distribution of the major GABAA receptor subunits were unaltered. The single channel main conductance level and the Hill coefficient were reduced to values consistent with recombinant GABAA receptors composed of alpha and beta subunits. The GABA response was potentiated by pentobarbital but not by flunitrazepam. Diazepam was inactive behaviorally. Thus, the gamma 2 subunit is dispensable for the assembly of functional GABAA receptors but is required for normal channel conductance and the formation of BZ sites in vivo. BZ sites are not essential for embryonic development, as suggested by the normal body weight and histology of newborn mice. Postnatally, however, the reduced GABAA receptor function is associated with retarded growth, sensorimotor dysfunction, and drastically reduced life-span. The lack of postnatal GABAA receptor regulation by endogenous ligands of BZ sites might contribute to this phenotype. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7644489

  5. Analgesia and unwanted benzodiazepine effects in point-mutated mice expressing only one benzodiazepine-sensitive GABAA receptor subtype

    PubMed Central

    Ralvenius, William T.; Benke, Dietmar; Acuña, Mario A.; Rudolph, Uwe; Zeilhofer, Hanns Ulrich

    2015-01-01

    Agonists at the benzodiazepine-binding site of GABAA receptors (BDZs) enhance synaptic inhibition through four subtypes (α1, α2, α3 and α5) of GABAA receptors (GABAAR). When applied to the spinal cord, they alleviate pathological pain; however, insufficient efficacy after systemic administration and undesired effects preclude their use in routine pain therapy. Previous work suggested that subtype-selective drugs might allow separating desired antihyperalgesia from unwanted effects, but the lack of selective agents has hitherto prevented systematic analyses. Here we use four lines of triple GABAAR point-mutated mice, which express only one benzodiazepine-sensitive GABAAR subtype at a time, to show that targeting only α2GABAARs achieves strong antihyperalgesia and reduced side effects (that is, no sedation, motor impairment and tolerance development). Additional pharmacokinetic and pharmacodynamic analyses in these mice explain why clinically relevant antihyperalgesia cannot be achieved with nonselective BDZs. These findings should foster the development of innovative subtype-selective BDZs for novel indications such as chronic pain. PMID:25865415

  6. Phytochemical profiling of Curcuma kwangsiensis rhizome extract, and identification of labdane diterpenoids as positive GABAA receptor modulators.

    PubMed

    Schramm, Anja; Ebrahimi, Samad Nejad; Raith, Melanie; Zaugg, Janine; Rueda, Diana C; Hering, Steffen; Hamburger, Matthias

    2013-12-01

    An ethyl acetate extract of Curcuma kwangsiensis S.G. Lee & C.F. Liang (Zingiberaceae) rhizomes (100 μg/ml) enhanced the GABA-induced chloride current (IGABA) through GABAA receptors of the α1β2γ2S subtype by 79.0±7.0%. Potentiation of IGABA was measured using the two-microelectrode voltage-clamp technique and Xenopus laevis oocytes. HPLC-based activity profiling of the crude extract led to the identification of 11 structurally related labdane diterpenoids, including four new compounds. Structure elucidation was achieved by comprehensive analysis of on-line (LC-PDA-ESI-TOF-MS) and off-line (microprobe 1D and 2D NMR) spectroscopic data. The absolute configuration of the compounds was established by comparison of experimental and calculated ECD spectra. Labdane diterpenes represent a new class of plant secondary metabolites eliciting positive GABAA receptor modulation. The highest efficiency was observed for zerumin A (maximum potentiation of IGABA by 309.4±35.6%, and EC50 of 24.9±8.8 μM).

  7. 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

  8. Reduced GABAA receptor α6 expression in the trigeminal ganglion alters inflammatory TMJ hypersensitivity

    PubMed Central

    Puri, Jyoti; Vinothini, Priya; Reuben, Jayne; Bellinger, Larry L.; Ailing, Li; Peng, Yuan B.; Kramer, Phillip R.

    2012-01-01

    Trigeminal ganglia neurons express the GABAA receptor subunit alpha 6 (Gabrα6) but the role of this particular subunit in orofacial hypersensitivity is unknown. In this report the function of Gabrα6 was tested by reducing its expression in the trigeminal ganglia and measuring the effect of this reduction on inflammatory temporomandibular joint (TMJ) hypersensitivity. Gabrα6 expression was reduced by infusing the trigeminal ganglia of male Sprague Dawley rats with small interfering RNA (siRNA) having homology to either the Gabrα6 gene (Gabrα6 siRNA) or no known gene (control siRNA). Sixty hours after siRNA infusion the rats received a bilateral TMJ injection of complete Freund’s adjuvant to induce an inflammatory response. Hypersensitivity was then quantitated by measuring meal duration, which lengthens when hypersensitivity increases. Neuronal activity in the trigeminal ganglia was also measured by quantitating the amount of phosphorylated ERK. Rats in a different group that did not have TMJ inflammation had an electrode placed in the spinal cord at the level of C1 sixty hours after siRNA infusion to record extracellular electrical activity of neurons that responded to TMJ stimulation. Our results show that Gabrα6 was expressed in both neurons and satellite glia of the trigeminal ganglia and that Gabrα6 positive neurons within the trigeminal ganglia have afferents in the TMJ. Gabrα6 siRNA infusion reduced Gabrα6 gene expression by 30% and significantly lengthened meal duration in rats with TMJ inflammation. Gabrα6 siRNA infusion also significantly increased p-ERK expression in the trigeminal ganglia of rats with TMJ inflammation and increased electrical activity in the spinal cord of rats without TMJ inflammation. These results suggest that maintaining Gabrα6 expression was necessary to inhibit primary sensory afferents in the trigeminal pathway and reduce inflammatory orofacial nociception. PMID:22521829

  9. Synthesis of GABAA Receptor Agonists and Evaluation of their α-Subunit Selectivity and Orientation in the GABA Binding Site

    PubMed Central

    Jansen, Michaela; Rabe, Holger; Strehle, Axelle; Dieler, Sandra; Debus, Fabian; Dannhardt, Gerd; Akabas, Myles H.; Lüddens, Hartmut

    2008-01-01

    Drugs used to treat various disorders target GABAA receptors. To develop α subunit selective compounds, we synthesized 5-(4-piperidyl)-3-isoxazolol (4-PIOL) derivatives. The 3-isoxazolol moiety was substituted by 1,3,5-oxadiazol-2-one, 1,3,5-oxadiazol-2-thione, and substituted 1,2,4-triazol-3-ol heterocycles with modifications to the basic piperidine substituent as well as substituents without basic nitrogen. Compounds were screened by [3H]muscimol binding and in patch-clamp experiments with heterologously expressed GABAA αiβ3γ2 receptors (i = 1–6). The effects of 5-aminomethyl-3H-[1,3,4]oxadiazol-2-one 5d were comparable to GABA for all α subunit isoforms. 5-piperidin-4-yl-3H-[1,3,4]oxadiazol-2-one 5a and 5-piperidin-4-yl-3H- [1,3,4]oxadiazol-2-thione 6a were weak agonists at α3–, α3–, and α5–containing receptors. When coapplied with GABA they were antagonistic inα2–, α4–, and α6–containing receptors and potentiated α3-containing receptors. 6a protected GABA binding site cysteine-substitution mutants α1F64C and α1S68C from reacting with methanethiosulfonate-ethylsulfonate. 6a specifically covalently modified the α1R66C thiol, in the GABA binding site, through its oxadiazolethione sulfur. These results demonstrate the feasibility of synthesizing α subtype selective GABA mimetic drugs. PMID:18651727

  10. Differential Modulation of GABAA Receptors Underlies Postsynaptic Depolarization- and Purinoceptor-Mediated Enhancement of Cerebellar Inhibitory Transmission: A Non-Stationary Fluctuation Analysis Study

    PubMed Central

    Ono, Yumie; Saitow, Fumihito; Konishi, Shiro

    2016-01-01

    Cerebellar GABAergic inhibitory transmission between interneurons and Purkinje cells (PCs) undergoes a long-lasting enhancement following different stimulations, such as brief depolarization or activation of purinergic receptors of postsynaptic PCs. The underlying mechanisms, however, are not completely understood. Using a peak-scaled non-stationary fluctuation analysis, we therefore aimed at characterizing changes in the electrophysiological properties of GABAA receptors in PCs of rat cerebellar cortex during depolarization-induced “rebound potentiation (RP)” and purinoceptor-mediated long-term potentiation (PM-LTP), because both RP and PM-LTP likely depend on postsynaptic mechanisms. Stimulation-evoked inhibitory postsynaptic currents (eIPSCs) were recorded from PCs in neonatal rat cerebellar slices. Our analysis showed that postsynaptic membrane depolarization induced RP of eIPSCs in association with significant increase in the number of synaptic GABAA receptors without changing the channel conductance. By contrast, bath application of ATP induced PM-LTP of eIPSCs with a significant increase of the channel conductance of GABAA receptors without affecting the receptor number. Pretreatment with protein kinase A (PKA) inhibitors, H-89 and cAMPS-Rp, completely abolished the PM-LTP. The CaMKII inhibitor KN-62 reported to abolish RP did not alter PM-LTP. These results suggest that the signaling mechanism underlying PM-LTP could involve ATP-induced phosphorylation of synaptic GABAA receptors, thereby resulting in upregulation of the channel conductance by stimulating adenylyl cyclase-PKA signaling cascade, possibly via activation of P2Y11 purinoceptor. Thus, our findings reveal that postsynaptic GABAA receptors at the interneuron-PC inhibitory synapses are under the control of two distinct forms of long-term potentiation linked with different second messenger cascades. PMID:26930485

  11. Blockage of A2A and A3 adenosine receptors decreases the desensitization of human GABAA receptors microtransplanted to Xenopus oocytes

    PubMed Central

    Roseti, Cristina; Palma, Eleonora; Martinello, Katiuscia; Fucile, Sergio; Morace, Roberta; Esposito, Vincenzo; Cantore, Gianpaolo; Arcella, Antonietta; Giangaspero, Felice; Aronica, Eleonora; Mascia, Addolorata; Di Gennaro, Giancarlo; Quarato, Pier Paolo; Manfredi, Mario; Cristalli, Gloria; Lambertucci, Catia; Marucci, Gabriella; Volpini, Rosaria; Limatola, Cristina; Eusebi, Fabrizio

    2009-01-01

    We previously found that the endogenous anticonvulsant adenosine, acting through A2A and A3 adenosine receptors (ARs), alters the stability of currents (IGABA) generated by GABAA receptors expressed in the epileptic human mesial temporal lobe (MTLE). Here we examined whether ARs alter the stability (desensitization) of IGABA expressed in focal cortical dysplasia (FCD) and in periglioma epileptic tissues. The experiments were performed with tissues from 23 patients, using voltage-clamp recordings in Xenopus oocytes microinjected with membranes isolated from human MTLE and FCD tissues or using patch-clamp recordings of pyramidal neurons in epileptic tissue slices. On repetitive activation, the epileptic GABAA receptors revealed instability, manifested by a large IGABA rundown, which in most of the oocytes (≈70%) was obviously impaired by the new A2A antagonists ANR82, ANR94, and ANR152. In most MTLE tissue-microtransplanted oocytes, a new A3 receptor antagonist (ANR235) significantly improved IGABA stability. Moreover, patch-clamped pyramidal neurons from human neocortical slices of periglioma epileptic tissues exhibited altered IGABA rundown on ANR94 treatment. Our findings indicate that antagonizing A2A and A3 receptors increases the IGABA stability in different epileptic tissues and suggest that adenosine derivatives may offer therapeutic opportunities in various forms of human epilepsy. PMID:19721003

  12. Enhancement by GABA of the association rate of picrotoxin and tert-butylbicyclophosphorothionate to the rat cloned alpha 1 beta 2 gamma 2 GABAA receptor subtype.

    PubMed Central

    Dillon, G. H.; Im, W. B.; Carter, D. B.; McKinley, D. D.

    1995-01-01

    1. We examined how gamma-aminobutyric acid (GABA) influences interaction of picrotoxin and tert-butylbicyclophosphorothionate (TBPS) with recombinant rat alpha 1 beta 2 gamma 2 GABAA receptors stably expressed in human embryonic kidney cells (HEK293), as monitored with changes in Cl- currents measured by the whole-cell patch clamp technique. 2. During application of GABA (5 microM) for 15 s, picrotoxin and TBPS dose-dependently accelerated the decay of inward GABA-induced currents (a holding potential of -60 mV under a symmetrical Cl- gradient). The drugs, upon preincubation with the receptors, also reduced the initial current amplitude in a preincubation time and concentration-dependent manner. This indicates their interaction with both GABA-bound and resting receptors. 3. The half maximal inhibitory concentration for picrotoxin and TBPS at the beginning of a 15 s GABA (5 microM) pulse was several times greater than that obtained at the end of the pulse. GABA thus appears to enhance picrotoxin and TBPS potency, but only at concentrations leading to occupancy of both high and low affinity GABA sites, i.e., 5 microM. Preincubation of the receptors with the drugs in the presence of GABA at 200 nM, which leads to occupancy of only high affinity GABA sites in the alpha 1 beta 2 gamma 2 subtype, produced no appreciable change in potency of picrotoxin or TBPS. This indicates that they preferentially interact with multiliganded, but not monoliganded receptors, unlike U-93631, a novel ligand to the picrotoxin site, which has higher affinity to both mono- and multiliganded receptors than resting receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7582470

  13. Gender and age differences in expression of GABAA receptor subunits in rat somatosensory thalamus and cortex in an absence epilepsy model.

    PubMed

    Li, Huifang; Huguenard, John R; Fisher, Robert S

    2007-03-01

    Absence epilepsy is more prevalent in females, but reasons for this gender asymmetry are unknown. We reported previously that perinatal treatment of Long-Evans Hooded rats with the cholesterol synthesis inhibitor (CSI) AY9944 causes a life-long increase in EEG spike-wave discharges (SWDs), correlated with decreased expression of GABA(A) receptor subunit gamma2 protein levels in thalamic reticular and ventrobasal nuclei (SS thalamus) [Li, H., Kraus, A., Wu, J., Huguenard, J.R., Fisher, R.S., 2006. Selective changes in thalamic and cortical GABA(A) receptor subunits in a model of acquired absence epilepsy in the rat. Neuropharmacology 51, 121-128]. In this study, we explored time course and gender different effects of perinatal AY9944 treatment on expression of GABA(A) receptor alpha1 and gamma2 subunits in SS thalamus and SS cortex. Perinatal AY9944 treatment-induced decreases in GABA(A) gamma2 receptor subunits in rat SS thalamus and increases in SS cortex are gender and age specific. The findings suggest a mechanism for the higher prevalence of absence epilepsy in female patients.

  14. 5-HT2 receptors mediate functional modulation of GABAa receptors and inhibitory synaptic transmissions in human iPS-derived neurons

    PubMed Central

    Wang, Haitao; Hu, Lingli; Liu, Chunhua; Su, Zhenghui; Wang, Lihui; Pan, Guangjin; Guo, Yiping; He, Jufang

    2016-01-01

    Neural progenitors differentiated from induced pluripotent stem cells (iPS) hold potentials for treating neurological diseases. Serotonin has potent effects on neuronal functions through multiple receptors, underlying a variety of neural disorders. Glutamate and GABA receptors have been proven functional in neurons differentiated from iPS, however, little is known about 5-HT receptor-mediated modulation in such neuronal networks. In the present study, human iPS were differentiated into cells possessing featured physiological properties of cortical neurons. Whole-cell patch-clamp recording was used to examine the involvement of 5-HT2 receptors in functional modulation of GABAergic synaptic transmission. We found that serotonin and DOI (a selective agonist of 5-HT2A/C receptor) reversibly reduced GABA-activated currents, and this 5-HT2A/C receptor mediated inhibition required G protein, PLC, PKC, and Ca2+ signaling. Serotonin increased the frequency of miniature inhibitory postsynaptic currents (mIPSCs), which could be mimicked by α-methylserotonin, a 5-HT2 receptor agonist. In contrast, DOI reduced both frequency and amplitude of mIPSCs. These findings suggested that in iPS-derived human neurons serotonin postsynaptically reduced GABAa receptor function through 5-HT2A/C receptors, but presynaptically other 5-HT2 receptors counteracted the action of 5-HT2A/C receptors. Functional expression of serotonin receptors in human iPS-derived neurons provides a pre-requisite for their normal behaviors after grafting. PMID:26837719

  15. Comparison of Steroid Modulation of Spontaneous Inhibitory Postsynaptic Currents in Cultured Hippocampal Neurons and Steady-State Single-Channel Currents from Heterologously Expressed α1β2γ2L GABA(A) Receptors.

    PubMed

    Chakrabarti, Sampurna; Qian, Mingxing; Krishnan, Kathiresan; Covey, Douglas F; Mennerick, Steven; Akk, Gustav

    2016-04-01

    Neuroactive steroids are efficacious modulators of γ-aminobutyric acid type A receptor (GABA(A)) receptor function. The effects of steroids on the GABA(A) 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 GABA(A) 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.

  16. Decreases in mitochondrial reactive oxygen species initiate GABAA receptor-mediated electrical suppression in anoxia-tolerant turtle neurons

    PubMed Central

    Hogg, David W; Pamenter, Matthew E; Dukoff, David J; Buck, Leslie T

    2015-01-01

    Key points Anoxia induces hyper-excitability and cell death in mammalian brain but in the western painted turtle (Chrysemys picta bellii) enhanced GABA transmission prevents injury. The mechanism responsible for increased GABA transmission is unknown; however, reactive oxygen species (ROS) generated by mitochondria may play a role because this is an oxygen-sensitive process. In this study, we show that inhibition of mitochondrial ROS production is sufficient to initiate a redox-sensitive GABA signalling cascade that suppresses pyramidal neuron action potential frequency. These results further our understanding of the turtle's unique strategy for reducing ATP consumption during anoxia and highlights a natural mechanism in which to explore therapies to protect mammalian brain from low-oxygen insults (e.g. cerebral stroke). Abstract Anoxia induces hyper-excitability and cell death in mammalian brain but in the anoxia-tolerant western painted turtle (Chrysemys picta bellii) neuronal electrical activity is suppressed (i.e. spike arrest), adenosine triphosphate (ATP) consumption is reduced, and cell death does not occur. Electrical suppression is primarily the result of enhanced γ-aminobutyric acid (GABA) transmission; however, the underlying mechanism responsible for initiating oxygen-sensitive GABAergic spike arrest is unknown. In turtle cortical pyramidal neurons there are three types of GABAA receptor-mediated currents: spontaneous inhibitory postsynaptic currents (IPSCs), giant IPSCs and tonic currents. The aim of this study was to assess the effects of reactive oxygen species (ROS) scavenging on these three currents since ROS levels naturally decrease with anoxia and may serve as a redox signal to initiate spike arrest. We found that anoxia, pharmacological ROS scavenging, or inhibition of mitochondrial ROS generation enhanced all three types of GABA currents, with tonic currents comprising ∼50% of the total current. Application of hydrogen peroxide inhibited

  17. Maternal restraint stress delays maturation of cation-chloride cotransporters and GABAA receptor subunits in the hippocampus of rat pups at puberty.

    PubMed

    Veerawatananan, Bovorn; Surakul, Pornprom; Chutabhakdikul, Nuanchan

    2016-06-01

    The GABAergic synapse undergoes structural and functional maturation during early brain development. Maternal stress alters GABAergic synapses in the pup's brain that are associated with the pathophysiology of neuropsychiatric disorders in adults; however, the mechanism for this is still unclear. In this study, we examined the effects of maternal restraint stress on the development of Cation-Chloride Cotransporters (CCCs) and the GABAA receptor α1 and α5 subunits in the hippocampus of rat pups at different postnatal ages. Our results demonstrate that maternal restraint stress induces a transient but significant increase in the level of NKCC1 (Sodium-Potassium Chloride Cotransporter 1) only at P14, followed by a brief, yet significant increase in the level of KCC2 (Potassium-Chloride Cotransporter 2) at P21, which then decreases from P28 until P40. Thus, maternal stress alters NKCC1 and KCC2 ratio in the hippocampus of rat pups, especially during P14 to P28. Maternal restraint stress also caused biphasic changes in the level of GABAA receptor subunits in the pup's hippocampus. GABAA receptor α1 subunit gradually increased at P14 then decreased thereafter. On the contrary, GABAA receptor α5 subunit showed a transient decrease followed by a long-term increase from P21 until P40. Altogether, our study suggested that the maternal restraint stress might delay maturation of the GABAergic system by altering the expression of NKCC1, KCC2 and GABAA receptor α1 and α5 subunits in the hippocampus of rat pups. These changes demonstrate the dysregulation of inhibitory neurotransmission during early life, which may underlie the pathogenesis of psychiatric diseases at adolescence. PMID:26844244

  18. Maternal restraint stress delays maturation of cation-chloride cotransporters and GABAA receptor subunits in the hippocampus of rat pups at puberty

    PubMed Central

    Veerawatananan, Bovorn; Surakul, Pornprom; Chutabhakdikul, Nuanchan

    2015-01-01

    The GABAergic synapse undergoes structural and functional maturation during early brain development. Maternal stress alters GABAergic synapses in the pup's brain that are associated with the pathophysiology of neuropsychiatric disorders in adults; however, the mechanism for this is still unclear. In this study, we examined the effects of maternal restraint stress on the development of Cation-Chloride Cotransporters (CCCs) and the GABAA receptor α1 and α5 subunits in the hippocampus of rat pups at different postnatal ages. Our results demonstrate that maternal restraint stress induces a transient but significant increase in the level of NKCC1 (Sodium–Potassium Chloride Cotransporter 1) only at P14, followed by a brief, yet significant increase in the level of KCC2 (Potassium-Chloride Cotransporter 2) at P21, which then decreases from P28 until P40. Thus, maternal stress alters NKCC1 and KCC2 ratio in the hippocampus of rat pups, especially during P14 to P28. Maternal restraint stress also caused biphasic changes in the level of GABAA receptor subunits in the pup's hippocampus. GABAA receptor α1 subunit gradually increased at P14 then decreased thereafter. On the contrary, GABAA receptor α5 subunit showed a transient decrease followed by a long-term increase from P21 until P40. Altogether, our study suggested that the maternal restraint stress might delay maturation of the GABAergic system by altering the expression of NKCC1, KCC2 and GABAA receptor α1 and α5 subunits in the hippocampus of rat pups. These changes demonstrate the dysregulation of inhibitory neurotransmission during early life, which may underlie the pathogenesis of psychiatric diseases at adolescence. PMID:26844244

  19. 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.

  20. The impact of tonic GABAA receptor-mediated inhibition on neuronal excitability varies across brain region and cell type

    PubMed Central

    Lee, Vallent; Maguire, Jamie

    2014-01-01

    The diversity of GABAA receptor (GABAAR) subunits and the numerous configurations during subunit assembly give rise to a variety of receptors with different functional properties. This heterogeneity results in variations in GABAergic conductances across numerous brain regions and cell types. Phasic inhibition is mediated by synaptically-localized receptors with a low affinity for GABA and results in a transient, rapidly desensitizing GABAergic conductance; whereas, tonic inhibition is mediated by extrasynaptic receptors with a high affinity for GABA and results in a persistent GABAergic conductance. The specific functions of tonic versus phasic GABAergic inhibition in different cell types and the impact on specific neural circuits are only beginning to be unraveled. Here we review the diversity in the magnitude of tonic GABAergic inhibition in various brain regions and cell types, and highlight the impact on neuronal excitability in different neuronal circuits. Further, we discuss the relevance of tonic inhibition in various physiological and pathological contexts as well as the potential of targeting these receptor subtypes for treatment of diseases, such as epilepsy. PMID:24550784

  1. 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

  2. The effects of the nonselective benzodiazepine lorazepam and the α2 /α3 subunit-selective GABAA receptor modulators AZD7325 and AZD6280 on plasma prolactin levels.

    PubMed

    Te Beek, Erik T; Chen, Xia; Jacobs, Gabriël E; Nahon, Kimberly J; de Kam, Marieke L; Lappalainen, Jaakko; Cross, Alan J; van Gerven, Joop M A; Hay, Justin L

    2015-03-01

    Compounds with selectivity for GABAA receptor subtypes may differ significantly from nonselective benzodiazepines in their dopaminergic effects in vivo. To explore the exact role of the GABAA receptor subtypes in the regulation of prolactin secretion and the differential effects of selective and nonselective GABA receptor modulators, the effects of the nonselective benzodiazepine lorazepam, as well as two novel α2 /α3 subunit-selective GABAA receptor modulators AZD7325 and AZD6280, on prolactin levels were measured in healthy male volunteers. Following administration of lorazepam at 2 mg doses and AZD6280 at 10 mg and 40 mg doses, prolactin levels increased significantly compared with placebo (difference 42.0%, 19.8%, and 32.8%, respectively), suggesting that the α2 and/or α3 receptor subtypes are involved in GABAergic modulation of prolactin secretion, although possible roles of the α1 and α5 receptor subtypes are not excluded. The increases in prolactin levels after administration of AZD7325 at 2 mg and 10 mg doses (difference 7.6% and 10.5%, respectively) did not reach statistical significance, suggesting that doses of AZD7325 or intrinsic efficacy at the α2 and α3 receptor subtypes may have been too low. PMID:27128218

  3. GABA-induced uncoupling of GABA/benzodiazepine site interactions is mediated by increased GABAA receptor internalization and associated with a change in subunit composition.

    PubMed

    Gutiérrez, M L; Ferreri, M C; Gravielle, M C

    2014-01-17

    Persistent activation of GABAA receptors triggers compensatory changes in receptor function that are relevant to physiological, pathological and pharmacological conditions. Chronic treatment of cultured neurons with GABA for 48h has been shown to produce a down-regulation of receptor number and an uncoupling of GABA/benzodiazepine site interactions with a half-time of 24-25h. Down-regulation is the result of a transcriptional repression of GABAA receptor subunit genes and depends on activation of L-type voltage-gated calcium channels. The mechanism of this uncoupling is currently unknown. We have previously demonstrated that a single brief exposure of rat primary neocortical cultures to GABA for 5-10min (t½=3min) initiates a process that results in uncoupling hours later (t½=12h) without a change in receptor number. Uncoupling is contingent upon GABAA receptor activation and independent of voltage-gated calcium influx. This process is accompanied by a selective decrease in subunit mRNA levels. Here, we report that the brief GABA exposure induces a decrease in the percentage of α3-containing receptors, a receptor subtype that exhibits a high degree of coupling between GABA and benzodiazepine binding sites. Initiation of GABA-induced uncoupling is prevented by co-incubation of GABA with high concentrations of sucrose suggesting that it is dependent on a receptor internalization step. Moreover, results from immunocytochemical and biochemical experiments indicate that GABA exposure causes an increase in GABAA receptor endocytosis. Together, these data suggest that the uncoupling mechanism involves an initial increase in receptor internalization followed by activation of a signaling cascade that leads to selective changes in receptor subunit levels. These changes might result in the assembly of receptors with altered subunit compositions that display a lower degree of coupling between GABA and benzodiazepine sites. Uncoupling might represent a homeostatic mechanism

  4. Assessment of Expression of Genes Coding GABAA Receptors during Chronic and Acute Intoxication of Laboratory Rats with Ethanol.

    PubMed

    Osechkina, N S; Ivanov, M B; Nazarov, G V; Batotsyrenova, E G; Lapina, N V; Babkin, A V; Berdinskikh, I S; Melekhova, A S; Voitsekhovich, K O; Lisitskii, D S; Kashina, T V

    2016-02-01

    Expression of genes encoding the individual subunits of ionotropic GABAA receptor was assessed after acute and chronic intoxication of rats with ethanol. The chronic 1-month-long exposure to ethanol signifi cantly decreased (by 38%) expression of Gabrb1 gene in the hippocampus. Acute exposure to ethanol elevated expression of genes Gabrb1 (by 1.7 times), Gabra1 (by 3.8 times), and Gabra4 (by 6.5 times), although it diminished expression of Gabra2 gene by 1.4 times. In preliminarily alcoholized rats, acute intoxication with ethanol enhanced expression of genes Gabrb1 and Gabra5 by 1.7 and 8.7 times, respectively. There was neither acute nor chronic effect of ethanol on expression of gene Gabra3. PMID:26902358

  5. Identification of an Inhibitory Alcohol Binding Site in GABAA ρ1 Receptors.

    PubMed

    Borghese, Cecilia M; Ruiz, Carlos I; Lee, Ui S; Cullins, Madeline A; Bertaccini, Edward J; Trudell, James R; Harris, R Adron

    2016-01-20

    Alcohols inhibit γ-aminobutyric acid type A ρ1 receptor function. After introducing mutations in several positions of the second transmembrane helix in ρ1, we studied the effects of ethanol and hexanol on GABA responses using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes. The 6' mutations produced the following effects on ethanol and hexanol responses: small increase or no change (T6'M), increased inhibition (T6'V), and small potentiation (T6'Y and T6'F). The 5' mutations produced mainly increases in hexanol inhibition. Other mutations produced small (3' and 9') or no changes (2' and L277 in the first transmembrane domain) in alcohol effects. These results suggest an inhibitory alcohol binding site near the 6' position. Homology models of ρ1 receptors based on the X-ray structure of GluCl showed that the 2', 5', 6', and 9' residues were easily accessible from the ion pore, with 5' and 6' residues from neighboring subunits facing each other; L3' and L277 also faced the neighboring subunit. We tested ethanol through octanol on single and double mutated ρ1 receptors [ρ1(I15'S), ρ1(T6'Y), and ρ1(T6'Y,I15'S)] to further characterize the inhibitory alcohol pocket in the wild-type ρ1 receptor. The pocket can only bind relatively short-chain alcohols and is eliminated by introducing Y in the 6' position. Replacing the bulky 15' residue with a smaller side chain introduced a potentiating binding site, more sensitive to long-chain than to short-chain alcohols. In conclusion, the net alcohol effect on the ρ1 receptor is determined by the sum of its actions on inhibitory and potentiating sites.

  6. Antinociceptive effects of fisetin against diabetic neuropathic pain in mice: Engagement of antioxidant mechanisms and spinal GABAA receptors.

    PubMed

    Zhao, Xin; Li, Xin-Lin; Liu, Xin; Wang, Chuang; Zhou, Dong-Sheng; Ma, Qing; Zhou, Wen-Hua; Hu, Zhen-Yu

    2015-12-01

    Peripheral painful neuropathy is one of the most common complications in diabetes and necessitates improved treatment. Fisetin, a naturally occurring flavonoid, has been reported to exert antidepressant-like effect in previous studies. As antidepressant drugs are employed clinically to treat neuropathic pain, this work aimed to investigate whether fisetin possess beneficial effect on diabetic neuropathic pain and explore the mechanism(s). We subjected mice to diabetes by a single intraperitoneal (i.p.) injection of streptozotocin (200mg/kg), and von Frey test or Hargreaves test was used to assess mechanical allodynia or thermal hyperalgesia, respectively. Chronic treatment of diabetic mice with fisetin not only ameliorated the established symptoms of thermal hyperalgesia and mechanical allodynia, but also arrested the development of neuropathic pain when given at low doses. Although chronic fisetin administration did not impact on the symptom of hyperglycemia in diabetic mice, it reduced exacerbated oxidative stress in tissues of spinal cord, dorsal root ganglion (DRG) and sciatic verve. Furthermore, the analgesic actions of fisetin were abolished by repetitive co-treatment with the reactive oxygen species (ROS) donor tert-butyl hydroperoxide (t-BOOH), but potentiated by the ROS scavenger phenyl-N-tert-butylnitrone (PBN). Finally, acute blockade of spinal GABAA receptors by bicuculline totally counteracted such fisetin analgesia. These findings indicate that chronic fisetin treatment can delay or correct neuropathic hyperalgesia and allodynia in mice with type 1 diabetes. Mechanistically, the present fisetin analgesia may be associated with its antioxidant activity, and spinal GABAA receptors are likely rendered as downstream targets. PMID:26520392

  7. Honokiol promotes non-rapid eye movement sleep via the benzodiazepine site of the GABAA receptor in mice

    PubMed Central

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

    2012-01-01

    BACKGROUND AND PURPOSE 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. EXPERIMENTAL APPROACH Honokiol was administered i.p. at 20:00 h in mice. Flumazenil, an antagonist at the benzodiazepine site of the GABAA 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. KEY RESULTS 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. CONCLUSION AND IMPLICATIONS Honokiol promoted NREM sleep by modulating the benzodiazepine site of the GABAA receptor, suggesting potential applications in the treatment of insomnia, especially for patients who experience difficulty in falling and staying asleep. PMID:22537192

  8. Antinociceptive effects of fisetin against diabetic neuropathic pain in mice: Engagement of antioxidant mechanisms and spinal GABAA receptors.

    PubMed

    Zhao, Xin; Li, Xin-Lin; Liu, Xin; Wang, Chuang; Zhou, Dong-Sheng; Ma, Qing; Zhou, Wen-Hua; Hu, Zhen-Yu

    2015-12-01

    Peripheral painful neuropathy is one of the most common complications in diabetes and necessitates improved treatment. Fisetin, a naturally occurring flavonoid, has been reported to exert antidepressant-like effect in previous studies. As antidepressant drugs are employed clinically to treat neuropathic pain, this work aimed to investigate whether fisetin possess beneficial effect on diabetic neuropathic pain and explore the mechanism(s). We subjected mice to diabetes by a single intraperitoneal (i.p.) injection of streptozotocin (200mg/kg), and von Frey test or Hargreaves test was used to assess mechanical allodynia or thermal hyperalgesia, respectively. Chronic treatment of diabetic mice with fisetin not only ameliorated the established symptoms of thermal hyperalgesia and mechanical allodynia, but also arrested the development of neuropathic pain when given at low doses. Although chronic fisetin administration did not impact on the symptom of hyperglycemia in diabetic mice, it reduced exacerbated oxidative stress in tissues of spinal cord, dorsal root ganglion (DRG) and sciatic verve. Furthermore, the analgesic actions of fisetin were abolished by repetitive co-treatment with the reactive oxygen species (ROS) donor tert-butyl hydroperoxide (t-BOOH), but potentiated by the ROS scavenger phenyl-N-tert-butylnitrone (PBN). Finally, acute blockade of spinal GABAA receptors by bicuculline totally counteracted such fisetin analgesia. These findings indicate that chronic fisetin treatment can delay or correct neuropathic hyperalgesia and allodynia in mice with type 1 diabetes. Mechanistically, the present fisetin analgesia may be associated with its antioxidant activity, and spinal GABAA receptors are likely rendered as downstream targets.

  9. Effect of the alpha subunit subtype on the macroscopic kinetic properties of recombinant GABA(A) receptors.

    PubMed

    Picton, Amber J; Fisher, Janet L

    2007-08-24

    The GABA(A) receptors (GABARs) are chloride-permeable ligand-gated ion channels responsible for fast inhibitory neurotransmission. These receptors are structurally heterogeneous, and in mammals can be formed from a combination of sixteen different subunit subtypes. Much of this variety comes from the six different alpha subunit subtypes. All neuronal GABARs contain an alpha subunit, and the identity of the alpha subtype affects the pharmacological properties of the receptors. The expression of each of the different alpha subtypes is regulated developmentally and regionally and changes with both normal physiological processes such development and synaptic plasticity, and pathological conditions such as epilepsy. In order to understand the functional significance of this structural heterogeneity, we examined the effect of the alpha subtype on the receptor's response to GABA. Each of the six alpha subtypes was transiently co-expressed with the beta3 and gamma2L subunits in mammalian cells. The sensitivity to GABA was measured with whole-cell recordings. We also determined the activation, deactivation, desensitization, and recovery kinetics for the six isoforms using rapid application recordings from excised macropatches. We found unique characteristics associated with each alpha subunit subtype. These properties would be expected to influence the post-synaptic response to GABA, creating functional diversity among neurons expressing different alpha subunits.

  10. GABAA and GABAB receptor-mediated effects on the spontaneous activity of the longitudinal layer in cat terminal ileum.

    PubMed

    Pencheva, N; Radomirov, R; Venkova, K

    1991-01-01

    1. GABA and GABAergic agonists-muscimol and (+/-)baclofen changed the spontaneous mechanical activity in isolated cat terminal ileum. 2. GABA at doses ranging from 5 microM to 2 mM produced concentration-dependent biphasic responses consisting of a transient relaxation followed by contractions with a tonic and a phasic components. 3. The GABA-induced relaxation was sensitive to bicuculline and picrotoxinin and was mimicked by muscimol, while the GABA-induced contractions were insensitive to bicuculline and picrotoxinin and were mimicked by (+/-)baclofen. Specific cross desensitization occurred between GABA and muscimol or GABA and (+/-)baclofen. 4. The bicuculline-sensitive relaxation induced by GABA and muscimol was abolished by atropine or tetrodotoxin (TTX), while the bicuculline-insensitive contractions induced by GABA and (+/-)baclofen were not antagonized by atropine or TTX, though they were slightly suppressed. 5. The GABA effects in the longitudinal layer of cat terminal ileum were mediated by the following receptors: -GABAA prejunctional receptors whose activation causes relaxation, probably through an inhibitory action on cholinergic neurons; -GABAB prejunctional receptors whose activation cause contractions; -GABAB postjunctional receptors located on the smooth muscle membrane whose activation induces tonic and phasic contractions.

  11. The heterogeneity in GABAA receptor-mediated IPSC kinetics reflects heterogeneity of subunit composition among inhibitory and excitatory interneurons in spinal lamina II

    PubMed Central

    Labrakakis, Charalampos; Rudolph, Uwe; De Koninck, Yves

    2014-01-01

    GABAergic inhibition displays rich functional diversity throughout the CNS, which arises from variations in the nature of inputs, subunit composition, subcellular localization of receptors and synapse geometry, or reuptake mechanisms. In the spinal dorsal horn (SDH), GABAA and glycine receptors play a major role in the control of excitability and accuracy of nociceptive processing. Identifying which components shape the properties of the inhibitory synapses in different cell types is necessary to understand how nociceptive information is integrated. To address this, we used transgenic mice where inhibitory interneurons express GAD65-EGFP. We found that GABAA, but not glycine receptor-mediated evoked IPSCs displayed slower kinetics in EGFP+ vs. EGFP− interneurons. GABAA miniature IPSC decay kinetics showed a large variability in both populations, however the distribution of decays differed between EGFP+ and EGFP− interneurons. The range of mIPSC decay kinetics observed was replicated in experiments using rapid application of GABA on outside-out patches taken from SDH neurons in slices. Furthermore, GABAA decay kinetics were not affected by uptake blockers and were not different in mice lacking δ or α5 subunits, indicating that intrinsic channel properties likely underlie the heterogeneity. To identify whether other α subunits shape the various kinetic properties observed we took advantage of knock-in mice carrying point mutations in either the α1, α2, or α3 subunits rendering Ro 15-4513 a selective agonist at the benzodiazepine modulatory site. We found that α1 and α2 subunit underlie the fast decaying component of IPSCs while the slow component is determined by the α3 subunit. The differential distribution of GABAA subunits at inhibitory synapses thus sculpts the heterogeneity of the SDH inhibitory circuitry. This diversity of inhibitory elements can be harnessed to selectively modulate different components of the spinal nociceptive circuitry for

  12. Modulation of spontaneous intracellular Ca²⁺ fluctuations and spontaneous cholinergic transmission in rat chromaffin cells in situ by endogenous GABA acting on GABAA receptors.

    PubMed

    Tzitzitlini, Alejandre-García; Pedro, Segura-Chama; Martha, Pérez-Armendáriz E; Rodolfo, Delgado-Lezama; Arturo, Hernández-Cruz

    2016-02-01

    Using fluorescence [Ca(2+)]i imaging in rat adrenal slices, we characterized the effects of agonists and antagonists of the GABAA receptor (GABAA-R) on resting intracellular Ca(2+) ([Ca(2+)]i) and spontaneous [Ca(2+)]i fluctuations (SCFs) in hundreds of individual chromaffin cells (CCs) recorded simultaneously in situ. Muscimol, a GABAA-R agonist (20 μM; 25 s), induced an increase of resting [Ca(2+)]i in 43 ± 3 % of CCs, a decrease in 26 ± 2 %, and no response in 30 ± 5 %. In Ca(2+)-free external medium, SCFs ceased completely and muscimol failed to elicit [Ca(2+)]i rises. All muscimol-induced [Ca(2+)]i changes were blocked by the GABAA-R antagonist bicuculline, suggesting that they result from changes in membrane potential depending on the cell's Cl(-) equilibrium potential. Unexpectedly, bicuculline increased the amplitude and frequency of SCFs in 54 % of CCs, revealing a tonic inhibition of SCFs by ambient GABA acting through GABAA-R. Mecamylamine (a specific nicotinic cholinergic blocker) decreased basal SCF activity in 18 % of CCs and inhibited bicuculline-induced SCF intensification, suggesting that spontaneous acetylcholine (ACh) release from nerve endings contributes to SCF generation in CCs in situ and that blockade of presynaptic GABAA-Rs intensifies SCFs in part through the disinhibition of spontaneous cholinergic transmission. Electrophysiological experiments confirmed that spontaneous excitatory postsynaptic currents recorded from CCs in situ were enhanced by bicuculline. To our knowledge, this is the first description of a regulatory effect of endogenous GABA on synaptic currents and SCFs of adrenal CCs. These findings denote a novel GABA-mediated presynaptic and postsynaptic regulatory mechanism of CC activity which may participate in the control of catecholamine secretion.

  13. Estrous cycle variations in GABA(A) receptor phosphorylation enable rapid modulation by anabolic androgenic steroids in the medial preoptic area.

    PubMed

    Oberlander, J G; Porter, D M; Onakomaiya, M M; Penatti, C A A; Vithlani, M; Moss, S J; Clark, A S; Henderson, L P

    2012-12-13

    Anabolic androgenic steroids (AAS), synthetic testosterone derivatives that are used for ergogenic purposes, alter neurotransmission and behaviors mediated by GABA(A) 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 GABA(A) receptor-mediated currents is dependent upon the phosphorylation state of the receptor complex. Here we show that phosphorylation of the GABA(A) receptor complex immunoprecipitated by β(2)/β(3) 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-methyltestosterone (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 β(3) subunit of the GABA(A) receptor. Although phosphorylation of these β(3) 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 a

  14. Orexin a phosphorylates the γ-Aminobutyric acid type A receptor β2 subunit on a serine residue and changes the surface expression of the receptor in SH-SY5Y cells exposed to propofol.

    PubMed

    Andersson, Henrik; Björnström, Karin; Eintrei, Christina; Sundqvist, Tommy

    2015-11-01

    Propofol activates the γ-aminobutyric acid type A receptor (GABAA R) and causes a reversible neurite retraction, leaving a thin, thread-like structure behind; it also reverses the transport of vesicles in rat cortical neurons. The awakening peptide orexin A (OA) inhibits this retraction via phospholipase D (PLD) and protein kinase Cɛ (PKCɛ). The human SH-SY5Y cells express both GABAA Rs and orexin 1 and 2 receptors. These cells are used to examine the interaction between OA and the GABAA R. The effects of OA are studied with flow cytometry and immunoblotting. This study shows that OA stimulates phosphorylation on the serine residues of the GABAA R β2 subunit and that the phosphorylation is caused by the activation of PLD and PKCɛ. OA administration followed by propofol reduces the cell surface expression of the GABAA R, whereas propofol stimulation before OA increases the surface expression. The GABAA R β2 subunit is important for receptor recirculation, and the effect of OA on propofol-stimulated cells may be due to a disturbed recirculation of the GABAA R.

  15. 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. PMID:25914173

  16. The anti-convulsant stiripentol acts directly on the GABA(A) receptor as a positive allosteric modulator.

    PubMed

    Fisher, Janet L

    2009-01-01

    Stiripentol (STP) has been used as co-therapy for treatment of epilepsy for many years. Its mechanism of action has long been considered to be indirect, as it inhibits the enzymes responsible for metabolism of other anti-convulsant agents. However, a recent report suggested that STP might also act at the neuronal level, increasing inhibitory GABAergic neurotransmission. We examined the effect of STP on the functional properties of recombinant GABA(A) receptors (GABARs) and found that it was a positive allosteric modulator of these ion channels. Its activity showed some dependence on subunit composition, with greater potentiation of alpha3-containing receptors and reduced potentiation when the beta1 or epsilon subunits were present. STP caused a leftward shift in the GABA concentration-response relationship, but did not increase the peak response of the receptors to a maximal GABA concentration. Although STP shares some functional characteristics with the neurosteroids, its activity was not inhibited by a neurosteroid site antagonist and was unaffected by a mutation in the alpha3 subunit that reduced positive modulation by neurosteroids. The differential effect of STP on beta1- and beta2/beta3-containing receptors was not altered by mutations within the second transmembrane domain that affect modulation by loreclezole. These findings suggest that STP acts as a direct allosteric modulator of the GABAR at a site distinct from many commonly used anti-convulsant, sedative and anxiolytic drugs. Its higher activity at alpha3-containing receptors as well as its activity at delta-containing receptors may provide a unique opportunity to target selected populations of GABARs.

  17. Inhibition by levetiracetam of a non-GABAA receptor-associated epileptiform effect of bicuculline in rat hippocampus

    PubMed Central

    Margineanu, Doru Georg; Wülfert, Ernst

    1997-01-01

    Extracellular recording of field potentials, evoked by commissural stimulation in hippocampal area CA3 of anaesthetized rats, was performed in order to study the mode of action of the novel antiepileptic drug levetiracetam (ucb LO59). The amplitude of orthodromic field population spike (PS2) markedly increased and repetitive population spikes appeared when the recording micropipette contained either bicuculline methiodide (BMI), or the specific GABAA antagonist gabazine (SR-95531). BMI-induced increases in PS2 were reduced in a dose-dependent manner by 1 to 320 μmol kg−1 levetiracetam i.v., with a U-shape dose-response relationship. However, levetiracetam did not reduce the increases in PS2 produced by gabazine. Clonazepam (1 mg kg−1, i.p.), carbamazepine (20 mg kg−1, i.p.) and valproate (200 mg kg−1, i.v.) were ineffective in preventing BMI-induced increases in PS2, while the calcium channel antagonist flunarizine, 50 μmol kg−1, i.p., reduced PS2 increments caused by BMI. The L-type calcium channel blocker nifedipine, 100 μmol kg−1, i.p., was without effect. Similar to levetiracetam, flunarizine did not reduce the increases in PS2 induced by gabazine. These data suggest that the increased excitability of CA3 neurones, caused by BMI administered in situ, involves calcium-dependent processes not associated with blockade of GABAA receptors. The inhibition by levetiracetam of this calcium-dependent effect of BMI might contribute to the antiepileptic effects of the drug. PMID:9401779

  18. Effect of extracellular pH on recombinant alpha1beta2gamma2 and alpha1beta2 GABAA receptors.

    PubMed

    Mercik, Katarzyna; Pytel, Maria; Cherubini, Enrico; Mozrzymas, Jerzy W

    2006-08-01

    Recently, we have reported that extracellular protons allosterically modulated neuronal GABA(A) receptors [Mozrzymas, J.W., Zarnowska, E.D., Pytel, M., Mercik, K., 2003a. Modulation of GABA(A) receptors by hydrogen ions reveals synaptic GABA transient and a crucial role of desensitiztion process. Journal of Neuroscience 23, 7981-7992]. However, GABAARs in neurons are heterogeneous and the effect of hydrogen ions depends on the receptor subtype. In particular, gamma2 subunit sets the receptor sensibility to several modulators including protons. However, the mechanisms whereby protons modulate gamma2-containing and gamma2-free GABAARs have not been fully elucidated. To this end, current responses to ultrafast GABA applications were recorded for alpha1beta2gamma2 and alpha1beta2 receptors at different pH values. For both receptor types, increase in pH induced a decrease in amplitudes of currents elicited by saturating [GABA] but this effect was stronger for alpha1beta2 receptors. In the case of alpha1beta2gamma2 receptors, protons strongly affected the current time course due to a down regulation of binding and desensitization rates. This effect was qualitatively similar to that described in neurons. Protons strongly influenced the amplitude of alpha1beta2 receptor-mediated currents but the effect on their kinetics was weak suggesting a predominant direct non-competitive inhibition with a minor allosteric modulation. In conclusion, we provide evidence that extracellular protons strongly affect GABAA receptors and that, depending on the presence of the gamma2 subunit, the modulatory mechanisms show profound quantitative and qualitative differences.

  19. GABA(A) receptors in the central amygdala are involved in memory retention deficits induced by cannabinoids in rats.

    PubMed

    Hasanein, Parisa; Sharifi, Maryam

    2015-11-01

    The central nucleus of the amygdala (CeA) as the main output of amygdala plays an important role in memory processes. In this study we first evaluated the effects of intra-CeA administrations of different doses of a cannabinoid CB1 agonist, WIN55, 212-2, GABA(A) receptor agonist and antagonist, muscimol and bicuculline, alone on memory retention using passive avoidance learning (PAL) test in rats. Then we examined the effects of GABA(A) receptor agents on the responses induced by intra-CeA microinjection of different doses of WIN55, 212-2. We found that administration of WIN55, 212-2 (0.05, 0.1, 0.2 and 0.4μg/rat) immediately after training impaired memory retrieval in a dose-dependent fashion. Although pre-test intra-CeA administration of muscimol (125, 250 and 500ng/rat) alone had no effect on the step-through latency, its co-administration (125ng/rat) with different doses of WIN55, 212-2 potentiated the amnesic effects of any doses of WIN55, 212-2. The results also showed that pre-test intra-CeA administration of bicuculline (200, 400 and 800ng/rat) alone had no significant effect, but at dose of 200ng/rat disrupted post-training WIN55, 212-2-induced amnesia in the retention test. Furthermore, the additional effect of muscimol (125ng/rat) on memory impairment induced by WIN55, 212-2 (0.1μg/rat) was prevented by intra-CeA co-injection of bicuculline (200ng/rat). We indicated that stimulating or blocking GAGA(A) receptors in the CeA by muscimol and bicuculline interfere with WIN55, 212-2-induced deficits in memory retention in a PAL task and therefore suggests an interaction between cannabinergic and GABAergic systems of the CeA in memory process.

  20. Compartmentation of alpha 1 and alpha 2 GABA(A) receptor subunits within rat extended amygdala: implications for benzodiazepine action.

    PubMed

    Kaufmann, Walter A; Humpel, Christian; Alheid, George F; Marksteiner, Josef

    2003-02-21

    The extended amygdala, a morphological and functional entity within the basal forebrain, is a neuronal substrate for emotional states like fear and anxiety. Anxiety disorders are commonly treated by benzodiazepines that mediate their action via GABA(A) receptors. The binding properties and action of benzodiazepines depend on the alpha-subunit profile of the hetero-pentameric receptors: whereas the alpha1 subunit is associated with benzodiazepine type I pharmacology and reportedly mediates sedative as well as amnesic actions of benzodiazepines, the alpha2 subunit confers benzodiazepine type II pharmacology and mediates the anxiolytic actions of benzodiazepines. We determined the localization of alpha1 and alpha2 subunits within the extended amygdala, identified by secretoneurin immunostaining, to define the morphological substrates for the diverse benzodiazepine actions. A moderate expression of the alpha1 subunit could be detected in compartments of the medial subdivision and a strong expression of the alpha2 subunit throughout the central subdivision. It is concluded that the alpha1 and alpha2 subunits are differentially expressed within the extended amygdala, indicating that this structure is compartmentalized with respect to function and benzodiazepine action. PMID:12573516

  1. Synaptic pruning in the female hippocampus is triggered at puberty by extrasynaptic GABAA receptors on dendritic spines

    PubMed Central

    Afroz, Sonia; Parato, Julie; Shen, Hui; Smith, Sheryl Sue

    2016-01-01

    Adolescent synaptic pruning is thought to enable optimal cognition because it is disrupted in certain neuropathologies, yet the initiator of this process is unknown. One factor not yet considered is the α4βδ GABAA receptor (GABAR), an extrasynaptic inhibitory receptor which first emerges on dendritic spines at puberty in female mice. Here we show that α4βδ GABARs trigger adolescent pruning. Spine density of CA1 hippocampal pyramidal cells decreased by half post-pubertally in female wild-type but not α4 KO mice. This effect was associated with decreased expression of kalirin-7 (Kal7), a spine protein which controls actin cytoskeleton remodeling. Kal7 decreased at puberty as a result of reduced NMDAR activation due to α4βδ-mediated inhibition. In the absence of this inhibition, Kal7 expression was unchanged at puberty. In the unpruned condition, spatial re-learning was impaired. These data suggest that pubertal pruning requires α4βδ GABARs. In their absence, pruning is prevented and cognition is not optimal. DOI: http://dx.doi.org/10.7554/eLife.15106.001 PMID:27136678

  2. The role of ovarian hormone-derived neurosteroids on the regulation of GABAA receptors in affective disorders

    PubMed Central

    MacKenzie, Georgina; Maguire, Jamie

    2014-01-01

    Rationale Neuroactive derivatives of steroid hormones, neurosteroids, can act on GABAA receptors (GABAARs) to potentiate the effects of GABA on these receptors. Neurosteroids become elevated to physiologically relevant levels under conditions characterized by increased steroid hormones. There is considerable evidence for plasticity of GABAARs associated with altered levels of neurosteroids which may counteract the fluctuations in the levels of these allosteric modulators. Objectives The objective of this review is to summarize the current literature on GABAAR plasticity under conditions characterized by alterations in neurosteroid levels, such as over the estrous cycle, during puberty, and throughout pregnancy and the postpartum period. Results The expression of specific GABAAR subunits are altered over the estrous cycle, at puberty, and throughout pregnancy and the postpartum period. Inability to regulate δ subunit-containing GABAARs throughout pregnancy and the postpartum period is associated with depression-like behavior restricted to the postpartum period. Conclusions GABAAR plasticity associated with alterations in neurosteroid levels represents a homeostatic compensatory mechanism to maintain an ideal level of inhibition to offset the potentiating effects of neurosteroids on GABAergic inhibition. Failure to properly regulate GABAARs under conditions of altered neurosteroid levels may increase vulnerability to mood disorders, such as premenstrual syndrome (PMS), premenstrual dysphoric disorder (PMDD), and postpartum depression. PMID:24402140

  3. Central PGE2 exhibits anxiolytic-like activity via EP1 and EP4 receptors in a manner dependent on serotonin 5-HT1A, dopamine D1 and GABAA receptors.

    PubMed

    Suzuki, Chihiro; Miyamoto, Chihiro; Furuyashiki, Tomoyuki; Narumiya, Shuh; Ohinata, Kousaku

    2011-07-21

    We found that centrally administered prostaglandin (PG) E(2) exhibited anxiolytic-like activity in the elevated plus-maze and open field test in mice. Agonists selective for EP(1) and EP(4) receptors, among four receptor subtypes for PGE(2), mimicked the anxiolytic-like activity of PGE(2). The anxiolytic-like activity of PGE(2) was blocked by an EP(1) or EP(4) antagonist, as well as in EP(4) but not EP(1) knockout mice. Central activation of either EP(1) or EP(4) receptors resulted in anxiolytic-like activity. The PGE(2)-induced anxiolytic-like activity was inhibited by antagonists for serotonin 5-HT(1A), dopamine D(1) and GABA(A) receptors. Taken together, PGE(2) exhibits anxiolytic-like activity via EP(1) and EP(4) receptors, with downstream involvement of 5-HT(1A), D(1) and GABA(A) receptor systems.

  4. Effect of low doses of progesterone in the expression of the GABA(A) receptor α4 subunit and procaspase-3 in the hypothalamus of female rats.

    PubMed

    Arbo, Bruno D; Andrade, Susie; Osterkamp, Gabriela; Gomez, Rosane; Ribeiro, Maria Flávia M

    2014-08-01

    Progesterone is a steroid which regulates neural function, thereby modulating neurotransmission, cell survival, and behavior. Previous studies by our group have shown that chronic administration of low doses of progesterone in diestrus II female rats has an antidepressant-like effect in the forced swimming test (FST). Depression is associated with the several neurotransmitters systems, including GABA and serotonin, and with neurodegeneration and cell death in some brain circuits. The aim of this study was to verify the effect of progesterone on the protein expression of the GABA(A) receptor α4 subunit, serotonin transporter (SERT), Akt, extracellular signal-regulated kinase (Erk), and caspase-3 in the hypothalamus of diestrus II female rats exposed to the FST. Female rats were treated with a daily injection of progesterone (0.4 mg/kg) or vehicle, during two complete oestrous cycles. On the day of the experiment, the animals were euthanized 30 min after the FST, the hypothalamus was dissected and protein expression of GABA(A) receptor α4 subunit, SERT, Akt, Erk, and caspase-3 was evaluated. Progesterone increased the expression of GABA(A) receptor α4 subunit but did not change the expression of SERT. Progesterone decreased the expression of procaspase-3 in the hypothalamus without changing the activation of Akt and Erk in this structure. In summary, our results suggest that progesterone acts to increase the expression of the GABA(A) receptor α4 subunit and decrease the expression of procaspase-3 in the hypothalamus of female rats. Such effects may be involved in the antidepressant-like effect of progesterone in female rats exposed to the FST.

  5. Plasticity of GABAA receptor-mediated neurotransmission in the nucleus accumbens of alcohol-dependent rats

    PubMed Central

    Liang, Jing; Lindemeyer, A. Kerstin; Suryanarayanan, Asha; Meyer, Edward M.; Marty, Vincent N.; Ahmad, S. Omar; Shao, Xuesi Max; Olsen, Richard W.

    2014-01-01

    Chronic alcohol exposure-induced changes in reinforcement mechanisms and motivational state are thought to contribute to the development of cravings and relapse during protracted withdrawal. The nucleus accumbens (NAcc) is a key structure of the mesolimbic dopaminergic reward system and plays an important role in mediating alcohol-seeking behaviors. Here we describe the long-lasting alterations of γ-aminobutyric acid type A receptors (GABAARs) of medium spiny neurons (MSNs) in the NAcc after chronic intermittent ethanol (CIE) treatment, a rat model of alcohol dependence. CIE treatment and withdrawal (>40 days) produced decreases in the ethanol and Ro15-4513 potentiation of extrasynaptic GABAARs, which mediate the picrotoxin-sensitive tonic current (Itonic), while potentiation of synaptic receptors, which give rise to miniature inhibitory postsynaptic currents (mIPSCs), was increased. Diazepam sensitivity of both Itonic and mIPSCs was decreased by CIE treatment. The average magnitude of Itonic was unchanged, but mIPSC amplitude and frequency decreased and mIPSC rise time increased after CIE treatment. Rise-time histograms revealed decreased frequency of fast-rising mIPSCs after CIE treatment, consistent with possible decreases in somatic GABAergic synapses in MSNs from CIE rats. However, unbiased stereological analysis of NeuN-stained NAcc neurons did not detect any decreases in NAcc volume, neuronal numbers, or neuronal cell body volume. Western blot analysis of surface subunit levels revealed selective decreases in α1 and δ and increases in α4, α5, and γ2 GABAAR subunits after CIE treatment and withdrawal. Similar, but reversible, alterations occurred after a single ethanol dose (5 g/kg). These data reveal CIE-induced long-lasting neuroadaptations in the NAcc GABAergic neurotransmission. PMID:24694935

  6. Tonic Inhibition of Accumbal Spiny Neurons by Extrasynaptic α4βδ GABAA Receptors Modulates the Actions of Psychostimulants

    PubMed Central

    Maguire, Edward P.; Macpherson, Tom; Swinny, Jerome D.; Dixon, Claire I.; Herd, Murray B.; Belelli, Delia; Stephens, David N.

    2014-01-01

    Within the nucleus accumbens (NAc), synaptic GABAA receptors (GABAARs) mediate phasic inhibition of medium spiny neurons (MSNs) and influence behavioral responses to cocaine. We demonstrate that both dopamine D1- and D2-receptor-expressing MSNs (D-MSNs) additionally harbor extrasynaptic GABAARs incorporating α4, β, and δ subunits that mediate tonic inhibition, thereby influencing neuronal excitability. Both the selective δ-GABAAR agonist THIP and DS2, a selective positive allosteric modulator, greatly increased the tonic current of all MSNs from wild-type (WT), but not from δ−/− or α4−/− mice. Coupling dopamine and tonic inhibition, the acute activation of D1 receptors (by a selective agonist or indirectly by amphetamine) greatly enhanced tonic inhibition in D1-MSNs but not D2-MSNs. In contrast, prolonged D2 receptor activation modestly reduced the tonic conductance of D2-MSNs. Behaviorally, WT and constitutive α4−/− mice did not differ in their expression of cocaine-conditioned place preference (CPP). Importantly, however, mice with the α4 deletion specific to D1-expressing neurons (α4D1−/−) showed increased CPP. Furthermore, THIP administered systemically or directly into the NAc of WT, but not α4−/− or α4D1−/− mice, blocked cocaine enhancement of CPP. In comparison, α4D2−/− mice exhibited normal CPP, but no cocaine enhancement. In conclusion, dopamine modulation of GABAergic tonic inhibition of D1- and D2-MSNs provides an intrinsic mechanism to differentially affect their excitability in response to psychostimulants and thereby influence their ability to potentiate conditioned reward. Therefore, α4βδ GABAARs may represent a viable target for the development of novel therapeutics to better understand and influence addictive behaviors. PMID:24431441

  7. Glycine and GABAA receptors mediate tonic and phasic inhibitory processes that contribute to prepulse inhibition in the goldfish startle network

    PubMed Central

    Curtin, Paul C. P.; Preuss, Thomas

    2015-01-01

    Prepulse inhibition (PPI) is understood as a sensorimotor gating process that attenuates sensory flow to the startle pathway during early stages (20–1000 ms) of information processing. Here, we applied in vivo electrophysiology and pharmacology to determine if PPI is mediated by glycine receptors (GlyRs) and/or GABAA receptors (GABAARs) in the goldfish auditory startle circuit. Specifically, we used selective antagonists to dissect the contributions of target receptors on sound-evoked postsynaptic potentials (PSPs) recorded in the neurons that initiate startle, the Mauthner-cells (M-cell). We found that strychnine, a GlyR antagonist, disrupted a fast-activated (5 ms) and rapidly (<50 ms) decaying (feed-forward) inhibitory process that contributes to PPI at 20 ms prepulse/pulse inter-stimulus intervals (ISI). Additionally we observed increases of the evoked postsynaptic potential (PSP) peak amplitude (+87.43 ± 21.53%, N = 9) and duration (+204 ± 48.91%, N = 9). In contrast, treatment with bicuculline, a GABAAR antagonist, caused a general reduction in PPI across all tested interstimulus intervals (ISIs) (20–500 ms). Bicuculline also increased PSP peak amplitude (+133.8 ± 10.3%, N = 5) and PSP duration (+284.95 ± 65.64%, N = 5). Treatment with either antagonist also tonically increased post-synaptic excitability in the M-cells, reflected by an increase in the magnitude of antidromically-evoked action potentials (APs) by 15.07 ± 3.21%, N = 7 and 16.23 ± 7.08%, N = 5 for strychnine and bicuculline, respectively. These results suggest that GABAARs and GlyRs are functionally segregated to short- and longer-lasting sound-evoked (phasic) inhibitory processes that contribute to PPI, with the mediation of tonic inhibition by both receptor systems being critical for gain control within the M-cell startle circuit. PMID:25852486

  8. Modulation by general anaesthetics of rat GABAA receptors comprised of α1β3 and β3 subunits expressed in human embryonic kidney 293 cells

    PubMed Central

    Davies, Paul A; Kirkness, Ewen F; Hales, Tim G

    1997-01-01

    Radioligand binding and patch-clamp techniques were used to study the actions of γ-aminobutyric acid (GABA) and the general anaesthetics propofol (2,6-diisopropylphenol), pentobarbitone and 5α-pregnan-3α-ol-20-one on rat α1 and β3 GABAA receptor subunits, expressed either alone or in combination.Membranes from HEK293 cells after transfection with α1 cDNA did not bind significant levels of [35S]-tert-butyl bicyclophosphorothionate ([35S]-TBPS) (<0.03 pmol mg−1 protein). GABA (100 μM) applied to whole-cells transfected with α1 cDNA and clamped at −60 mV, also failed to activate discernible currents.The membranes of cells expressing β3 cDNAs bound [35S]-TBPS (∼1 pmol mg−1 protein). However, the binding was not influenced by GABA (10 nM–100 μM). Neither GABA (100 μM) nor picrotoxin (10 μM) affected currents recorded from cells expressing β3 cDNA, suggesting that β3 subunits do not form functional GABAA receptors or spontaneously active ion channels.GABA (10 nM–100 μM) modulated [35S]-TBPS binding to the membranes of cells transfected with both α1 and β3 cDNAs. GABA (0.1 μM–1 mM) also dose-dependently activated inward currents with an EC50 of 9 μM recorded from cells transfected with α1 and β3 cDNAs, clamped at −60 mV.Propofol (10 nM–100 μM), pentobarbitone (10 nM–100 μM) and 5α-pregnan-3α-ol-20-one (1 nM–30 μM) modulated [35S]-TBPS binding to the membranes of cells expressing either α1β3 or β3 receptors. Propofol (100 μM), pentobarbitone (1 mM) and 5α-pregnan-3α-ol-20-one (10 μM) also activated currents recorded from cells expressing α1β3 receptors.Propofol (1 μM–1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing β3 homomers. In contrast, application of 5α-pregnan-3α-ol-20-one (10 μM) failed to activate detectable currents.Propofol (100 μM)-activated currents recorded from cells expressing either α1β3 or β3

  9. A tight coupling between β₂Y97 and β₂F200 of the GABA(A) receptor mediates GABA binding.

    PubMed

    Tran, Phu N; Laha, Kurt T; Wagner, David A

    2011-10-01

    The GABA(A) receptor is an oligopentameric chloride channel that is activated via conformation changes induced upon the binding of the endogenous ligand, GABA, to the extracellular inter-subunit interfaces. Although dozens of amino acid residues at the α/β interface have been implicated in ligand binding, the structural elements that mediate ligand binding and receptor activation are not yet fully described. In this study, double-mutant cycle analysis was employed to test for possible interactions between several arginines (α₁R67, α₁R120, α₁R132, and β₂R207) and two aromatic residues (β₂Y97 and β₂F200) that are present in the ligand-binding pocket and are known to influence GABA affinity. Our results show that neither α₁R67 nor α₁R120 is functionally coupled to either of the aromatics, whereas a moderate coupling exists between α₁R132 and both aromatic residues. Significant functional coupling between β₂R207 and both β₂Y97 and β₂F200 was found. Furthermore, we identified an even stronger coupling between the two aromatics, β₂Y97 and β₂F200, and for the first time provided direct evidence for the involvement of β₂Y97 and β₂F200 in GABA binding. As these residues are tightly linked, and mutation of either has similar, severe effects on GABA binding and receptor kinetics, we believe they form a single functional unit that may directly coordinate GABA.

  10. Synaptic-Type α1β2γ2L GABAA Receptors Produce Large Persistent Currents in the Presence of Ambient GABA and Anesthetic Drugs

    PubMed Central

    Li, Ping

    2015-01-01

    Synaptic GABAA receptors respond to synaptically released GABA and are considered to be unaffected by the low levels of ambient transmitter in the brain. We show that synaptic-type α1β2γ2L GABAA receptors expressed in HEK293 cells respond with large steady-state currents to combinations of a low concentration (0.5 μM) of GABA and clinically used GABAergic modulators propofol, etomidate, or pentobarbital or the steroid alphaxalone. At a maximally effective concentration of modulator, the current levels at the end of 2-minute applications of drug combinations were >10% of the peak response to saturating GABA. In the absence of modulators, 0.5 μM GABA generated a steady-state response of 1% of the peak response to saturating GABA. The concentration-response curves for enhancement of steady-state currents by propofol, etomidate, pentobarbital, or alphaxalone were at similar or lower drug concentrations compared with concentration-response relationships for enhancement of peak responses. We propose that modulation of tonically activated synaptic-type GABAA receptors contributes to the clinical actions of sedative drugs. PMID:25667223

  11. [The contribution of glycine and GABA(A) receptors to generation of the inhibitory postsynaptic potentials in the frog spinal cord motoneurones].

    PubMed

    Kurchavyĭ, G G; Kalinina, N I; Veselkin, N P

    2010-06-01

    The contribution of glycine and GABA(A) receptors to generation of the inhibitory postsynaptic potentials (IPSPs) evoked by microstimulation of the inhibitory fibers was studied intracellularly in the motoneurones of the isolated frog spinal cord. IPSPs were isolated by bloking EPSPs with kynurenate or CNQX and AP-5. The reversion under the small depolarising current (1-10) nA was used for the identification of IPSPs. The selective GlyR antagonist strychnine (1-5 microM) reduced the amplitude of IPSPs by a factor of 4.7 on the average in all studied motoneurones, while the selective GABA(A)R antagonist bicuculline (50-70 microM)--only by a factor of 1.6 and had no effect in 44% of motoneurones. Sequential applications of strychnine and bicuculline completely blocked the IPSPs. The results suggest that postsynaptic inhibition in the frog motoneurones is mediated by glycine (predominantly) and GABA(A) (to a smaller extent) receptors. It is possible the GABA(A) receptors are partly extrasynaptic. PMID:20795472

  12. The effects of GABAA and NMDA receptors in the shell-accumbens on spatial memory of METH-treated rats.

    PubMed

    Heysieattalab, Soomaayeh; Naghdi, Nasser; Zarrindast, Mohammad-Reza; Haghparast, Abbas; Mehr, Shahram Ejtemaei; Khoshbouei, Habibeh

    2016-03-01

    Methamphetamine (METH) is a highly addictive and neurotoxic psychostimulant. Its use in humans is often associated with neurocognitive impairment and deficits in hippocampal plasticity. Striatal dopamine system is one of the main targets of METH. The dopamine neurons in the striatum directly or indirectly regulate the GABA and glutamatergic signaling in this region and thus their outputs. This is consistent with previous reports showing modification of neuronal activity in the striatum modulates the expression of hippocampal LTP and hippocampal-dependent memory tasks such as Morris water maze (MWM). Therefore, reversing or preventing METH-induced synaptic modifications via pharmacological manipulations of the shell-nucleus accumbens (shell-NAc) may introduce a viable therapeutic target to attenuate the METH-induced memory deficits. This study is designed to investigate the role of intra-shell NAc manipulation of GABAA and NMDA receptors and their interaction with METH on memory performance in MWM task. Pharmacological manipulations were performed in rats received METH or saline. We found systemic saline plus intra-shell NAc infusions of muscimol dose-dependently impaired performance, while bicuculline had no effect. Surprisingly, the intra-NAc infusions of 0.005μg/rat muscimol that has no effect on memory performance (ineffective dose) prevented METH-induced memory impairment. In the contrary, the intra-NAc infusions of bicuculline (0.2μg/rat) increased METH-induced memory impairment. However, pre-training intra-NAc infusions of D-AP5 dose-dependently impaired performance, while NMDA had no effect in rats received systemic saline (control group). The intra-NAc infusions with an ineffective dose of NMDA (0.1μg/rat) increased METH-induced memory impairment. Furthermore, intra-NAc infusions of D-AP5 with an ineffective dose (0.1μg/rat) prevented METH-induced memory impairment. Our result is consistent with the interpretation that METH-mediated learning deficit

  13. Silencing the α2 subunit of GABAA receptors in rat dorsal root ganglia reveals its major role in antinociception post-traumatic nerve injury

    PubMed Central

    Obradović, Aleksandar LJ; Scarpa, Joseph; Osuru, Hari P; Weaver, Janelle L; Park, Ji-Yong; Pathirathna, Sriyani; Peterkin, Alexander; Lim, Yunhee; Jagodic, Miljenko M; Todorovic, Slobodan M; Jevtovic-Todorovic, Vesna

    2015-01-01

    Background Neuropathic pain is likely the result of repetitive high frequency bursts of peripheral afferent activity leading to long-lasting changes in synaptic plasticity in the spinal dorsal horn (DH). Drugs that promote GABA activity in the DH provide partial relief of neuropathic symptoms. We examined how in vivo silencing of the GABAA α2 gene in DRG controls of NPP. Methods After crush injury to the right sciatic nerve of female rats, the α2 GABAA antisense and mismatch oligodeoxynucleotides or NO-711 (a GABA uptake inhibitor) were applied to the L5 DRG. In vivo behavioral assessment of nociception was conducted prior to the injury and ensuing 10 days (n=4–10). In vitro quantification of α2 GABAA protein and electrophysiology studies of GABAA currents were performed on acutely dissociated L5 DRG neurons at relevant time-points (n=6–14). Results NPP post-crush injury of a sciatic nerve in adult female rats coincides with significant down-regulation of the α2 subunit expression in the ipsilateral DRG (about 30%). Selective down-regulation of α2 expression in DRGs significantly worsens mechanical (2.55±0.75 to 5.16±1.16) and thermal (7.97±0.96 to 5.51±0.75) hypersensitivity in crush-injured animals and causes development of significant mechanical (2.33±0.40 to 5.00±0.33) and thermal (10.80±0.29 to 7.34±0.81) hypersensitivity in sham animals (data shown as MEAN±SD). Conversely, up-regulation of endogenous GABA via blockade of its uptake in DRG alleviates NPP. Conclusions The GABAA receptor in the DRG plays an important role in pathophysiology of NPP caused by sciatic nerve injury and represent promising target for novel pain therapies. PMID:26164299

  14. Lability of GABAA receptor function in human partial epilepsy: possible relationship to hypometabolism.

    PubMed

    Pumain, René; Ahmed, Mounia Sid; Kurcewicz, Irène; Trottier, Suzanne; Louvel, Jacques; Turak, Baris; Devaux, Bertrand; Laschet, Jacques

    2008-11-01

    The function of the gamma-aminobutyric acid type A receptor (GABA(A)R) is maintained by endogenous phosphorylation. We have shown that the corresponding kinase is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using the locally produced glycolytic ATP. In addition, using cerebral tissue obtained during curative surgery for epilepsy, we showed that both the endogenous phosphorylation and the GABA(A)R function are significantly reduced in the "epileptogenic" cerebral cortex when compared to "control" tissue. This dysfunction likely contributes to seizure generation and/or transition from the interictal to the ictal state. Glucose utilization is decreased in the epileptogenic cortex of patients with partial epilepsy in the interictal state, but the relationship to the disorder remains unclear. We propose that this hypometabolism is related to the deficiency in the endogenous phosphorylation of GABA(A)R and the resulting greater lability of GABAergic inhibition. Several lines of evidences indeed suggest that GABAergic inhibition is costly in terms of metabolic consumption. The deficiency of this glycolysis-dependent mechanism may thus link epileptogenicity to glucose hypometabolism. The antiepileptic effect of ketogenic diets may be mediated by the subsequent rise in the NADH/NAD(+) index, which favors GABA(A)R endogenous phosphorylation and should contribute to restoration of GABAergic inhibition in the epileptogenic zone.

  15. Repeated neonatal handling with maternal separation permanently alters hippocampal GABAA receptors and behavioral stress responses

    PubMed Central

    Hsu, Fu-Chun; Zhang, Guo-Jun; Raol, Yogendra Sinh H.; Valentino, Rita J.; Coulter, Douglas A.; Brooks-Kayal, Amy R.

    2003-01-01

    Increasing evidence suggests that postnatal events, such as handling or maternal separation, can produce long-term changes in brain function. These are often expressed as changes in the profile of endocrine or behavioral responses to stress. Changes in γ-aminobutyric acid type A receptors (GABARs), which mediate the majority of fast synaptic inhibition in adult brain, have been proposed as one potential mediator of these behavioral effects. In the current article, we use a combination of single-cell electrophysiology and antisense mRNA amplification to demonstrate permanent molecular and functional differences in GABARs within hippocampal dentate granule neurons after as few as two episodes of neonatal handling with brief maternal separation. Adult animals that as pups experienced handling with maternal separation maintained a more immature GABAR phenotype and exhibited increased activity in response to swim stress. These findings demonstrate the exquisite sensitivity of the developing GABAergic system to even subtle environmental manipulations and provide an unique molecular mechanism by which postnatal handling with maternal separation may alter stress-related behavior. PMID:14530409

  16. Zolpidem is a potent stoichiometry-selective modulator of α1β3 GABAA receptors: evidence of a novel benzodiazepine site in the α1-α1 interface

    PubMed Central

    Che Has, Ahmad Tarmizi; Absalom, Nathan; van Nieuwenhuijzen, Petra S.; Clarkson, Andrew N.; Ahring, Philip K.; Chebib, Mary

    2016-01-01

    Zolpidem is not a typical GABAA receptor hypnotic. Unlike benzodiazepines, zolpidem modulates tonic GABA currents in the rat dorsal motor nucleus of the vagus, exhibits residual effects in mice lacking the benzodiazepine binding site, and improves speech, cognitive and motor function in human patients with severe brain injury. The receptor by which zolpidem mediates these effects is not known. In this study we evaluated binary α1β3 GABAA receptors in either the 3α1:2β3 or 2α1:3β3 subunit stoichiometry, which differ by the existence of either an α1-α1 interface, or a β3-β3 interface, respectively. Both receptor stoichiometries are readily expressed in Xenopus oocytes, distinguished from each other by using GABA, zolpidem, diazepam and Zn2+. At the 3α1:2β3 receptor, clinically relevant concentrations of zolpidem enhanced GABA in a flumazenil-sensitive manner. The efficacy of diazepam was significantly lower compared to zolpidem. No modulation by either zolpidem or diazepam was detected at the 2α1:3β3 receptor, indicating that the binding site for zolpidem is at the α1-α1 interface, a site mimicking the classical α1-γ2 benzodiazepine site. Activating α1β3 (3α1:2β3) receptors may, in part, mediate the physiological effects of zolpidem observed under distinct physiological and clinical conditions, constituting a potentially attractive drug target. PMID:27346730

  17. The influence of stress at puberty on mood and learning: role of the α4βδ GABAA receptor.

    PubMed

    Smith, S S

    2013-09-26

    It is well-known that the onset of puberty is associated with changes in mood as well as cognition. Stress can have an impact on these outcomes, which in many cases, can be more influential in females, suggesting that gender differences exist. The adolescent period is a vulnerable time for the onset of certain psychopathologies, including anxiety disorders, depression and eating disorders, which are also more prevalent in females. One factor which may contribute to stress-triggered anxiety at puberty is the GABAA receptor (GABAR), which is known to play a pivotal role in anxiety. Expression of α4βδ GABARs increases on the dendrites of CA1 pyramidal cells at the onset of puberty in the hippocampus, part of the limbic circuitry which governs emotion. This receptor is a sensitive target for the stress steroid 3α-OH-5[α]β-pregnan-20-one or [allo]pregnanolone, which paradoxically reduces inhibition and increases anxiety during the pubertal period (post-natal day ∼35-44) of female mice in contrast to its usual effect to enhance inhibition and reduce anxiety. Spatial learning and synaptic plasticity are also adversely impacted at puberty, likely a result of increased expression of α4βδ GABARs on the dendritic spines of CA1 hippocampal pyramidal cells, which are essential for consolidation of memory. This review will focus on the role of these receptors in mediating behavioral changes at puberty. Stress-mediated changes in mood and cognition in early adolescence may have relevance for the expression of psychopathologies in adulthood.

  18. Oxytocin prevents ethanol actions at δ subunit-containing GABAA receptors and attenuates ethanol-induced motor impairment in rats.

    PubMed

    Bowen, Michael T; Peters, Sebastian T; Absalom, Nathan; Chebib, Mary; Neumann, Inga D; McGregor, Iain S

    2015-03-10

    Even moderate doses of alcohol cause considerable impairment of motor coordination, an effect that substantially involves potentiation of GABAergic activity at δ subunit-containing GABA(A) receptors (δ-GABA(A)Rs). Here, we demonstrate that oxytocin selectively attenuates ethanol-induced motor impairment and ethanol-induced increases in GABAergic activity at δ-GABA(A)Rs and that this effect does not involve the oxytocin receptor. Specifically, oxytocin (1 µg i.c.v.) given before ethanol (1.5 g/kg i.p.) attenuated the sedation and ataxia induced by ethanol in the open-field locomotor test, wire-hanging test, and righting-reflex test in male rats. Using two-electrode voltage-clamp electrophysiology in Xenopus oocytes, oxytocin was found to completely block ethanol-enhanced activity at α4β1δ and α4β3δ recombinant GABA(A)Rs. Conversely, ethanol had no effect when applied to α4β1 or α4β3 cells, demonstrating the critical presence of the δ subunit in this effect. Oxytocin had no effect on the motor impairment or in vitro effects induced by the δ-selective GABA(A)R agonist 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, which binds at a different site on δ-GABA(A)Rs than ethanol. Vasopressin, which is a nonapeptide with substantial structural similarity to oxytocin, did not alter ethanol effects at δ-GABA(A)Rs. This pattern of results confirms the specificity of the interaction between oxytocin and ethanol at δ-GABA(A)Rs. Finally, our in vitro constructs did not express any oxytocin receptors, meaning that the observed interactions occur directly at δ-GABA(A)Rs. The profound and direct interaction observed between oxytocin and ethanol at the behavioral and cellular level may have relevance for the development of novel therapeutics for alcohol intoxication and dependence.

  19. 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 Central

    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

    2016-01-01

    Summary Background 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. Methods 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. Findings 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

  20. Molecular cloning and expression analysis of GABA(A) receptor-associated protein (GABARAP) from small abalone, Haliotis diversicolor.

    PubMed

    Bai, Rongyao; You, Weiwei; Chen, Jun; Huang, Heqing; Ke, Caihuan

    2012-10-01

    GABA(A) receptor-associated protein (GABARAP), a multifunctional protein participating in autophagy process, is evolutionarily conserved and involves in innate immunity in eukaryotic cells, but currently there is no research on the relationship between GABARAP and innate immunity in mollusc. In the present study, the GABARAP full-length cDNA and its genomic DNA were firstly cloned from small abalone (Haliotis diversicolor), which was named as saGABARAP. Its full-length cDNA is 963 bp with a 354 bp open reading frame encoding a protein of 117 aa, a 276 bp 5'-UTR, and a 333 bp 3'-UTR including a poly(A) tail, two typical polyadenylation signals (AATAA) and two RNA instability motifs (ATTTA). The deduced protein has an estimated molecular weight of 13.9 kDa and a predicted PI of 8.73. Its genomic DNA comprises 4352 bp, containing three exons and two introns. Quantitative real-time PCR analysis revealed that saGABARAP was constitutively expressed in all examined tissues, with the highest expression level in hepatopancreas, and was upregulated in hepatopancreas and hemocytes after bacterial challenge. In addition, saGABARAP was ubiquitously expressed at all examined embryonic and larval development stages. These results suggested that saGABARAP could respond to bacteria challenge and may play a vital role in the adult innate immune system against pathogens and the development process of abalone embryo and larvae.

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

    PubMed

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

    2013-12-01

    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. PMID:24012744

  2. Activity blockade and GABAA receptor blockade produce synaptic scaling through chloride accumulation in embryonic spinal motoneurons and interneurons.

    PubMed

    Lindsly, Casie; Gonzalez-Islas, Carlos; Wenner, Peter

    2014-01-01

    Synaptic scaling represents a process whereby the distribution of a cell's synaptic strengths are altered by a multiplicative scaling factor. Scaling is thought to be a compensatory response that homeostatically controls spiking activity levels in the cell or network. Previously, we observed GABAergic synaptic scaling in embryonic spinal motoneurons following in vivo blockade of either spiking activity or GABAA receptors (GABAARs). We had determined that activity blockade triggered upward GABAergic scaling through chloride accumulation, thus increasing the driving force for these currents. To determine whether chloride accumulation also underlies GABAergic scaling following GABAAR blockade we have developed a new technique. We expressed a genetically encoded chloride-indicator, Clomeleon, in the embryonic chick spinal cord, which provides a non-invasive fast measure of intracellular chloride. Using this technique we now show that chloride accumulation underlies GABAergic scaling following blockade of either spiking activity or the GABAAR. The finding that GABAAR blockade and activity blockade trigger scaling via a common mechanism supports our hypothesis that activity blockade reduces GABAAR activation, which triggers synaptic scaling. In addition, Clomeleon imaging demonstrated the time course and widespread nature of GABAergic scaling through chloride accumulation, as it was also observed in spinal interneurons. This suggests that homeostatic scaling via chloride accumulation is a common feature in many neuronal classes within the embryonic spinal cord and opens the possibility that this process may occur throughout the nervous system at early stages of development.

  3. Simultaneous optical recording in multiple cells by digital holographic microscopy of chloride current associated to activation of the ligand-gated chloride channel GABA(A) receptor.

    PubMed

    Jourdain, Pascal; Boss, Daniel; Rappaz, Benjamin; Moratal, Corinne; Hernandez, Maria-Clemencia; Depeursinge, Christian; Magistretti, Pierre Julius; Marquet, Pierre

    2012-01-01

    Chloride channels represent a group of targets for major clinical indications. However, molecular screening for chloride channel modulators has proven to be difficult and time-consuming as approaches essentially rely on the use of fluorescent dyes or invasive patch-clamp techniques which do not lend themselves to the screening of large sets of compounds. To address this problem, we have developed a non-invasive optical method, based on digital holographic microcopy (DHM), allowing monitoring of ion channel activity without using any electrode or fluorescent dye. To illustrate this approach, GABA(A) mediated chloride currents have been monitored with DHM. Practically, we show that DHM can non-invasively provide the quantitative determination of transmembrane chloride fluxes mediated by the activation of chloride channels associated with GABA(A) receptors. Indeed through an original algorithm, chloride currents elicited by application of appropriate agonists of the GABA(A) receptor can be derived from the quantitative phase signal recorded with DHM. Finally, chloride currents can be determined and pharmacologically characterized non-invasively simultaneously on a large cellular sampling by DHM.

  4. Valerian inhibits rat hepatocarcinogenesis by activating GABA(A) receptor-mediated signaling.

    PubMed

    Kakehashi, Anna; Kato, Ayumi; Ishii, Naomi; Wei, Min; Morimura, Keiichirou; Fukushima, Shoji; Wanibuchi, Hideki

    2014-01-01

    Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA) A receptor (GABA(A)R) system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(A)R agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis) at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN). Formation of glutathione S-transferase placental form positive (GST-P(+)) foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2'-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P(+) foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21(Waf1/Cip1), p53 and Bax mRNA expression. Interestingly, expression of the GABA(A)R alpha 1 subunit was observed in GST-P(+) foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P(+) foci by activating GABA(A)R-mediated signaling. PMID:25419570

  5. Chronic Intermittent Ethanol Regulates Hippocampal GABA(A) Receptor Delta Subunit Gene Expression

    PubMed Central

    Follesa, Paolo; Floris, Gabriele; Asuni, Gino P.; Ibba, Antonio; Tocco, Maria G.; Zicca, Luca; Mercante, Beniamina; Deriu, Franca; Gorini, Giorgio

    2015-01-01

    Chronic ethanol consumption causes structural and functional reorganization in the hippocampus and induces alterations in the gene expression of gamma-aminobutyric acid type A receptors (GABAARs). Distinct forced intermittent exposure models have been used previously to investigate changes in GABAAR expression, with contrasting results. Here, we used repeated cycles of a Chronic Intermittent Ethanol paradigm to examine the relationship between voluntary, dependence-associated ethanol consumption, and GABAAR gene expression in mouse hippocampus. Adult male C57BL/6J mice were exposed to four 16-h ethanol vapor (or air) cycles in inhalation chambers alternated with limited-access two-bottle choice between ethanol (15%) and water consumption. The mice exposed to ethanol vapor showed significant increases in ethanol consumption compared to their air-matched controls. GABAAR alpha4 and delta subunit gene expression were measured by qRT-PCR at different stages. There were significant changes in GABAAR delta subunit transcript levels at different time points in ethanol-vapor exposed mice, while the alpha4 subunit levels remained unchanged. Correlated concurrent blood ethanol concentrations suggested that GABAAR delta subunit mRNA levels fluctuate depending on ethanol intoxication, dependence, and withdrawal state. Using a vapor-based Chronic Intermittent Ethanol procedure with combined two-bottle choice consumption, we corroborated previous evidences showing that discontinuous ethanol exposure affects GABAAR delta subunit expression but we did not observe changes in alpha4 subunit. These findings indicate that hippocampal GABAAR delta subunit expression changes transiently over the course of a Chronic Intermittent Ethanol paradigm associated with voluntary intake, in response to ethanol-mediated disturbance of GABAergic neurotransmission. PMID:26617492

  6. Ethanol-Induced Plasticity of GABAA Receptors in the Basolateral Amygdala

    PubMed Central

    Lindemeyer, A. Kerstin; Liang, Jing; Marty, Vincent N.; Meyer, Edward M.; Suryanarayanan, Asha; Olsen, Richard W.

    2014-01-01

    Acute and chronic ethanol (EtOH) administration is known to affect function, surface expression, and subunit composition of γ-aminobutyric acid (A) receptors (GABAARs) in different parts of the brain, which is believed to play a major role in alcohol dependence and withdrawal symptoms. The basolateral amygdala (BLA) participates in anxiety-like behaviors including those induced by alcohol withdrawal. In the present study we assessed the changes in cell surface levels of select GABAAR subunits in the BLA of a rat model of alcohol dependence induced by chronic intermittent EtOH (CIE) treatment and long-term (>40 days) withdrawal and investigated the time-course of such changes after a single dose of EtOH (5 g/kg, gavage). We found an early decrease in surface expression of α4 and γ subunits at 1 h following single dose EtOH treatment. At 48 h post-EtOH and after CIE treatment there was an increase in α4 and γ2, while α1, α2, and γ surface expression were decreased. To relate functional changes in GABAARs to changes in their subunit composition we analyzed miniature inhibitory postsynaptic currents (mIPSCs) and the picrotoxin-sensitive tonic current (Itonic) 48 h after EtOH intoxication. The Itonic magnitude and most of the mIPSC kinetic parameters (except faster mIPSC decay) were unchanged at 48 h post-EtOH. At the same time, Itonic potentiation by acute EtOH was greatly reduced, whereas mIPSCs became significantly more sensitive to potentiation by acute EtOH. These results suggest that EtOH intoxication-induced GABAAR plasticity in the BLA might contribute to the diminished sedative/hypnotic and maintained anxiolytic effectiveness of EtOH. PMID:24710789

  7. PWZ-029, an inverse agonist selective for α₅ GABAA receptors, improves object recognition, but not water-maze memory in normal and scopolamine-treated rats.

    PubMed

    Milić, Marija; Timić, Tamara; Joksimović, Srđan; Biawat, Poonam; Rallapalli, Sundari; Divljaković, Jovana; Radulović, Tamara; Cook, James M; Savić, Miroslav M

    2013-03-15

    Inverse agonism at the benzodiazepine site of α(5) subunit-containing GABA(A) receptors is an attractive approach for the development of putative cognition-enhancing compounds, which are still far from clinical application. Several ligands with binding and/or functional selectivity for α(5) GABA(A) receptors have been synthesized and tested in a few animal models. PWZ-029 is an α(5) GABA(A) selective inverse agonist whose memory enhancing effects were demonstrated in the passive avoidance task in rats and in Pavlovian fear conditioning in mice. In the present study we investigated the effects of PWZ-029 administration in novel object recognition test and Morris water maze, in normal and scopolamine-treated rats. All the three doses of PWZ-029 (2, 5 and 10 mg/kg) improved object recognition after the 24-h delay period, as shown by significant differences between the exploration times of the novel and old object, and the respective discrimination indices. PWZ-029 (2 mg/kg) also successfully reversed the 0.3 mg/kg scopolamine-induced deficit in recognition memory after the 1-h delay. In the Morris water maze test, PWZ-029 (5, 10 and 15 mg/kg) did not significantly influence swim patterns, either during five acquisition days or during the treatment-free probe trial. PWZ-029 (2, 5 and 10 mg/kg) also proved to be ineffective in the reversal of the 1mg/kg scopolamine-induced memory impairment in the water maze. The present mixed results encourage use of a variety of tests and experimental conditions in order to increase the predictability of preclinical testing of selective α(5) GABA(A) inverse agonists.

  8. Comparison of kinetic and pharmacological profiles of recombinant α1γ2L and α1β2γ2L GABAA receptors - A clue to the role of intersubunit interactions.

    PubMed

    Brodzki, Marek; Rutkowski, Radoslaw; Jatczak, Magdalena; Kisiel, Magdalena; Czyzewska, Marta M; Mozrzymas, Jerzy W

    2016-08-01

    The fastest inhibitory mechanism in the CNS is mediated by ionotropic GABAA receptors and it is known that subunit composition critically determines their properties. While a typical GABAA receptor consists of two α, two β and one γ/δ subunit, there are some exceptions, e.g. αβ receptors. Functional α1γ2 GABAA receptors can be expressed in recombinant model (Verdoorn et al., 1990) and although their role remains unknown, it seems appealing to extend their characterization to further explore the structure-function relationship of GABAA receptors. Intriguingly, this receptor is lacking canonical GABA binding sites but it can be activated by GABA and dose-response relationships for α1β2γ2L and α1γ2L receptors overlap. Deactivation kinetics was similar for both receptors but the percentage of the fast component was smaller in the case of α1γ2L receptors and, consequently, the mean deactivation time constant was slower. The rate and extent of macroscopic desensitization were smaller in the case of α1γ2L receptors but they showed slower recovery. Both receptor types had a similar proton sensitivity showing only subtle but significant differences in pH effects on deactivation. Flurazepam exerted a similar effect on both receptors but the rapid deactivation components were differently affected and an opposite effect was observed on desensitization extent. Rebound currents evoked by pentobarbital were undistinguishable for both receptor types. Taking altogether, although some significant differences were found, α1β2γ2L and α1γ2L receptors showed unforeseen similarity. We propose that functioning of GABAA receptors might rely on subunit-subunit cooperative interactions to a larger extent than believed so far. PMID:27179992

  9. Altered Cortical GABAA Receptor Composition, Physiology, and Endocytosis in a Mouse Model of a Human Genetic Absence Epilepsy Syndrome*

    PubMed Central

    Zhou, Chengwen; Huang, Zhiling; Ding, Li; Deel, M. Elizabeth; Arain, Fazal M.; Murray, Clark R.; Patel, Ronak S.; Flanagan, Christopher D.; Gallagher, Martin J.

    2013-01-01

    Patients with generalized epilepsy exhibit cerebral cortical disinhibition. Likewise, mutations in the inhibitory ligand-gated ion channels, GABAA receptors (GABAARs), cause generalized epilepsy syndromes in humans. Recently, we demonstrated that heterozygous knock-out (Hetα1KO) of the human epilepsy gene, the GABAAR α1 subunit, produced absence epilepsy in mice. Here, we determined the effects of Hetα1KO on the expression and physiology of GABAARs in the mouse cortex. We found that Hetα1KO caused modest reductions in the total and surface expression of the β2 subunit but did not alter β1 or β3 subunit expression, results consistent with a small reduction of GABAARs. Cortices partially compensated for Hetα1KO by increasing the fraction of residual α1 subunit on the cell surface and by increasing total and surface expression of α3, but not α2, subunits. Co-immunoprecipitation experiments revealed that Hetα1KO increased the fraction of α1 subunits, and decreased the fraction of α3 subunits, that associated in hybrid α1α3βγ receptors. Patch clamp electrophysiology studies showed that Hetα1KO layer VI cortical neurons exhibited reduced inhibitory postsynaptic current peak amplitudes, prolonged current rise and decay times, and altered responses to benzodiazepine agonists. Finally, application of inhibitors of dynamin-mediated endocytosis revealed that Hetα1KO reduced base-line GABAAR endocytosis, an effect that probably contributes to the observed changes in GABAAR expression. These findings demonstrate that Hetα1KO exerts two principle disinhibitory effects on cortical GABAAR-mediated inhibitory neurotransmission: 1) a modest reduction of GABAAR number and 2) a partial compensation with GABAAR isoforms that possess physiological properties different from those of the otherwise predominant α1βγ GABAARs. PMID:23744069

  10. Pubertal Expression of α4βδ GABAA Receptors Reduces Seizure-Like Discharges in CA1 Hippocampus.

    PubMed

    Yang, Lie; Shen, Hui; Merlin, Lisa R; Smith, Sheryl S

    2016-01-01

    More than half of children with epilepsy outgrow their seizures, yet the underlying mechanism is unknown. GABAergic inhibition increases at puberty in female mice due to expression of extrasynaptic α4βδ GABAA receptors (GABARs). Therefore, we tested the role of these receptors in regulating seizure-like discharges in CA1 hippocampus using a high K(+) (8.5 mM) seizure model. Spontaneous field potentials were recorded from hippocampus of pre-pubertal (~28-32 PND) and pubertal (~35-44 PND) female wild-type or α4-/- mice. The coastline length, a measure of burst intensity, was assessed. 8.5 mM K(+) induced seizure-like discharges in over 60% of pre-pubertal slices, but only in 7% of pubertal slices, where the coastline length was reduced by 70% (P = 0.04). However, the pubertal decrease in seizure-like discharges was not seen in the α4-/-, implicating α4βδ GABARs as the cause of the decreased seizure-like activity during puberty. Administration of THIP or DS2, to selectively increase α4βδ current, reduced activity in 8.5 mM K(+) at puberty, while blockade of α5-GABARs had no effect. GABAergic current was depolarizing but inhibitory in 8.5 mM K(+), suggesting a mechanism for the effects of α4βδ and α5-GABARs, which exhibit different polarity-dependent desensitization. These data suggest that α4βδ GABARs are anti-convulsant during adolescence. PMID:27561815

  11. Pubertal Expression of α4βδ GABAA Receptors Reduces Seizure-Like Discharges in CA1 Hippocampus.

    PubMed

    Yang, Lie; Shen, Hui; Merlin, Lisa R; Smith, Sheryl S

    2016-01-01

    More than half of children with epilepsy outgrow their seizures, yet the underlying mechanism is unknown. GABAergic inhibition increases at puberty in female mice due to expression of extrasynaptic α4βδ GABAA receptors (GABARs). Therefore, we tested the role of these receptors in regulating seizure-like discharges in CA1 hippocampus using a high K(+) (8.5 mM) seizure model. Spontaneous field potentials were recorded from hippocampus of pre-pubertal (~28-32 PND) and pubertal (~35-44 PND) female wild-type or α4-/- mice. The coastline length, a measure of burst intensity, was assessed. 8.5 mM K(+) induced seizure-like discharges in over 60% of pre-pubertal slices, but only in 7% of pubertal slices, where the coastline length was reduced by 70% (P = 0.04). However, the pubertal decrease in seizure-like discharges was not seen in the α4-/-, implicating α4βδ GABARs as the cause of the decreased seizure-like activity during puberty. Administration of THIP or DS2, to selectively increase α4βδ current, reduced activity in 8.5 mM K(+) at puberty, while blockade of α5-GABARs had no effect. GABAergic current was depolarizing but inhibitory in 8.5 mM K(+), suggesting a mechanism for the effects of α4βδ and α5-GABARs, which exhibit different polarity-dependent desensitization. These data suggest that α4βδ GABARs are anti-convulsant during adolescence.

  12. Functional characterization of ivermectin binding sites in α1β2γ2L GABA(A) receptors

    PubMed Central

    Estrada-Mondragon, Argel; Lynch, Joseph W.

    2015-01-01

    GABAA receptors (GABAARs) are the major inhibitory neurotransmitter receptors in the brain and are therapeutic targets for many indications including sedation, anesthesia and anxiolysis. There is, however, considerable scope for the development of new therapeutics with improved beneficial effects and reduced side-effect profiles. The anthelminthic drug, ivermectin, activates the GABAAR although its binding site is not known. The molecular site of action of ivermectin has, however, been defined by crystallography in the homologous glutamate-gated chloride channel. Resolving the molecular mechanisms of ivermectin binding to α1β2γ2L GABAARs may provide insights into the design of improved therapeutics. Given that ivermectin binds to subunit interfaces, we sought to define (1) which subunit interface sites it binds to, (2) whether these sites are equivalent in terms of ivermectin sensitivity or efficacy, and (3) how many must be occupied for maximal efficacy. Our approach involved precluding ivermectin from binding to particular interfaces by introducing bulky M3 domain 36′F sidechains to the “+” side of those interfaces. We thereby demonstrated that ivermectin produces irreversible channel activation only when it binds to the single γ2L-β2 interface site. When it binds to α1-β2 sites it elicits potentiation of GABA-gated currents but has no irreversible activating effect. Ivermectin cannot bind to the β2-α1 interface site due to its endogenous bulky 36′ methionine. Replacing this with an alanine creates a functional site at this interface, but surprisingly it is inhibitory. Molecular docking simulations reveal that the γ2L-β2 interface forms more contacts with ivermectin than the other interfaces, possibly explaining why ivermectin appears to bind irreversibly at this interface. This study demonstrates unexpectedly stark pharmacological differences among GABAAR ivermectin binding sites. PMID:26441518

  13. Pubertal Expression of α4βδ GABAA Receptors Reduces Seizure-Like Discharges in CA1 Hippocampus

    PubMed Central

    Yang, Lie; Shen, Hui; Merlin, Lisa R.; Smith, Sheryl S.

    2016-01-01

    More than half of children with epilepsy outgrow their seizures, yet the underlying mechanism is unknown. GABAergic inhibition increases at puberty in female mice due to expression of extrasynaptic α4βδ GABAA receptors (GABARs). Therefore, we tested the role of these receptors in regulating seizure-like discharges in CA1 hippocampus using a high K+ (8.5 mM) seizure model. Spontaneous field potentials were recorded from hippocampus of pre-pubertal (~28–32 PND) and pubertal (~35–44 PND) female wild-type or α4−/− mice. The coastline length, a measure of burst intensity, was assessed. 8.5 mM K+ induced seizure-like discharges in over 60% of pre-pubertal slices, but only in 7% of pubertal slices, where the coastline length was reduced by 70% (P = 0.04). However, the pubertal decrease in seizure-like discharges was not seen in the α4−/−, implicating α4βδ GABARs as the cause of the decreased seizure-like activity during puberty. Administration of THIP or DS2, to selectively increase α4βδ current, reduced activity in 8.5 mM K+ at puberty, while blockade of α5-GABARs had no effect. GABAergic current was depolarizing but inhibitory in 8.5 mM K+, suggesting a mechanism for the effects of α4βδ and α5-GABARs, which exhibit different polarity-dependent desensitization. These data suggest that α4βδ GABARs are anti-convulsant during adolescence. PMID:27561815

  14. The terpenoids Myrtenol and Verbenol act on δ subunit-containing GABAA receptors and enhance tonic inhibition in dentate gyrus granule cells.

    PubMed

    van Brederode, Johannes; Atak, Sinem; Kessler, Artur; Pischetsrieder, Monika; Villmann, Carmen; Alzheimer, Christian

    2016-08-15

    Sideritis plants and their extracts have been used in traditional medicine as sedatives, anxiolytics and anticonvulsant agents. Pinenes are the most prevalent of the volatile aroma components in Siderites extracts and the pinene metabolites myrtenol and verbenol have been identified as the most potent positive allosteric modulators of synaptic GABAA receptors composed of α1β2 and α1β2γ2 subunits. In view of their therapeutic spectrum, we wondered whether these two terpenoids would also augment tonic GABA currents mediated by extrasynaptic GABAA receptors containing the δ subunit. When we expressed α4β2δ receptors in HEK293 cells, we found that co-application of myrtenol or verbenol enhanced whole-cell current responses to GABA by up to 100%. Consistent with their effects on heterologous α1β2γ2 receptors, we found that myrtenol and verbenol, when co-applied with GABA via local perfusion, increased the amplitude and area of miniature inhibitory postsynaptic potentials (mIPSCs) recorded in whole-cell voltage-clamp recordings from granule cells in the dentate gyrus of mouse hippocampal brain slices. In addition, co-application of terpenoids with GABA was also able to enhance tonic GABA current, measured from the change in baseline current and current noise, compared to GABA perfusion alone. Our results suggest that myrtenol and verbenol act as positive allosteric modulators at synaptic and extrasynaptic GABAA receptors, thereby augmenting phasic and tonic GABAergic inhibition. Thus, our study reveals an important pharmacological and therapeutic target of bicyclic monoterpenoids.

  15. Rapid Antidepressant Action and Restoration of Excitatory Synaptic Strength After Chronic Stress by Negative Modulators of Alpha5-Containing GABAA Receptors

    PubMed Central

    Fischell, Jonathan; Van Dyke, Adam M; Kvarta, Mark D; LeGates, Tara A; Thompson, Scott M

    2015-01-01

    Selective serotonin reuptake inhibitors (SSRIs) are the primary pharmacological treatment for depression, but SSRIs are effective in only half of the patients and typically take several weeks to relieve symptoms. The NMDA receptor antagonist ketamine exerts a rapid antidepressant action, but has troubling side effects. We hypothesized that negative allosteric modulators of GABAA receptors would exert similar effects on brain activity as ketamine, but would not exert as many side effects if targeted only to GABAA receptors containing α5 subunits, which are enriched in the hippocampus and prefrontal cortex. Here, we show that the α5-selective negative modulator L-655,708 reversed the alterations in hedonic behavior in the sucrose preference and social interaction tests produced by two different chronic stress paradigms in rats within 24 h of systemic administration. Similar effects were observed with another α5-selective negative modulator, MRK-016. L-655,708 had no effect on hedonic or open-field behavior in unstressed animals. Within 24 h, L-655,708 injection also restored the strength of pathologically weakened excitatory synaptic transmission at the stress-sensitive temporoammonic-CA1 synapse, measured electrophysiologically, and increased levels of the GluA1 subunit of the AMPA receptor, measured with western blotting. We suggest that the ability of L-655,708 to restore excitatory synaptic strength rapidly may underlie its ability to restore stress-induced behavioral alterations rapidly, supporting evidence that dysfunction of multiple excitatory synapses in cortico-mesolimbic reward pathways contributes, in part, to the genesis of depression. Negative allosteric modulators of α5 subunit-containing GABAA receptors represent a promising novel class of fast-acting and clinically viable antidepressant compounds. PMID:25900119

  16. ASSESSMENT OF SUBUNIT-DEPENDENT DIRECT GATING AND ALLOSTERIC MODULATORY EFFECTS OF CARISOPRODOL AT GABAA RECEPTORS

    PubMed Central

    Kumar, Manoj; González, Lorie A.; Dillon, Glenn H.

    2016-01-01

    Carisoprodol is a widely prescribed muscle relaxant, abuse of which has grown considerably in recent years. It directly activates and allosterically modulates α1β2γ2 GABAARs, although the site(s) of action are unknown. To gain insight into the actions of carisoprodol, subunit-dependent effects of this drug were assessed. Whole-cell patch clamp recordings were obtained from HEK293 cells expressing α1β2, α1β3 or αxβzγ2 (where x = 1–6 and z = 1–3) GABAARs, and in receptors incorporating the δ subunit (modeling extrasynaptic receptors). The ability to directly gate and allosterically potentiate GABA-gated currents was observed for all configurations. Presence or absence of the γ2 subunit did not affect the ability of carisoprodol to directly gate or allosterically modulate the receptor. Presence of the β1 subunit conferred highest efficacy for direct activation relative to maximum GABA currents, while presence of the β2 subunit conferred highest efficacy for allosteric modulation of the GABA response. With regard to α subunits, carisoprodol was most efficacious at enhancing the actions of GABA in receptors incorporating the α1 subunit. The ability to directly gate the receptor was generally comparable regardless of the α subunit isoform, although receptors incorporating the α3 subunit showed significantly reduced direct gating efficacy and affinity. In extrasynaptic (α1β3δ and α4β3δ) receptors, carisoprodol had greater efficacy than GABA as a direct gating agonist. In addition, carisoprodol allosterically potentiated both EC20 and saturating GABA concentrations in these receptors. In assessing voltage-dependence, we found direct gating and inhibitory effects were insensitive to membrane voltage, whereas allosteric modulatory effects were affected by membrane voltage. Our findings demonstrate direct and allosteric effects of carisoprodol at synaptic and extrasynpatic GABAARs and that subunit isoform influences these effects. PMID:25896767

  17. γ-Aminbuturic Acid A Receptor Mitigates Homocysteine-Induced Endothelial Cell Permeability

    PubMed Central

    Tyagi, Neetu; Moshal, Karni S.; Tyagi, Suresh C.; Lominadze, David

    2010-01-01

    Many cerebrovascular disorders are accompanied by an increased homocysteine (Hcy) levels. We have previously shown that acute hyperhomocysteinemia (HHcy) leads to an increased microvascular permeability in the mouse brain. Hcy competitively binds to γ -aminbuturic acid (GABA) receptors and may increase vascular permeability by acting as an excitatory neurotransmitter. However, the role of GABA-A (GABAA) receptor in Hcy-induced endothelial cell (EC) permeability remains unclear. In the present study we attempted to determine the role of GABAA receptor and the possible mechanisms involved in Hcy-induced EC layer permeability. Mouse aortic and brain ECs were grown in Transwells and treated with 50 μM Hcy in the presence or absence of GABAA-specific agonist muscimol. Role of matrix metalloproteinase-9 (MMP-9) was determined using its activity inhibitor GM-6001. Involvement of extracellular signal-regulated kinase (ERK) signaling was assessed using its kinase activity inhibitors PD98059 or U0126. EC permeability to the known content of bovine serum albumin (BSA)-conjugated with Alexa Flour-488 was assessed by measuring fluorescence intensity of the solutes in the Transwell's lower chambers. It was found that Hcy induced the formation of filamentous actin (F-actin). Hcy-induced EC permeability to BSA was significantly decreased by GABA and muscimol treatments. Presence of MMP-9 or ERK kinase activity inhibitors restored the Hcy-induced EC permeability to its baseline level. The mediation BSA leakage through the ECs was further confirmed in the experiments where Hcy-induced alterations in transendothelial electrical resistance of confluent ECs were assessed. The data suggest that Hcy increases EC layer permeability through inhibition of GABAA receptor and F-actin formation, in part, by transducing ERK and MMP-9 activation. PMID:18080868

  18. 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.

  19. The essential role of hippocampal alpha6 subunit-containing GABAA receptors in maternal separation stress-induced adolescent depressive behaviors.

    PubMed

    Yang, Linjie; Xu, Ting; Zhang, Ke; Wei, Zhisheng; Li, Xuran; Huang, Mingfa; Rose, Gregory M; Cai, Xiang

    2016-10-15

    Exposure to early stressful adverse life events such as maternal separation severely impacts the development of the nervous system. Using immunohistochemistry, quantitative PCR and Western blot approaches, we found that alpha6 subunit-containing GABAA receptors (Gabra6-containing GABAA Rs) were expressed on hippocampal interneurons of adolescent rats. Maternal separation stress (MS) from postnatal day 2 to15 significantly reduced Gabra6 expression and provoked depressive behaviors such as anhedonia. Furosemide, the selective antagonist of Gabra6-containing GABAARs, strongly increased peak amplitude of evoked IPSCs at CA3-CA1 synapses and the frequency of miniature IPSPs recorded from CA1 pyramidal cells in naive control animals, and this effect was occluded in MS animals. Knockdown of Gabra6 expression in hippocampus mimicked furosemide's effect and was sufficient to produce similar depressive symptoms that were observed in MS animals. These results indicate that the Gabra6-containing GABAA R is a key modulator of hippocampal synaptic transmission and likely plays a crucial role in the pathophysiology of maternal separation-induced depression. PMID:27388150

  20. Setting the time course of inhibitory synaptic currents by mixing multiple GABA(A) receptor α subunit isoforms.

    PubMed

    Eyre, Mark D; Renzi, Massimiliano; Farrant, Mark; Nusser, Zoltan

    2012-04-25

    The kinetics of IPSCs influence many neuronal processes, such as the frequencies of oscillations and the duration of shunting inhibition. The subunit composition of recombinant GABA(A) receptors (GABA(A)Rs) strongly affects the deactivation kinetics of GABA-evoked currents. However, for GABAergic synapses, the relationship between subunit composition and IPSC decay is less clear. Here we addressed this by combining whole-cell recordings of miniature IPSCs (mIPSCs) and quantitative immunolocalization of synaptic GABA(A)R subunits. In cerebellar stellate, thalamic relay, and main olfactory bulb (MOB) deep short-axon cells of Wistar rats, the only synaptic α subunit was α1, and zolpidem-sensitive mIPSCs had weighted decay time constants (τ(w)) of 4-6 ms. Nucleus reticularis thalami neurons expressed only α3 as the synaptic α subunit and exhibited slow (τ(w) = 28 ms), zolpidem-insensitive mIPSCs. By contrast, MOB external tufted cells contained two α subunit types (α1 and α3) at their synapses. Quantitative analysis of multiple immunolabeled images revealed small within-cell, but large between-cell, variability in synaptic α1/α3 ratios. This corresponded to large cell-to-cell variability in the decay (τ(w) = 3-30 ms) and zolpidem sensitivity of mIPSCs. Currents evoked by rapid application of GABA to patches excised from HEK cells expressing different mixtures of α1 and α3 subunits displayed highly variable deactivation times that correlated with the α1/α3 cDNA ratio. Our results demonstrate that diversity in the decay of IPSCs can be generated by varying the expression of different GABA(A)R subunits that alone confer different decay kinetics, allowing the time course of inhibition to be tuned to individual cellular requirements.

  1. To what extent is it possible to dissociate the anxiolytic and sedative/hypnotic properties of GABAA receptors modulators?

    PubMed

    Chagraoui, A; Skiba, M; Thuillez, C; Thibaut, F

    2016-11-01

    The relatively common view indicates a possible dissociation between the anxiolytic and sedative/hypnotic properties of benzodiazepines (BZs). Indeed, GABAA receptor (GABAAR) subtypes have specific cerebral distribution in distinct neural circuits. Thus, GABAAR subtype-selective drugs may be expected to perform distinct functions. However, standard behavioral test assays provide limited direction towards highlighting new action mechanisms of ligands targeting GABAARs. Automated behavioral tests, lack sensitivity as some behavioral characteristics or subtle behavioral changes of drug effects or that are not considered in the overall analysis (Ohl et al., 2001) and observation-based analyses are not always performed. In addition, despite the use of genetically engineered mice, any possible dissociation between the anxiolytic and sedative properties of BZs remains controversial. Moreover, the involvement the different subtypes of GABAAR subtypes in the anxious behavior and the mechanism of action of anxiolytic agents remains unclear since there has been little success in the pharmacological investigations so far. This raises the question of the involvement of the different subunits in anxiolytic-like and/or sedative effects; and the actual implication of these subunits, particularly, α-subunits in the modulation of sedation and/or anxiety-related disorders. This present review was prompted by several conflicting studies on the degree of involvement of these subunits in anxiolytic-like and/or sedative effects. To this end, we explored the GABAergic system, particularly, the role of different subunits containing synaptic GABAARs. We report herein the targeting gene encoding the different subunits and their contribution in anxiolytic-like and/or sedative actions, as well as, the mechanism underlying tolerance to BZs. PMID:27495357

  2. Docking of 1,4-Benzodiazepines in the α1/γ2 GABAA Receptor Modulator SiteS⃞

    PubMed Central

    Berezhnoy, D.; Gibbs, T. T.; Farb, D. H.

    2009-01-01

    Positive allosteric modulation of the GABAA receptor (GABAAR) via the benzodiazepine recognition site is the mechanism whereby diverse chemical classes of therapeutic agents act to reduce anxiety, induce and maintain sleep, reduce seizures, and induce conscious sedation. The binding of such therapeutic agents to this allosteric modulatory site increases the affinity of GABA for the agonist recognition site. A major unanswered question, however, relates to how positive allosteric modulators dock in the 1,4-benzodiazepine (BZD) recognition site. In the present study, the X-ray structure of an acetylcholine binding protein from the snail Lymnea stagnalis and the results from site-directed affinity-labeling studies were used as the basis for modeling of the BZD binding pocket at the α1/γ2 subunit interface. A tethered BZD was introduced into the binding pocket, and molecular simulations were carried out to yield a set of candidate orientations of the BZD ligand in the binding pocket. Candidate orientations were refined based on known structure-activity and stereospecificity characteristics of BZDs and the impact of the α1H101R mutation. Results favor a model in which the BZD molecule is oriented such that the C5-phenyl substituent extends approximately parallel to the plane of the membrane rather than parallel to the ion channel. Application of this computational modeling strategy, which integrates site-directed affinity labeling with structure-activity knowledge to create a molecular model of the docking of active ligands in the binding pocket, may provide a basis for the design of more selective GABAAR modulators with enhanced therapeutic potential. PMID:19483108

  3. Two types of functionally different GABAA receptors mediate GABA modulation of cholinergic transmission in cat terminal ileum.

    PubMed

    Radomirov, R; Pencheva, N

    1995-08-01

    1. The effects of GABA (1 microM-2 mM) on longitudinally or circularly oriented organ bath preparations of cat terminal ileum consisted of a relaxation phase with an inhibition of the rhythmic spontaneous phasic contractions, followed by a phase of contractions characterized by an elevation in basal tone and an increase in amplitude of the spontaneous phasic contractions. 2. Muscimol (100 microM), but not baclofen (100 microM), mimicked the relaxation phase of the response to applied GABA (100 microM) in all tissue preparations. In addition, muscimol induced a phase of contractile activity in the circular muscle layer whilst baclofen exerted a 'GABA-like' contractile effect on the longitudinal muscle layer. Bicuculline (30 microM) or picrotoxinin (30 microM) antagonized the GABA- or muscimol-induced relaxations in all preparations and decreased the GABA- but not the baclofen-induced contractions of the longitudinal muscle layer. 3. Tetrodotoxin (0.5 microM) or atropine (0.1 microM) prevented the bicuculline-sensitive phases of the GABA or muscimol effects on both muscle layers but not the contractile effect of baclofen on the longitudinal muscle layer. 4. The bicuculline-sensitive phases of the GABA effect on both muscle layers were almost completely eliminated by 1 nM pirenzepine. At this concentration pirenzepine did not affect the electrically-evoked cholinergic twitch contractions or contractile responses to applied acetylcholine of both muscle layers. 5. During electrically-evoked cholinergic twitch contractions of both muscle layers, GABA (100 microM) had an inhibitory effect. The inhibition occurred in the presence of pirenzepine (1 nM) but not of bicuculline (30 microM). 6. It is suggested that two types of functionally different bicuculline-sensitive GABAA receptors mediate an exitatory presynaptic and an inhibitory prejunctional action of GABA on the cholinergic transmission in cat terminal ileum.

  4. Inhibition of microglia in the basolateral amygdala enhanced morphine-induced antinociception: Possible role of GABAA receptors.

    PubMed

    Kosarmadar, Nastaran; Ghasemzadeh, Zahra; Rezayof, Ameneh

    2015-10-15

    In clinical medicine, morphine is widely used to relieve many types of pain, but it has several side effects such as the development of tolerance and dependence. In order to decrease the side effects of morphine administration for the treatment of pain, the combination of minocycline as a glial inhibitor and morphine has been suggested in previous studies. It is important to understand which synaptic mechanisms are involved in the potentiative effect of minocycline on morphine antinociception. To this aim, male Wistar rats were bilaterally cannulated in the basolateral amygdala by srereotaxic instrument. A tail-flick apparatus was used to measure the pain threshold. The results revealed that intraperitoneal injection of morphine (2.5-7.5 mg/kg) induced antinociception. Intra-basolateral amygdala microinjection of minocycline (5-10 µg/rat) by itself had no effect on tail-flick latency, while the microinjection of the same doses of minocycline with an ineffective dose of morphine (5 mg/kg) induced antinociception. Intra-basolateral amygdala microinjection of different doses of muscimol (0.001-0.005 µg/rat) increased the minocycline-induced potentioation on morphine response in the tail-flick test. Intra-basolateral amygdala microinjection of muscimol by itself had no effect on tail-flick latency. On the other hand, intra-basolateral amygdala microinjection of bicuculline (0.01-0.1 µg/rat) inhibited minocycline-induced potentiation of morphine antinociception. It should be noted that intra-basolateral amygdala bicucculine by itself had no effect on tail-flick latency. It can thus be concluded that intra-basolateral amygdala minocycline potentiates morphine response in the tail-flick test. Moreover, basolateral amygdala GABAergic system may be involved in the minocycline-induced potentiation of morphine response via GABAA receptors.

  5. Etomidate Impairs Long-Term Potentiation In Vitro by Targeting α5-Subunit Containing GABAA Receptors on Nonpyramidal Cells

    PubMed Central

    Rodgers, F. Clifford; Zarnowska, Ewa D.; Laha, Kurt T.; Engin, Elif; Zeller, Anja; Keist, Ruth; Rudolph, Uwe

    2015-01-01

    Previous experiments using genetic and pharmacological manipulations have provided strong evidence that etomidate impairs synaptic plasticity and memory by modulating α5-subunit containing GABAA receptors (α5-GABAARs). Because α5-GABAARs mediate tonic inhibition (TI) in hippocampal CA1 pyramidal cells and etomidate enhances TI, etomidate enhancement of TI in pyramidal cells has been proposed as the underlying mechanism (Martin et al., 2009). Here we tested this hypothesis by selectively removing α5-GABAARs from pyramidal neurons (CA1–pyr–α5–KO) and comparing the ability of etomidate to enhance TI and block LTP in fl–α5 (WT), global–α5–KO (gl–α5–KO), and CA1–pyr–α5–KO mice. Etomidate suppressed LTP in slices from WT and CA1–pyr–α5–KO but not gl–α5–KO mice. There was a trend toward reduced TI in both gl–α5–KO and CA1–pyr–α5–KO mice, but etomidate enhanced TI to similar levels in all genotypes. The dissociation between effects of etomidate on TI and LTP in gl–α5–KO mice indicates that increased TI in pyramidal neurons is not the mechanism by which etomidate impairs LTP and memory. Rather, the ability of etomidate to block LTP in WT and CA1–pyr–α5–KO mice, but not in gl–α5–KO mice, points toward α5-GABAARs on nonpyramidal cells as the essential effectors controlling plasticity in this in vitro model of learning and memory. PMID:26134653

  6. Gene Expression Analysis of CL-20-induced Reversible Neurotoxicity Reveals GABAA Receptors as Potential Target in the Earthworm Eisenia fetida

    PubMed Central

    Gong, Ping; Guan, Xin; Pirooznia, Mehdi; Liang, Chun; Perkins, Edward J.

    2012-01-01

    The earthworm Eisenia fetida is one of the most used species in standardized soil ecotoxicity tests. Endpoints such as survival, growth and reproduction are eco-toxicologically relevant but provide little mechanistic insight into toxicity pathways, especially at the molecular level. Here we applied a toxicogenomic approach to investigate the mode of action underlying the reversible neurotoxicity of hexanitrohexaazaisowurtzitane (CL-20), a cyclic nitroamine explosives compound. We developed an E. fetida-specific shotgun microarray targeting 15119 unique E. fetida transcripts. Using this array we profiled gene expression in E. fetida in response to exposure to CL-20. Eighteen earthworms were exposed for 6 days to 0.2 μg/cm2 of CL-20 on filter paper, half of which were allowed to recover in a clean environment for 7 days. Nine vehicle control earthworms were sacrificed at day 6 and 13, separately. Electrophysiological measurements indicated that the conduction velocity of earthworm medial giant nerve fiber decreased significantly after 6-day exposure to CL-20, but was restored after 7 days of recovery. Total RNA was isolated from the four treatment groups including 6-day control, 6-day exposed, 13-day control and 13-day exposed (i.e. 6-day exposure followed by 7-day recovery), and was hybridized to the 15K shot-gun oligo array. Statistical and bioinformatic analyses suggest that CL-20 initiated neurotoxicity by non-competitively blocking the ligand-gated GABAA receptor ion channel, leading to altered expression of genes involved in GABAergic, cholinergic, and Agrin-MuSK pathways. In the recovery phase, expression of affected genes returned to normality, possibly as a result of autophagy and CL-20 dissociation/metabolism. This study provides significant insights into potential mechanisms of CL-20-induced neurotoxicity and the recovery of earthworms from transient neurotoxicity stress. PMID:22191394

  7. The general anaesthetic etomidate inhibits the excitability of mouse thalamocortical relay neurons by modulating multiple modes of GABAA receptor-mediated inhibition

    PubMed Central

    Herd, Murray B; Lambert, Jeremy J; Belelli, Delia

    2014-01-01

    Modulation of thalamocortical (TC) relay neuron function has been implicated in the sedative and hypnotic effects of general anaesthetics. Inhibition of TC neurons is mediated predominantly by a combination of phasic and tonic inhibition, together with a recently described ‘spillover’ mode of inhibition, generated by the dynamic recruitment of extrasynaptic γ-aminobutyric acid (GABA)A receptors (GABAARs). Previous studies demonstrated that the intravenous anaesthetic etomidate enhances tonic and phasic inhibition in TC relay neurons, but it is not known how etomidate may influence spillover inhibition. Moreover, it is unclear how etomidate influences the excitability of TC neurons. Thus, to investigate the relative contribution of synaptic (α1β2γ2) and extrasynaptic (α4β2δ) GABAARs to the thalamic effects of etomidate, we performed whole-cell recordings from mouse TC neurons lacking synaptic (α10/0) or extrasynaptic (δ0/0) GABAARs. Etomidate (3 μm) significantly inhibited action-potential discharge in a manner that was dependent on facilitation of both synaptic and extrasynaptic GABAARs, although enhanced tonic inhibition was dominant in this respect. Additionally, phasic inhibition evoked by stimulation of the nucleus reticularis exhibited a spillover component mediated by δ-GABAARs, which was significantly prolonged in the presence of etomidate. Thus, etomidate greatly enhanced the transient suppression of TC spike trains by evoked inhibitory postsynaptic potentials. Collectively, these results suggest that the deactivation of thalamus observed during etomidate-induced anaesthesia involves potentiation of tonic and phasic inhibition, and implicate amplification of spillover inhibition as a novel mechanism to regulate the gating of sensory information through the thalamus during anaesthetic states. PMID:24773078

  8. Alternative-Splicing in the Exon-10 Region of GABAA Receptor β2 Subunit Gene: Relationships between Novel Isoforms and Psychotic Disorders

    PubMed Central

    Zhao, Cunyou; Xu, Zhiwen; Wang, Feng; Chen, Jianhuan; Ng, Siu-Kin; Wong, Pak-Wing; Yu, Zhiliang; Pun, Frank W.; Ren, Lihuan; Lo, Wing-Sze; Tsang, Shui-Ying; Xue, Hong

    2009-01-01

    Background Non-coding single nucleotide polymorphisms (SNPs) in GABRB2, the gene for β2-subunit of gamma-aminobutyric acid type A (GABAA) receptor, have been associated with schizophrenia (SCZ) and quantitatively correlated to mRNA expression and alternative splicing. Methods and Findings Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an “alternative splicing hotspot” that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, β2S1 and β2S2, bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased β2S1 expression and decreased β2S2 expression in both SCZ and bipolar disorder (BPD) compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both β2S1 and β2S2 expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for β2S2 expression. Moreover, site-directed mutagenesis indicated that Thr365, a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down. Conclusion This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to β2-subunit splicing diversity and the etiologies of SCZ and BPD. PMID:19763268

  9. Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

    PubMed Central

    Brown, Laura E.; Fuchs, Celine; Nicholson, Martin W.; Stephenson, F. Anne; Thomson, Alex M.; Jovanovic, Jasmina N.

    2014-01-01

    Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed

  10. Single-cell genetic expression of mutant GABAA receptors causing Human genetic epilepsy alters dendritic spine and GABAergic bouton formation in a mutation-specific manner

    PubMed Central

    Lachance-Touchette, Pamela; Choudhury, Mayukh; Stoica, Ana; Di Cristo, Graziella; Cossette, Patrick

    2014-01-01

    Mutations in genes encoding for GABAA receptor subunits is a well-established cause of genetic generalized epilepsy. GABA neurotransmission is implicated in several developmental processes including neurite outgrowth and synapse formation. Alteration in excitatory/inhibitory synaptic activities plays a critical role in epilepsy, thus here we investigated whether mutations in α1 subunit of GABAA receptor may affect dendritic spine and GABAergic bouton formation. In particular, we examined the effects of three mutations of the GABRA1 gene (D219N, A322D and K353delins18X) that were found in a cohort of French Canadian families with genetic generalized epilepsy. We used a novel single-cell genetic approach, by preparing cortical organotypic cultures from GABRA1flox/flox mice and simultaneously inactivating endogenous GABRA1 and transfecting mutant α1 subunits in single glutamatergic pyramidal cells and basket GABAergic interneurons by biolistic transfection. We found that GABRA1−/− GABAergic cells showed reduced innervation field, which was rescued by co-expressing α1-A322D and α1-WT but not α1-D219N. We further found that the expression of the most severe GABRA1 missense mutation (α1-A322D) induced a striking increase of spine density in pyramidal cells along with an increase in the number of mushroom-like spines. In addition, α1-A322D expression in GABAergic cells slightly increased perisomatic bouton density, whereas other mutations did not alter bouton formation. All together, these results suggest that the effects of different GABAAR mutations on GABAergic bouton and dendritic spine formation are specific to the mutation and cannot be always explained by a simple loss-of-function gene model. The use of single cell genetic manipulation in organotypic cultures may provide a better understanding of the specific and distinct neural circuit alterations caused by different GABAA receptor subunit mutations and will help define the pathophysiology of genetic

  11. Both alpha2 and alpha3 GABAA receptor subtypes mediate the anxiolytic properties of benzodiazepine site ligands in the conditioned emotional response paradigm.

    PubMed

    Morris, H V; Dawson, G R; Reynolds, D S; Atack, J R; Stephens, D N

    2006-05-01

    Mice with point-mutated alpha2 GABAA receptor subunits (rendering them diazepam insensitive) are resistant to the anxiolytic-like effects of benzodiazepines (BZs) in unconditioned models of anxiety. We investigated the role of the alpha2 GABAA subtype in a model of conditioned anxiety. alpha2(H101R) and wildtype 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 conditioned stimulus (CS+) that predicted footshock. The ability of diazepam, ethanol and pentobarbital to reduce suppression during the CS+ was interpreted as an anxiolytic response. Diazepam (0, 0.5, 1, 2, 4 and 8 mg/kg) induced a dose-dependent anxiolytic-like effect in wildtype mice. At high doses, diazepam (2, 4 and 8 mg/kg) was sedative in alpha2(H101R) mice. Analysis of the anxiolytic properties of nonsedative diazepam doses (0.5 and 1 mg/kg), showed that alpha2(H101R) mice were resistant to the anxiolytic effects of diazepam. Equivalent anxiolytic properties of pentobarbital (20 mg/kg) and ethanol (1 and 2 g/kg) were seen in both genotypes. These findings confirm the critical importance of the alpha2 GABAA subtype in mediating BZ anxiolysis. However, as a compound, L-838417, with agonist properties at alpha2, alpha3 and alpha5-containing receptors, gave rise to anxiolytic-like activity in alpha2(H101R) mice in the CER test, alpha3-containing GABA receptors are also likely to contribute to anxiolysis. Observations that alpha2(H101R) mice were more active, and displayed a greater suppression of lever pressing in response to fear-conditioned stimuli than wildtype mice, suggests that the alpha2(H101R) mutation may not be behaviourally silent.

  12. 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.

  13. Snake neurotoxin α-bungarotoxin is an antagonist at native GABAA receptors

    PubMed Central

    Hannan, Saad; Mortensen, Martin; Smart, Trevor G.

    2015-01-01

    The snake neurotoxin α-bungarotoxin (α-Bgtx) is a competitive antagonist at nicotinic acetylcholine receptors (nAChRs) and is widely used to study their function and cell-surface expression. Increasingly, α-Bgtx is also used as an imaging tool for fluorophore-labelling studies, and given the structural conservation within the pentameric ligand-gated ion channel family, we assessed whether α-Bgtx could bind to recombinant and native γ-aminobutyric type-A receptors (GABAARs). Applying fluorophore-linked α-Bgtx to recombinant αxβ1/2γ2 GABAARs expressed in HEK-293 cells enabled clear cell-surface labelling of α2β1/2γ2 contrasting with the weaker staining of α1/4β1/2γ2, and no labelling for α3/5/6β1/2γ2. The labelling of α2β2γ2 was abolished by bicuculline, a competitive antagonist at GABAARs, and by d-tubocurarine (d-Tc), which acts in a similar manner at nAChRs and GABAARs. Labelling by α-Bgtx was also reduced by GABA, suggesting that the GABA binding site at the receptor β–α subunit interface forms part of the α-Bgtx binding site. Using whole-cell recording, high concentrations of α-Bgtx (20 μM) inhibited GABA-activated currents at all αxβ2γ2 receptors examined, but at lower concentrations (5 μM), α-Bgtx was selective for α2β2γ2. Using α-Bgtx, at low concentrations, permitted the selective inhibition of α2 subunit-containing GABAARs in hippocampal dentate gyrus granule cells, reducing synaptic current amplitudes without affecting the GABA-mediated tonic current. In conclusion, α-Bgtx can act as an inhibitor at recombinant and native GABAARs and may be used as a selective tool to inhibit phasic but not tonic currents in the hippocampus. PMID:25634239

  14. Loss of Ethanol Conditioned Taste Aversion and Motor Stimulation in Knockin Mice with Ethanol-Insensitive α2-Containing GABAA Receptors

    PubMed Central

    Borghese, C. M.; McCracken, M. L.; Benavidez, J. M.; Geil, C. R.; Osterndorff-Kahanek, E.; Werner, D. F.; Iyer, S.; Swihart, A.; Harrison, N. L.; Homanics, G. E.; Harris, R. A.

    2011-01-01

    GABA type A receptors (GABAA-Rs) are potential targets of ethanol. However, there are multiple subtypes of this receptor, and, thus far, individual subunits have not been definitively linked with specific ethanol behavioral actions. Interestingly, though, a chromosomal cluster of four GABAA-R subunit genes, including α2 (Gabra2), was associated with human alcoholism (Am J Hum Genet 74:705–714, 2004; Pharmacol Biochem Behav 90:95–104, 2008; J Psychiatr Res 42:184–191, 2008). The goal of our study was to determine the role of receptors containing this subunit in alcohol action. We designed an α2 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity in a recombinant expression system. Knockin mice containing this mutant subunit were tested in a range of ethanol behavioral tests. These mutant mice did not develop the typical conditioned taste aversion in response to ethanol and showed complete loss of the motor stimulant effects of ethanol. Conversely, they also demonstrated changes in ethanol intake and preference in multiple tests. The knockin mice showed increased ethanol-induced hypnosis but no difference in anxiolytic effects or recovery from acute ethanol-induced motor incoordination. Overall, these studies demonstrate that the effects of ethanol at GABAergic synapses containing the α2 subunit are important for specific behavioral effects of ethanol that may be relevant to the genetic linkage of this subunit with human alcoholism. PMID:20876231

  15. Paired Burst Stimulation Causes GABAA Receptor-Dependent Spike Firing Facilitation in CA1 of Rat Hippocampal Slices.

    PubMed

    Tominaga, Takashi; Tominaga, Yoko

    2016-01-01

    The theta oscillation (4-8 Hz) is a pivotal form of oscillatory activity in the hippocampus that is intermittently concurrent with gamma (25-100 Hz) burst events. In in vitro preparation, a stimulation protocol that mimics the theta oscillation, theta burst stimulation (TBS), is used to induce long-term potentiation. Thus, TBS is thought to have a distinct role in the neural network of the hippocampal slice preparation. However, the specific mechanisms that make TBS induce such neural circuit modifications are still unknown. Using electrophysiology and voltage-sensitive dye imaging (VSDI), we have found that TBS induces augmentation of spike firing. The augmentation was apparent in the first couple of brief burst stimulation (100 Hz four pulses) on a TBS-train in a presence of NMDA receptor blocker (APV 50 μM). In this study, we focused on the characterizes of the NMDA independent augmentation caused by a pair of the brief burst stimulation (the first pair of the TBS; paired burst stimulation-PBS). We found that PBS enhanced membrane potential responses on VSDI signal and intracellular recordings while it was absent in the current recording under whole-cell clamp condition. The enhancement of the response accompanied the augmentation of excitatory postsynaptic potential (EPSP) to spike firing (E-S) coupling. The paired burst facilitation (PBF) reached a plateau when the number of the first burst stimulation (priming burst) exceeds three. The interval between the bursts of 150 ms resulted in the maximum PBF. Gabazine (a GABAA receptor antagonist) abolished PBF. The threshold for spike generation of the postsynaptic cells measured with a current injection to cells was not lowered by the priming burst of PBS. These results indicate that PBS activates the GABAergic system to cause short-term E-S augmentation without raising postsynaptic excitability. We propose that a GABAergic system of area CA1 of the hippocampus produce the short-term E-S plasticity that could

  16. Low-dose alcohol actions on α4β3δ GABAA receptors are reversed by the behavioral alcohol antagonist Ro15-4513

    PubMed Central

    Wallner, M.; Hanchar, H. J.; Olsen, R. W.

    2006-01-01

    Although it is now more than two decades since it was first reported that the imidazobenzodiazepine Ro15-4513 reverses behavioral alcohol effects, the molecular target(s) of Ro15-4513 and the mechanism of alcohol antagonism remain elusive. Here, we show that Ro15-4513 blocks the alcohol enhancement on recombinant “extrasynaptic” α4/6β3δ GABAA receptors at doses that do not reduce the GABA-induced Cl− current. At low ethanol concentrations (≤30 mM), the Ro15-4513 antagonism is complete. However, at higher ethanol concentrations (≥100 mM), there is a Ro15-4513-insensitive ethanol enhancement that is abolished in receptors containing a point mutation in the second transmembrane region of the β3 subunit (β3N265M). Therefore, α4/6β3δ GABA receptors have two distinct alcohol modulation sites: (i) a low-dose ethanol site present in α4/6β3δ receptors that is antagonized by the behavioral alcohol antagonist Ro15-4513 and (ii) a site activated at high (anesthetic) alcohol doses, defined by mutations in membrane-spanning regions. Receptors composed of α4β3N265Mδ subunits that lack the high-dose alcohol site show a saturable ethanol dose-response curve with a half-maximal enhancement at 16 mM, close to the legal blood alcohol driving limit in most U.S. states (17.4 mM). Like in behavioral experiments, the alcohol antagonist effect of Ro15-4513 on recombinant α4β3δ receptors is blocked by flumazenil and β-carboline-ethyl ester (β-CCE). Our findings suggest that ethanol/Ro15-4513-sensitive GABAA receptors are important mediators of behavioral alcohol effects. PMID:16698930

  17. Direct protein-protein coupling enables cross-talk between dopamine D5 and gamma-aminobutyric acid A receptors.

    PubMed

    Liu, F; Wan, Q; Pristupa, Z B; Yu, X M; Wang, Y T; Niznik, H B

    2000-01-20

    GABA(A) (gamma-aminobutyric-acid A) and dopamine D1 and D5 receptors represent two structurally and functionally divergent families of neurotransmitter receptors. The former comprises a class of multi-subunit ligand-gated channels mediating fast interneuronal synaptic transmission, whereas the latter belongs to the seven-transmembrane-domain single-polypeptide receptor superfamily that exerts its biological effects, including the modulation of GABA(A) receptor function, through the activation of second-messenger signalling cascades by G proteins. Here we show that GABA(A)-ligand-gated channels complex selectively with D5 receptors through the direct binding of the D5 carboxy-terminal domain with the second intracellular loop of the GABA(A) gamma2(short) receptor subunit. This physical association enables mutually inhibitory functional interactions between these receptor systems. The data highlight a previously unknown signal transduction mechanism whereby subtype-selective G-protein-coupled receptors dynamically regulate synaptic strength independently of classically defined second-messenger systems, and provide a heuristic framework in which to view these receptor systems in the maintenance of psychomotor disease states.

  18. Ethanol, not metabolized in brain, significantly reduces brain metabolism, probably via specific GABA(A) receptors

    PubMed Central

    Rae, Caroline D.; Davidson, Joanne E.; Maher, Anthony D.; Rowlands, Benjamin D.; Kashem, Mohammed A.; Nasrallah, Fatima A.; Rallapalli, Sundari K.; Cook, James M; Balcar, Vladimir J.

    2014-01-01

    Ethanol is a known neuromodulatory agent with reported actions at a range of neurotransmitter receptors. Here, we used an indirect approach, measuring the effect of alcohol on metabolism of [3-13C]pyruvate in the adult Guinea pig brain cortical tissue slice and comparing the outcomes to those from a library of ligands active in the GABAergic system as well as studying the metabolic fate of [1,2-13C]ethanol. Ethanol (10, 30 and 60 mM) significantly reduced metabolic flux into all measured isotopomers and reduced all metabolic pool sizes. The metabolic profiles of these three concentrations of ethanol were similar and clustered with that of the α4β3δ positive allosteric modulator DS2 (4-Chloro-N-[2-(2-thienyl)imidazo[1,2a]-pyridin-3-yl]benzamide). Ethanol at a very low concentration (0.1 mM) produced a metabolic profile which clustered with those from inhibitors of GABA uptake, and ligands showing affinity for α5, and to a lesser extent, α1-containing GABA(A)R. There was no measureable metabolism of [1,2-13C]ethanol with no significant incorporation of 13C from [1,2-13C]ethanol into any measured metabolite above natural abundance, although there were measurable effects on total metabolite sizes similar to those seen with unlabeled ethanol. The reduction in metabolism seen in the presence of ethanol is therefore likely to be due to its actions at neurotransmitter receptors, particularly α4β3δ receptors, and not because ethanol is substituting as a substrate or because of the effects of ethanol catabolites acetaldehyde or acetate. We suggest that the stimulatory effects of very low concentrations of ethanol are due to release of GABA via GAT1 and the subsequent interaction of this GABA with local α5-containing, and to a lesser extent, α1-containing GABA(A)R. PMID:24313287

  19. Ethanol potently and competitively inhibits binding of the alcohol antagonist Ro15-4513 to α4/6β3δ GABAA receptors

    PubMed Central

    Hanchar, H. Jacob; Chutsrinopkun, Panida; Meera, Pratap; Supavilai, Porntip; Sieghart, Werner; Wallner, Martin; Olsen, Richard W.

    2006-01-01

    Although GABAA receptors have long been implicated in mediating ethanol (EtOH) actions, receptors containing the “nonsynaptic” δ subunit only recently have been shown to be uniquely sensitive to EtOH. Here, we show that δ subunit-containing receptors bind the imidazo-benzodiazepines (BZs) flumazenil and Ro15-4513 with high affinity (Kd < 10 nM), contrary to the widely held belief that these receptors are insensitive to BZs. In immunopurified native cerebellar and recombinant δ subunit-containing receptors, binding of the alcohol antagonist [3H]Ro15-4513 is inhibited by low concentrations of EtOH (Ki ≈ 8 mM). Also, Ro15-4513 binding is inhibited by BZ-site ligands that have been shown to reverse the behavioral alcohol antagonism of Ro15-4513 (i.e., flumazenil, β-carbolinecarboxylate ethyl ester (β-CCE), and N-methyl-β-carboline-3-carboxamide (FG7142), but not including any classical BZ agonists like diazepam). Experiments that were designed to distinguish between a competitive and allosteric mechanism suggest that EtOH and Ro15-4513 occupy a mutually exclusive binding site. The fact that only Ro15-4513, but not flumazenil, can inhibit the EtOH effect, and that Ro15-4513 differs from flumazenil by only a single group in the molecule (an azido group at the C7 position of the BZ ring) suggest that this azido group in Ro15-4513 might be the area that overlaps with the alcohol-binding site. Our findings, combined with previous observations that Ro15-4513 is a behavioral alcohol antagonist, suggest that many of the behavioral effects of EtOH at relevant physiological concentrations are mediated by EtOH/Ro15-4513-sensitive GABAA receptors. PMID:16581914

  20. Brainstem node for loss of consciousness due to GABA(A) receptor-active anesthetics.

    PubMed

    Minert, Anne; Devor, Marshall

    2016-01-01

    The molecular agents that induce loss of consciousness during anesthesia are classically believed to act by binding to cognate transmembrane receptors widely distributed in the CNS and critically suppressing local processing and network connectivity. However, previous work has shown that microinjection of anesthetics into a localized region of the brainstem mesopontine tegmentum (MPTA) rapidly and reversibly induces anesthesia in the absence of global spread. This implies that functional extinction is determined by neural pathways rather than vascular distribution of the anesthetic agent. But does clinical (systemic-induced) anesthesia employ MPTA-linked circuitry? Here we show that cell-selective lesioning of the MPTA in rats does not, in itself, induce anesthesia or coma. However, it increases the systemic dose of pentobarbital required to induce anesthesia, in a manner proportional to the extent of the lesion. Such lesions also affect emergence, extending the duration of anesthesia. Off-target and sham lesions were ineffective. Combined with the prior microinjection data, we conclude that drug delivery to the MPTA is sufficient to induce loss-of-consciousness and that neurons in this locus are necessary for anesthetic induction at clinically relevant doses. Together, the results support an architecture for anesthesia with the MPTA serving as a key node in an endogenous network of dedicated pathways that switch between wake and unconsciousness. As such, the MPTA might also play a role in syncope, concussion and sleep.

  1. GABA(A) receptors containing (alpha)5 subunits in the CA1 and CA3 hippocampal fields regulate ethanol-motivated behaviors: an extended ethanol reward circuitry.

    PubMed

    June, H L; Harvey, S C; Foster, K L; McKay, P F; Cummings, R; Garcia, M; Mason, D; Grey, C; McCane, S; Williams, L S; Johnson, T B; He, X; Rock, S; Cook, J M

    2001-03-15

    GABA receptors within the mesolimbic circuitry have been proposed to play a role in regulating alcohol-seeking behaviors in the alcohol-preferring (P) rat. However, the precise GABA(A) receptor subunit(s) mediating the reinforcing properties of EtOH remains unknown. We examined the capacity of intrahippocampal infusions of an alpha5 subunit-selective ( approximately 75-fold) benzodiazepine (BDZ) inverse agonist [i.e., RY 023 (RY) (tert-butyl 8-(trimethylsilyl) acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a] [1,4] benzodiazepine-3-carboxylate)] to alter lever pressing maintained by concurrent presentation of EtOH (10% v/v) and a saccharin solution (0.05% w/v). Bilateral (1.5-20 microgram) and unilateral (0.01-40 microgram) RY dose-dependently reduced EtOH-maintained responding, with saccharin-maintained responding being reduced only with the highest doses (e.g., 20 and 40 microgram). The competitive BDZ antagonist ZK 93426 (ZK) (7 microgram) reversed the RY-induced suppression on EtOH-maintained responding, confirming that the effect was mediated via the BDZ site on the GABA(A) receptor complex. Intrahippocampal modulation of the EtOH-maintained responding was site-specific; no antagonism by RY after intra-accumbens [nucleus accumbens (NACC)] and intraventral tegmental [ventral tegmental area (VTA)] infusions was observed. Because the VTA and NACC contain very high densities of alpha1 and alpha2 subunits, respectively, we determined whether RY exhibited a "negative" or "neutral" pharmacological profile at recombinant alpha1beta3gamma2, alpha2beta3gamma2, and alpha5beta3gamma2 receptors expressed in Xenopus oocytes. RY produced "classic" inverse agonism at all alpha receptor subtypes; thus, a neutral efficacy was not sufficient to explain the failure of RY to alter EtOH responding in the NACC or VTA. The results provide the first demonstration that the alpha5-containing GABA(A) receptors in the hippocampus play an important role in regulating Et

  2. Glycine and GABA(A) ultra-sensitive ethanol receptors as novel tools for alcohol and brain research.

    PubMed

    Naito, Anna; Muchhala, Karan H; Asatryan, Liana; Trudell, James R; Homanics, Gregg E; Perkins, Daya I; Davies, Daryl L; Alkana, Ronald L

    2014-12-01

    A critical obstacle to developing effective medications to prevent and/or treat alcohol use disorders is the lack of specific knowledge regarding the plethora of molecular targets and mechanisms underlying alcohol (ethanol) action in the brain. To identify the role of individual receptor subunits in ethanol-induced behaviors, we developed a novel class of ultra-sensitive ethanol receptors (USERs) that allow activation of a single receptor subunit population sensitized to extremely low ethanol concentrations. USERs were created by mutating as few as four residues in the extracellular loop 2 region of glycine receptors (GlyRs) or γ-aminobutyric acid type A receptors (GABA(A)Rs), which are implicated in causing many behavioral effects linked to ethanol abuse. USERs, expressed in Xenopus oocytes and tested using two-electrode voltage clamp, demonstrated an increase in ethanol sensitivity of 100-fold over wild-type receptors by significantly decreasing the threshold and increasing the magnitude of ethanol response, without altering general receptor properties including sensitivity to the neurosteroid, allopregnanolone. These profound changes in ethanol sensitivity were observed across multiple subunits of GlyRs and GABA(A)Rs. Collectively, our studies set the stage for using USER technology in genetically engineered animals as a unique tool to increase understanding of the neurobiological basis of the behavioral effects of ethanol.

  3. GR3027 antagonizes GABAA receptor-potentiating neurosteroids and restores spatial learning and motor coordination in rats with chronic hyperammonemia and hepatic encephalopathy.

    PubMed

    Johansson, Maja; Agusti, Ana; Llansola, Marta; Montoliu, Carmina; Strömberg, Jessica; Malinina, Evgenya; Ragagnin, Gianna; Doverskog, Magnus; Bäckström, Torbjörn; Felipo, Vicente

    2015-09-01

    Hepatic encephalopathy (HE) is one of the primary complications of liver cirrhosis. Current treatments for HE, mainly directed to reduction of ammonia levels, are not effective enough because they cannot completely eliminate hyperammonemia and inflammation, which induce the neurological alterations. Studies in animal models show that overactivation of GABAA receptors is involved in cognitive and motor impairment in HE and that reducing this activation restores these functions. We have developed a new compound, GR3027, that selectively antagonizes the enhanced activation of GABAA receptors by neurosteroids such as allopregnanolone and 3α,21-dihydroxy-5α-pregnan-20-one (THDOC). This work aimed to assess whether GR3027 improves motor incoordination, spatial learning, and circadian rhythms of activity in rats with HE. GR3027 was administered subcutaneously to two main models of HE: rats with chronic hyperammonemia due to ammonia feeding and rats with portacaval shunts (PCS). Motor coordination was assessed in beam walking and spatial learning and memory in the Morris water maze and the radial maze. Circadian rhythms of ambulatory and vertical activity were also assessed. In both hyperammonemic and PCS rats, GR3027 restores motor coordination, spatial memory in the Morris water maze, and spatial learning in the radial maze. GR3027 also partially restores circadian rhythms of ambulatory and vertical activity in PCS rats. GR3027 is a novel approach to treatment of HE that would normalize neurological functions altered because of enhanced GABAergic tone, affording more complete normalization of cognitive and motor function than current treatments for HE.

  4. GR3027 antagonizes GABAA receptor-potentiating neurosteroids and restores spatial learning and motor coordination in rats with chronic hyperammonemia and hepatic encephalopathy

    PubMed Central

    Johansson, Maja; Agusti, Ana; Llansola, Marta; Montoliu, Carmina; Strömberg, Jessica; Malinina, Evgenya; Ragagnin, Gianna; Doverskog, Magnus; Bäckström, Torbjörn

    2015-01-01

    Hepatic encephalopathy (HE) is one of the primary complications of liver cirrhosis. Current treatments for HE, mainly directed to reduction of ammonia levels, are not effective enough because they cannot completely eliminate hyperammonemia and inflammation, which induce the neurological alterations. Studies in animal models show that overactivation of GABAA receptors is involved in cognitive and motor impairment in HE and that reducing this activation restores these functions. We have developed a new compound, GR3027, that selectively antagonizes the enhanced activation of GABAA receptors by neurosteroids such as allopregnanolone and 3α,21-dihydroxy-5α-pregnan-20-one (THDOC). This work aimed to assess whether GR3027 improves motor incoordination, spatial learning, and circadian rhythms of activity in rats with HE. GR3027 was administered subcutaneously to two main models of HE: rats with chronic hyperammonemia due to ammonia feeding and rats with portacaval shunts (PCS). Motor coordination was assessed in beam walking and spatial learning and memory in the Morris water maze and the radial maze. Circadian rhythms of ambulatory and vertical activity were also assessed. In both hyperammonemic and PCS rats, GR3027 restores motor coordination, spatial memory in the Morris water maze, and spatial learning in the radial maze. GR3027 also partially restores circadian rhythms of ambulatory and vertical activity in PCS rats. GR3027 is a novel approach to treatment of HE that would normalize neurological functions altered because of enhanced GABAergic tone, affording more complete normalization of cognitive and motor function than current treatments for HE. PMID:26138462

  5. GR3027 antagonizes GABAA receptor-potentiating neurosteroids and restores spatial learning and motor coordination in rats with chronic hyperammonemia and hepatic encephalopathy.

    PubMed

    Johansson, Maja; Agusti, Ana; Llansola, Marta; Montoliu, Carmina; Strömberg, Jessica; Malinina, Evgenya; Ragagnin, Gianna; Doverskog, Magnus; Bäckström, Torbjörn; Felipo, Vicente

    2015-09-01

    Hepatic encephalopathy (HE) is one of the primary complications of liver cirrhosis. Current treatments for HE, mainly directed to reduction of ammonia levels, are not effective enough because they cannot completely eliminate hyperammonemia and inflammation, which induce the neurological alterations. Studies in animal models show that overactivation of GABAA receptors is involved in cognitive and motor impairment in HE and that reducing this activation restores these functions. We have developed a new compound, GR3027, that selectively antagonizes the enhanced activation of GABAA receptors by neurosteroids such as allopregnanolone and 3α,21-dihydroxy-5α-pregnan-20-one (THDOC). This work aimed to assess whether GR3027 improves motor incoordination, spatial learning, and circadian rhythms of activity in rats with HE. GR3027 was administered subcutaneously to two main models of HE: rats with chronic hyperammonemia due to ammonia feeding and rats with portacaval shunts (PCS). Motor coordination was assessed in beam walking and spatial learning and memory in the Morris water maze and the radial maze. Circadian rhythms of ambulatory and vertical activity were also assessed. In both hyperammonemic and PCS rats, GR3027 restores motor coordination, spatial memory in the Morris water maze, and spatial learning in the radial maze. GR3027 also partially restores circadian rhythms of ambulatory and vertical activity in PCS rats. GR3027 is a novel approach to treatment of HE that would normalize neurological functions altered because of enhanced GABAergic tone, affording more complete normalization of cognitive and motor function than current treatments for HE. PMID:26138462

  6. Inhibitory nature of tiagabine-augmented GABAA receptor-mediated depolarizing responses in hippocampal pyramidal cells.

    PubMed

    Jackson, M F; Esplin, B; Capek, R

    1999-03-01

    . Independently of the stimulus intensity with which they were evoked, the charge transferred to the soma by excitatory postsynaptic currents (EPSCs), elicited in the presence of tiagabine (20 microM) during the large (1,428 +/- 331 pA) inward currents that underlie the DRs, was decreased on the average by 90.8 +/- 1.7%. Such inhibition occurred despite the presence of the GABAB receptor antagonist, CGP 52 432 (10 microM), indicating that GABAB heteroreceptors, located on glutamatergic terminals, do not mediate the observed reduction in the amplitude of excitatory postsynaptic responses. The present results suggest that despite facilitating the induction of GABA-mediated depolarizations, tiagabine application may nevertheless increase the effectiveness of synaptic inhibition during the synchronous high-frequency activation of inhibitory interneurons by enhanced shunting.

  7. Memory impairment due to fipronil pesticide exposure occurs at the GABAA receptor level, in rats.

    PubMed

    Godinho, Antonio Francisco; de Oliveira Souza, Ana Carolina; Carvalho, Caio Cristóvão; Horta, Daniel França; De Fraia, Daniel; Anselmo, Fabio; Chaguri, João Leandro; Faria, Caique Aparecido

    2016-10-15

    Fipronil (F) a pesticide considered of second generation cause various toxic effects in target and non-target organisms including humans in which provoke neurotoxicity, having the antagonism of gamma-amino butyric acid (GABA) as their main mechanism for toxic action. GABAergic system has been involved in processes related to the memory formation and consolidation. The present work studied the importance of GABA to the mechanisms involved in the very early development of fipronil-induced memory impairment in rats. Memory behavior was assessed using new object recognition task (ORT) and eight radial arm maze task (8-RAM) to study effects on cognitive and spatial memory. Locomotor behavior was assessed using open field task (OF). The dose of fipronil utilized was studied through a pilot experiment. The GABA antagonist picrotoxin (P) was used to enhance fipronil effects on GABAergic system. Fipronil or picrotoxin decrease memory studied in ORT and 8-RAM tasks. Additionally, F and P co-exposure enhanced effects on memory compared to controls, F, and P, suggesting strongly a GABAergic effect. Weight gain modulation and fipronil in blood were utilized as animal's intoxication indicators. In conclusion, here we report that second-generation pesticides, such as fipronil, can have toxic interactions with the CNS of mammals and lead to memory impairment by modulating the GABAergic system. PMID:27374426

  8. Memory impairment due to fipronil pesticide exposure occurs at the GABAA receptor level, in rats.

    PubMed

    Godinho, Antonio Francisco; de Oliveira Souza, Ana Carolina; Carvalho, Caio Cristóvão; Horta, Daniel França; De Fraia, Daniel; Anselmo, Fabio; Chaguri, João Leandro; Faria, Caique Aparecido

    2016-10-15

    Fipronil (F) a pesticide considered of second generation cause various toxic effects in target and non-target organisms including humans in which provoke neurotoxicity, having the antagonism of gamma-amino butyric acid (GABA) as their main mechanism for toxic action. GABAergic system has been involved in processes related to the memory formation and consolidation. The present work studied the importance of GABA to the mechanisms involved in the very early development of fipronil-induced memory impairment in rats. Memory behavior was assessed using new object recognition task (ORT) and eight radial arm maze task (8-RAM) to study effects on cognitive and spatial memory. Locomotor behavior was assessed using open field task (OF). The dose of fipronil utilized was studied through a pilot experiment. The GABA antagonist picrotoxin (P) was used to enhance fipronil effects on GABAergic system. Fipronil or picrotoxin decrease memory studied in ORT and 8-RAM tasks. Additionally, F and P co-exposure enhanced effects on memory compared to controls, F, and P, suggesting strongly a GABAergic effect. Weight gain modulation and fipronil in blood were utilized as animal's intoxication indicators. In conclusion, here we report that second-generation pesticides, such as fipronil, can have toxic interactions with the CNS of mammals and lead to memory impairment by modulating the GABAergic system.

  9. CRH receptor antagonism reverses the effect of social subordination upon central GABAA receptor binding in estradiol-treated ovariectomized female rhesus monkeys.

    PubMed

    Michopoulos, V; Embree, M; Reding, K; Sanchez, M M; Toufexis, D; Votaw, J R; Voll, R J; Goodman, M M; Rivier, J; Wilson, M E; Berga, S L

    2013-10-10

    Persistent exposure to environmental stressors causes dysregulation of the limbic-hypothalamic-pituitary-adrenal (LHPA) axis and alters GABAA receptor (GABAAR) levels throughout the brain. Social subordination in socially housed female rhesus results in distinctive stress-related physiological and behavioral phenotypes that are dependent on the ovarian hormone estradiol (E2). In the present study, we utilized ovariectomized adult female rhesus monkeys undergoing hormone replacement with E2 to test the hypothesis that the chronic psychosocial stress of subordination alters GABAAR binding potential (GABAAR BPND) in limbic regions implicated in emotional processing including the prefrontal cortex, temporal lobe (amygdala and hippocampus), and hypothalamus. Furthermore, we tested the hypothesis that peripheral administration of a corticotropin-releasing hormone (CRH) receptor antagonist (astressin B) would reverse the alterations in GABAAR binding within these regions in subordinate females. After subjects received astressin B or saline for three consecutive days, GABAAR BPND was determined by positron emission tomography (PET) using (18)F-flumazenil as a radioligand. T1-weighted structural magnetic resonance imaging scans were also acquired for PET scan co-registration, in order to perform a region of interest analysis using the pons as a reference region. Compared to socially dominant females, subordinate females exhibited increased GABAAR BPND in the prefrontal cortex but not in the temporal lobe or the hypothalamus. Administration of astressin B eliminated the status difference in GABAAR BPND in the prefrontal cortex, suggesting that the chronic stressor of social subordination modulates GABAergic tone via effects on CRH and the LHPA axis, at least in prefrontal regions.

  10. Eugenol inhibits the GABAA current in trigeminal ganglion neurons.

    PubMed

    Lee, Sang Hoon; Moon, Jee Youn; Jung, Sung Jun; Kang, Jin Gu; Choi, Seung Pyo; Jang, Jun Ho

    2015-01-01

    Eugenol has sedative, antioxidant, anti-inflammatory, and analgesic effects, but also serves as an irritant through the regulation of a different set of ion channels. Activation of gamma aminobutyric acid (GABA) receptors on sensory neurons leads to the stabilization of neuronal excitability but contributes to formalin-induced inflammatory pain. In this study, we examined the effect of eugenol on the GABA-induced current in rat trigeminal ganglia (TG) neurons and in human embryonic kidney (HEK) 293 cells expressing the GABAA receptor α1β2γ2 subtype using the whole-cell patch clamp technique. RT-PCR and Western blot analysis were used to confirm the expression of GABAA receptor γ2 subunit mRNA and protein in the TG and hippocampus. Eugenol decreased the amplitude ratio of the GABA-induced current to 27.5 ± 3.2% (p < 0.05) in TG neurons, which recovered after a 3-min washout. In HEK 293 cells expressing the α1β2γ2 subtype, eugenol inhibited GABA-induced currents in a dose-dependent manner. Application of eugenol also decreased the GABA response in the presence of a G-protein blocker. Eugenol pretreatment with different concentrations of GABA resulted in similar inhibition of the GABA-induced current in a non-competitive manner. In conclusion, eugenol inhibits the GABA-induced current in TG neurons and HEK 293 cells expressing the GABAA receptor in a reversible, dose-dependent, and non-competitive manner, but not via the G-protein pathway. We suggest that the GABAA receptor could be a molecular target for eugenol in the modulation of nociceptive information.

  11. Deleterious Rare Variants Reveal Risk for Loss of GABAA Receptor Function in Patients with Genetic Epilepsy and in the General Population

    PubMed Central

    Hernandez, Ciria C.; Klassen, Tara L.; Jackson, Laurel G.; Gurba, Katharine; Hu, Ningning; Macdonald, Robert L.

    2016-01-01

    Genetic epilepsies (GEs) account for approximately 50% of all seizure disorders, and familial forms include mutations in single GABAA receptor subunit genes (GABRs). In 144 sporadic GE cases (GECs), exome sequencing of 237 ion channel genes identified 520 GABR variants. Among these variants, 33 rare variants in 11 GABR genes were present in 24 GECs. To assess functional risk of variants in GECs, we selected 8 variants found in GABRA, 3 in GABRB, and 3 in GABRG and compared them to 18 variants found in the general population for GABRA1 (n = 9), GABRB3 (n = 7), and GABRG2 (n = 2). To identify deleterious variants and gain insight into structure-function relationships, we studied the gating properties, surface expression and structural perturbations of the 32 variants. Significant reduction of GABAA receptor function was strongly associated with variants scored as deleterious and mapped within the N-terminal and transmembrane domains. In addition, 12 out of 17 variants mapped along the β+/α- GABA binding interface, were associated with reduction in channel gating and were predicted to cause structural rearrangements of the receptor by in silico simulations. Missense or nonsense mutations of GABRA1, GABRB3 and GABRG2 primarily impair subunit biogenesis. In contrast, GABR variants affected receptor function by impairing gating, suggesting that different mechanisms are operating in GABR epilepsy susceptibility variants and disease-causing mutations. The functional impact of single GABR variants found in individuals with sporadic GEs warrants the use of molecular diagnosis and will ultimately improve the treatment of genetic epilepsies by using a personalized approach. PMID:27622563

  12. Deleterious Rare Variants Reveal Risk for Loss of GABAA Receptor Function in Patients with Genetic Epilepsy and in the General Population.

    PubMed

    Hernandez, Ciria C; Klassen, Tara L; Jackson, Laurel G; Gurba, Katharine; Hu, Ningning; Noebels, Jeffrey L; Macdonald, Robert L

    2016-01-01

    Genetic epilepsies (GEs) account for approximately 50% of all seizure disorders, and familial forms include mutations in single GABAA receptor subunit genes (GABRs). In 144 sporadic GE cases (GECs), exome sequencing of 237 ion channel genes identified 520 GABR variants. Among these variants, 33 rare variants in 11 GABR genes were present in 24 GECs. To assess functional risk of variants in GECs, we selected 8 variants found in GABRA, 3 in GABRB, and 3 in GABRG and compared them to 18 variants found in the general population for GABRA1 (n = 9), GABRB3 (n = 7), and GABRG2 (n = 2). To identify deleterious variants and gain insight into structure-function relationships, we studied the gating properties, surface expression and structural perturbations of the 32 variants. Significant reduction of GABAA receptor function was strongly associated with variants scored as deleterious and mapped within the N-terminal and transmembrane domains. In addition, 12 out of 17 variants mapped along the β+/α- GABA binding interface, were associated with reduction in channel gating and were predicted to cause structural rearrangements of the receptor by in silico simulations. Missense or nonsense mutations of GABRA1, GABRB3 and GABRG2 primarily impair subunit biogenesis. In contrast, GABR variants affected receptor function by impairing gating, suggesting that different mechanisms are operating in GABR epilepsy susceptibility variants and disease-causing mutations. The functional impact of single GABR variants found in individuals with sporadic GEs warrants the use of molecular diagnosis and will ultimately improve the treatment of genetic epilepsies by using a personalized approach. PMID:27622563

  13. Synergistic action of GABA-A and NMDA receptors in the induction of long-term depression in glutamatergic synapses in the newborn rat hippocampus.

    PubMed

    Pavlov, Ivan; Riekki, Ruusu; Taira, Tomi

    2004-12-01

    We show that activation of GABA(A) receptors (GABA(A)Rs) promotes induction of N-methyl-D-aspartate (NMDA) receptor (NMDAR)-dependent long-term depression (LTD) of glutamatergic synapses in the newborn rat hippocampal area CA1 in a developmentally restricted manner. In the newborn rat hippocampus two mechanistically different types of LTD of glutamatergic synapses could be induced under similar experimental conditions. The form of the LTD induced depended on the stimulation protocol and on the age of the animal. Low-frequency stimulation (1 Hz) with 100 stimuli induced a robust homosynaptic, reversible LTD at postnatal days 2-8 (P2-P8) but not at P14. This LTD was blocked by the NMDAR antagonist AP5 or by the GABA(A)R antagonist picrotoxin. Use of a low-chloride solution in the patch pipette resulting in E(GABA-A) < -70 mV blocked the NMDAR-dependent LTD, whereas clamping the cell to -40 mV during induction rescued it. In addition, it was possible to induce LTD at P14 with 100 stimuli if the cells were clamped to -40 mV during induction. Low-frequency stimulation with 900 stimuli induced a robust homosynaptic, reversible LTD both at P2-P8 and at P14. However, neither AP5 nor picrotoxin affected the LTD induced by 900 pulses at P2-P8. Instead, the 900 stimuli-induced LTD was blocked by the metabotropic glutamate receptor antagonists when co-applied with AP5. We suggest that during the first postnatal week postsynaptic depolarization provided by the activation of GABA(A)Rs shifts the threshold for the LTD induction, making the synapses more prone to activity-induced plasticity. From the second postnatal week onwards, when the GABA(A) responses are already hyperpolarizing, different mechanisms for LTD induction prevail. PMID:15579156

  14. Synergistic action of GABA-A and NMDA receptors in the induction of long-term depression in glutamatergic synapses in the newborn rat hippocampus.

    PubMed

    Pavlov, Ivan; Riekki, Ruusu; Taira, Tomi

    2004-12-01

    We show that activation of GABA(A) receptors (GABA(A)Rs) promotes induction of N-methyl-D-aspartate (NMDA) receptor (NMDAR)-dependent long-term depression (LTD) of glutamatergic synapses in the newborn rat hippocampal area CA1 in a developmentally restricted manner. In the newborn rat hippocampus two mechanistically different types of LTD of glutamatergic synapses could be induced under similar experimental conditions. The form of the LTD induced depended on the stimulation protocol and on the age of the animal. Low-frequency stimulation (1 Hz) with 100 stimuli induced a robust homosynaptic, reversible LTD at postnatal days 2-8 (P2-P8) but not at P14. This LTD was blocked by the NMDAR antagonist AP5 or by the GABA(A)R antagonist picrotoxin. Use of a low-chloride solution in the patch pipette resulting in E(GABA-A) < -70 mV blocked the NMDAR-dependent LTD, whereas clamping the cell to -40 mV during induction rescued it. In addition, it was possible to induce LTD at P14 with 100 stimuli if the cells were clamped to -40 mV during induction. Low-frequency stimulation with 900 stimuli induced a robust homosynaptic, reversible LTD both at P2-P8 and at P14. However, neither AP5 nor picrotoxin affected the LTD induced by 900 pulses at P2-P8. Instead, the 900 stimuli-induced LTD was blocked by the metabotropic glutamate receptor antagonists when co-applied with AP5. We suggest that during the first postnatal week postsynaptic depolarization provided by the activation of GABA(A)Rs shifts the threshold for the LTD induction, making the synapses more prone to activity-induced plasticity. From the second postnatal week onwards, when the GABA(A) responses are already hyperpolarizing, different mechanisms for LTD induction prevail.

  15. Identification of clathrin heavy chain as a direct interaction partner for the gamma-aminobutyric acid type A receptor associated protein.

    PubMed

    Mohrlüder, Jeannine; Hoffmann, Yvonne; Stangler, Thomas; Hänel, Karen; Willbold, Dieter

    2007-12-18

    Gamma-aminobutyric acid type A receptors (GABAA receptors) are the major sites of GABA-mediated fast synaptic inhibition in the central nervous system. Variation of the cell surface receptor count is postulated to be of importance in modulating inhibitory synaptic transmission. The GABAA receptor associated protein (GABARAP) is a ubiquitin-like modifier, implicated in GABAA receptor clustering, trafficking, and turnover. GABARAP pull-down experiments with brain lysate identified clathrin heavy chain to be GABARAP-associated. Phage display screening of a randomized peptide library for GABARAP ligands yielded a sequence motif which characterizes the peptide binding specificity of GABARAP. Sequence database searches with this motif revealed clathrin heavy chain as a protein containing the identified sequence motif within its residues 510-522, supporting the result of the pull-down experiments. Calreticulin, which was identified recently as a GABARAP ligand, contains a very similar sequence motif. We demonstrate that calreticulin indeed competes with clathrin heavy chain for GABARAP binding. Finally, employing nuclear magnetic resonance spectroscopy, we mapped the GABARAP residues responsible for binding to clathrin. The hereby mapped GABARAP regions overlap very well with the homologue residues in yeast Atg8 that were recently shown to be important for autophagy. Together with the knowledge that GABARAP and clathrin are known to be involved in GABAA receptor trafficking within the cell, this strongly suggests a clear physiological relevance of the direct interaction of GABARAP with clathrin heavy chain. PMID:18027972

  16. How to Properly Measure a Current-Voltage Relation?—Interpolation vs. Ramp Methods Applied to Studies of GABAA Receptors

    PubMed Central

    Yelhekar, Tushar D.; Druzin, Michael; Karlsson, Urban; Blomqvist, Erii; Johansson, Staffan

    2016-01-01

    The relation between current and voltage, I-V relation, is central to functional analysis of membrane ion channels. A commonly used method, since the introduction of the voltage-clamp technique, to establish the I-V relation depends on the interpolation of current amplitudes recorded at different steady voltages. By a theoretical computational approach as well as by experimental recordings from GABAA-receptor mediated currents in mammalian central neurons, we here show that this interpolation method may give reversal potentials and conductances that do not reflect the properties of the channels studied under conditions when ion flux may give rise to concentration changes. Therefore, changes in ion concentrations may remain undetected and conclusions on changes in conductance, such as during desensitization, may be mistaken. In contrast, an alternative experimental approach, using rapid voltage ramps, enable I-V relations that much better reflect the properties of the studied ion channels. PMID:26869882

  17. A tryptic hydrolysate from bovine milk αs1-casein enhances pentobarbital-induced sleep in mice via the GABAA receptor.

    PubMed

    Dela Peña, Irene Joy I; Kim, Hee Jin; de la Peña, June Bryan; Kim, Mikyung; Botanas, Chrislean Jun; You, Kyung Yi; Woo, Taeseon; Lee, Yong Soo; Jung, Jae-Chul; Kim, Kyung-Mi; Cheong, Jae Hoon

    2016-10-15

    Studies have shown that enzymatic hydrolysis of casein, the primary protein component of cow's milk, produces peptides with various biological activities, and some of these peptides may have sleep-promoting effects. In the present study, we evaluated the sedative and sleep-promoting effects of bovine αS1-casein tryptic hydrolysate (CH), containing a decapeptide αS1-casein known as alpha-casozepine. CH was orally administered to ICR mice at various concentrations (75, 150, 300, or 500mg/kg). An hour after administration, assessment of its sedative (open-field and rota-rod tests) and sleep-potentiating effects (pentobarbital-induced sleeping test and EEG monitoring) were conducted. Although a trend can be observed, CH treatment did not significantly alter the spontaneous locomotor activity and motor function of mice in the open-field and rota-rod tests. On the other hand, CH (150mg/kg, respectively) enhanced the sleep induced by pentobarbital sodium in mice. It also promoted slow-wave (delta) EEG activity in rats; a pattern indicative of sleep or relaxation. These behavioral results indicate that CH has sleep-promoting effects, but no or has minimal sedative effects. To elucidate the probable mechanism behind the effects of CH, we examined its action on intracellular chloride ion influx in cultured human neuroblastoma cells. CH dose-dependently increased chloride ion influx, which was blocked by co-administration of bicuculline, a competitive GABAA receptor antagonist. Taken together, the results of the present study suggest that CH has sleep-promoting properties which are probably mediated through the GABAA receptor-chloride ion channel complex. PMID:27401107

  18. GABA(A) receptor activation in the CA1 area of the dorsal hippocampus impairs consolidation of conditioned contextual fear in C57BL/6J mice.

    PubMed

    Misane, Ilga; Kruis, Ayla; Pieneman, Anton W; Ögren, Sven Ove; Stiedl, Oliver

    2013-02-01

    Local infusion of the GABA(A) receptor agonist muscimol is used for reversible inactivation of septohippocampal brain structures associated with cognitive functions. However, information on the effective duration, affected processes and site(s) of action of muscimol in the hippocampus is lacking. Therefore, the dose- and time-dependent effects of bilateral dorsohippocampal infusion of muscimol (0.01-2.0 μg/mouse) below the CA1 area were examined on processing of fear memory in male C57BL/6J mice. Infusion of muscimol 15 min-6 h but not 9 h or 24 h before training impaired conditioned context-dependent fear tested 24 h or 48 h after training. Post-training infusion of muscimol also impaired context-dependent fear when applied either 4 h or 6 h after training, although with lower efficacy. Muscimol was ineffective when administered immediately, 1 h or 24 h after training. Infusion of muscimol 15 min before training impaired context-dependent fear 4-6 h after training indicating preserved short-term but impaired long-term memory. Regardless of infusion time and dose, muscimol had no effect on tone-dependent (cued) fear memory. The impairment by the fluorescently-labeled muscimol-bodipy (5.3 μg/mouse) were similar to those of an equimolar dose of muscimol (1 μg/mouse). The distribution profile after local infusion indicated that muscimol-bodipy (5.3 μg/mouse) was confined to the CA1 area of the dorsal hippocampus. These results demonstrated that GABA(A) receptor activation in the CA1 area of the dorsal hippocampus causes a long-term memory impairment of conditioned context-dependent fear mediated by a long-lasting (≥6 h) muscimol action most likely affecting consolidation processes.

  19. Assessing the role of inferior olivary sensory signaling in the expression of conditioned eyeblinks using a combined glutamate/GABAA receptor antagonist protocol

    PubMed Central

    Zbarska, Svitlana

    2012-01-01

    The inferior olive (IO) is a major component of the eyeblink conditioning neural network. The cerebellar learning hypothesis assumes that the IO supplies the cerebellum with a “teaching” unconditioned stimulus input required for the acquisition of the conditioned response (CR) and predicts that inactivating this input leads to the extinction of CRs. Previous tests of this prediction attempted to block the teaching input by blocking glutamatergic sensory inputs in the IO. These tests were inconclusive because blocking glutamate neurotransmission in the IO produces a nonspecific tonic malfunction of cerebellar circuits. The purpose of the present experiment was to examine whether the behavioral outcomes of blocking glutamate receptors in the IO could be counterbalanced by reducing GABA-mediated inhibition in the IO. We found that injecting the IO with the glutamate antagonist γ-d-glutamylglycine (DGG) abolished previously learned CRs, whereas injecting the GABAA receptor antagonist gabazine at the same site did not affect CR incidence but shortened CR latencies and produced tonic eyelid closure. To test whether the glutamate antagonist-induced behavioral deficit could be offset by elevating IO activity with GABAA antagonists, rabbits were first injected with DGG and then with gabazine in the same training session. While DGG abolished CRs, follow-up injections of gabazine accelerated their recovery. These findings suggest that the level of IO neuronal activity is critical for the performance of CRs, and that combined pharmacological approaches that maintain spontaneous activity at near normal levels hold tremendous potential for unveiling the role of IO-mediated signals in eyeblink conditioning. PMID:21975449

  20. GABAA receptor-mediated feedforward and feedback inhibition differentially modulate the gain and the neural code transformation in hippocampal CA1 pyramidal cells.

    PubMed

    Jang, Hyun Jae; Park, Kyerl; Lee, Jaedong; Kim, Hyuncheol; Han, Kyu Hun; Kwag, Jeehyun

    2015-12-01

    Diverse variety of hippocampal interneurons exists in the CA1 area, which provides either feedforward (FF) or feedback (FB) inhibition to CA1 pyramidal cell (PC). However, how the two different inhibitory network architectures modulate the computational mode of CA1 PC is unknown. By investigating the CA3 PC rate-driven input-output function of CA1 PC using in vitro electrophysiology, in vitro-simulation of inhibitory network, and in silico computational modeling, we demonstrated for the first time that GABAA receptor-mediated FF and FB inhibition differentially modulate the gain, the spike precision, the neural code transformation and the information capacity of CA1 PC. Recruitment of FF inhibition buffered the CA1 PC spikes to theta-frequency regardless of the input frequency, abolishing the gain and making CA1 PC insensitive to its inputs. Instead, temporal variability of the CA1 PC spikes was increased, promoting the rate-to-temporal code transformation to enhance the information capacity of CA1 PC. In contrast, the recruitment of FB inhibition sub-linearly transformed the input rate to spike output rate with high gain and low spike temporal variability, promoting the rate-to-rate code transformation. These results suggest that GABAA receptor-mediated FF and FB inhibitory circuits could serve as network mechanisms for differentially modulating the gain of CA1 PC, allowing CA1 PC to switch between different computational modes using rate and temporal codes ad hoc. Such switch will allow CA1 PC to efficiently respond to spatio-temporally dynamic inputs and expand its computational capacity during different behavioral and neuromodulatory states in vivo.

  1. Allopregnanolone reduces immobility in the forced swimming test and increases the firing rate of lateral septal neurons through actions on the GABAA receptor in the rat.

    PubMed

    Rodrìguez-Landa, Juan Francisco; Contreras, Carlos M; Bernal-Morales, Blandina; Gutièrrez-Garcìa, Ana G; Saavedra, Margarita

    2007-01-01

    Since allopregnanolone reduces the total time of immobility in rats submitted to the forced swimming test, we decided to explore whether this neuroactive steroid shares other antidepressant-like actions, such as increasing the neuronal firing rate in the lateral septal nucleus (LSN). In order to discard the influence of the oestrous cycle on immobility and on the firing rate of LSN neurons, all Wistar rats used in the study underwent ovariectomy before treatments. A group of rats received different doses of allopregnanolone (0.5, 1.0, 2.0 and 3.0 mg/kg, i.p.) 1 hour before being forced to swim in order to identify the minimum effective dose diminishing immobility. None of the tested doses of allopregnanolone produced significant changes in motor activity in the open-field test. The minimum dose of allopregnanolone producing a significant reduction in the total time of immobility (p<0.05) against the vehicle was 1.0 mg/kg, while 2.0 mg/kg and above also increased the latency to the first period of immobility (p<0.05). The minimum effective dose of allopregnanolone reducing immobility in the forced swimming test (1.0 mg/kg) significantly (p <0.05) produced a higher (twofold) neuronal firing rate in LSN neurons, but did not produce any change in septofimbrial nucleus neurons, which fired at a rate similar to that of vehicle-treated rats. The pretreatment with the non-competitive GABAA receptor antagonist, picrotoxin (1.0 mg/kg), blocked the aforementioned actions of allopregnanolone on both immobility and LSN firing rate. In conclusion, allopregnanolone produces an antidepressant-like effect in the forced swimming test, associated with an increase in the LSN neuronal firing rate, seemingly mediated by the GABAA receptor.

  2. Developmental stability of taurine's activation on glycine receptors in cultured neurons of rat auditory cortex.

    PubMed

    Tang, Zheng-Quan; Lu, Yun-Gang; Chen, Lin

    2008-01-01

    Taurine is an endogenous amino acid that can activate glycine and/or gamma-aminobutyric acid type A (GABA(A)) receptors in the central nervous system. During natural development, taurine's receptor target undergoes a shift from glycine receptors to GABA(A) receptors in cortical neurons. Here, we demonstrate that taurine's receptor target in cortical neurons remains stable during in vitro development. With whole-cell patch-clamp recordings, we found that taurine always activated glycine receptors, rather than GABA(A) receptors, in neurons of rat auditory cortex cultured for 5-22 days. Our results suggest that the functional sensitivity of glycine and GABA(A) receptors to taurine is critically regulated by their developmental environments.

  3. Reducing GABAA α5 receptor-mediated inhibition rescues functional and neuromorphological deficits in a mouse model of down syndrome.

    PubMed

    Martínez-Cué, Carmen; Martínez, Paula; Rueda, Noemí; Vidal, Rebeca; García, Susana; Vidal, Verónica; Corrales, Andrea; Montero, Juan A; Pazos, Ángel; Flórez, Jesús; Gasser, Rodolfo; Thomas, Andrew W; Honer, Michael; Knoflach, Frédéric; Trejo, Jose Luis; Wettstein, Joseph G; Hernández, Maria-Clemencia

    2013-02-27

    Down syndrome (DS) is associated with neurological complications, including cognitive deficits that lead to impairment in intellectual functioning. Increased GABA-mediated inhibition has been proposed as a mechanism underlying deficient cognition in the Ts65Dn (TS) mouse model of DS. We show that chronic treatment of these mice with RO4938581 (3-bromo-10-(difluoromethyl)-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]diazepine), a selective GABA(A) α5 negative allosteric modulator (NAM), rescued their deficits in spatial learning and memory, hippocampal synaptic plasticity, and adult neurogenesis. We also show that RO4938581 normalized the high density of GABAergic synapse markers in the molecular layer of the hippocampus of TS mice. In addition, RO4938581 treatment suppressed the hyperactivity observed in TS mice without inducing anxiety or altering their motor abilities. These data demonstrate that reducing GABAergic inhibition with RO4938581 can reverse functional and neuromorphological deficits of TS mice by facilitating brain plasticity and support the potential therapeutic use of selective GABA(A) α5 NAMs to treat cognitive dysfunction in DS. PMID:23447605

  4. Computational prediction of MicroRNAs targeting GABA receptors and experimental verification of miR-181, miR-216 and miR-203 targets in GABA-A receptor

    PubMed Central

    2012-01-01

    Background GABA receptors are well known as the inhibitory receptors in the central nervous system and are also found in peripheral tissues. We have previously shown that GABA receptors are involved in lung development and fluid homeostasis. However, the microRNAs that regulate GABA receptors have not yet been identified. Results In this study, we used the online software, TargetScan and miRanda, to query the microRNAs that directly target GABA receptors and then selected some of them to verify experimentally using 3'-UTR reporter assays. Computational approaches predict many microRNA binding sites on the 3'-UTR of GABAA receptors, but not on GABAC receptors. 3'-UTR reporter assays only verified miR-181, miR-216, and miR-203 as the microRNAs that target GABA receptor α1-subunit among 10 microRNAs tested. Conclusions Our studies reinforce that microRNA target prediction needs to be verified experimentally. The identification of microRNAs that target GABA receptors provides a basis for further studies of post-transcriptional regulation of GABA receptors. PMID:22321448

  5. Maternal Separation during Breastfeeding Induces Gender-Dependent Changes in Anxiety and the GABA-A Receptor Alpha-Subunit in Adult Wistar Rats

    PubMed Central

    León Rodríguez, Diego Armando; Dueñas, Zulma

    2013-01-01

    Different models of rodent maternal separation (MS) have been used to investigate long-term neurobiological and behavioral changes, associated with early stress. However, few studies have involved the analysis of sex-related differences in central anxiety modulation. This study investigated whether MS during breastfeeding affected adult males and females in terms of anxiety and brain GABA-A receptor-alpha-subunit immunoreactivity. The brain areas analyzed were the amygdale (AM), hippocampus (HP), medial prefrontal cortex (mPFC), medial preoptic area (POA) and paraventricular nucleus (PVN). Rats were housed under a reversed light/dark cycle (lights off at 7∶00 h) with access to water and food ad libitum. Animals underwent MS twice daily during the dark cycle from postnatal day 1 to postnatal day 21. Behavior was tested when rats were 65–70 days old using the elevated plus maze and after brains were treated for immunohistochemistry. We found that separated females spent more time in the open arms and showed more head dipping behavior compared with controls. The separated males spent more time in the center of the maze and engaged in more stretching behavior than the controls. Immunohistochemistry showed that separated females had less immunostained cells in the HP, mPFC, PVN and POA, while separated males had fewer immunolabeled cells in the PFC, PVN and AM. These results could indicate that MS has gender-specific effects on anxiety behaviors and that these effects are likely related to developmental alterations involving GABA-A neurotransmission. PMID:23826356

  6. The apparent voltage dependence of GABAA receptor activation and modulation is inversely related to channel open probability.

    PubMed

    O'Toole, Kate K; Jenkins, Andrew

    2012-02-01

    The GABA type A receptor (GABA(A)R) is expressed ubiquitously throughout the brain and is a target for many therapeutic agents, including general anesthetics and benzodiazepines, which enhance receptor function by increasing the open probability (P(o)) of the ion channel. It is commonplace for in vitro studies of receptor pharmacological characteristics to use negative membrane holding potentials to mimic the resting potential of neurons and symmetrical chloride to eliminate Goldman rectification, which results in chloride flow in the opposite direction, compared with in vivo conditions. This critical difference is usually overlooked because the GABA(A)R has been reported to behave as an ohmic pore, but our results show that the current-voltage relationship is nonlinear with respect to P(o). Specifically, we found that currents were outwardly rectifying at low P(o) and linear at high P(o). We confirmed the correlation between P(o) and rectification with a partial agonist, piperidine-4-sulfonic acid, and a gating-impaired mutation, α1(L277A); both exhibited enhanced outward rectification. Furthermore, this correlation was independent of Goldman rectification and persisted under altered chloride gradient conditions, which suggests that rectification is linked to the direction of chloride flux. Finally, our results showed that the degree of potentiation by general anesthetics (etomidate, propofol, and isoflurane) was greater at negative membrane potentials. Traditional in vitro experiments thus overestimate the action of positive allosteric modulators of the GABA(A)R. Our results show that the direction of the driving force on the permeant ion, as well as P(o), must be considered together for a complete understanding of drug actions on ligand-gated ion channels.

  7. A question of balance--positive versus negative allosteric modulation of GABA(A) receptor subtypes as a driver of analgesic efficacy in rat models of inflammatory and neuropathic pain.

    PubMed

    Munro, Gordon; Erichsen, Helle K; Rae, Mark G; Mirza, Naheed R

    2011-01-01

    After injury GABA(A) receptor positive allosteric modulators (PAMs) mediate robust analgesia in animals via putative restoration of post-synaptic GABA(A)-α2 and -α3 receptor function within the spinal cord. GABA can also act at GABA(A) receptors localized on primary afferent neurones to inhibit presynaptic neurotransmitter release and produce analgesia via a process called primary afferent depolarization (PAD). Some forms of injury might sufficiently enhance PAD to shift it into a net excitatory process. Thus, negative allosteric modulators (NAMs) might also possess analgesic activity. We have compared compounds capable of either positively or negatively modulating GABA(A) receptors in rat models associated with injury-induced central sensitization. The subtype-selective PAMs NS11394 (1-10 mg/kg) and TPA023 (3-30 mg/kg) attenuated formalin-induced nocifensive behaviours. Similarly, both compounds reversed hindpaw mechanical hypersensitivity and weight bearing deficits in carrageenan-inflamed and nerve-injured rats. The non-selective PAM diazepam (1-5 mg/kg) was ineffective in all models. Surprisingly, both the non-selective NAM FG-7142 (3-30 mg/kg) and the α5-selective NAM α5IA-II (10-60 mg/kg) also attenuated formalin-induced nocifensive behaviours. In carrageenan-inflamed rats α5IA-II reversed mechanical hypersensitivity and weight bearing deficits whilst FG-7142 only attenuated weight bearing deficits. This picture was essentially reversed in nerve-injured rats for these two NAMs. With the exception of NS11394, all compounds attenuated exploratory motility behaviour in rats, either as a consequence of sedative or anxiogenic-like side-effects. These data indicate that the preferred selectivity and activity profiles for mediating analgesia upon activation of GABA(A) receptors might be more complex than previously anticipated, and is worthy of further exploration.

  8. Mutagenesis and computational docking studies support the existence of a histamine binding site at the extracellular β3+β3- interface of homooligomeric β3 GABAA receptors.

    PubMed

    Hoerbelt, Paul; Ramerstorfer, Joachim; Ernst, Margot; Sieghart, Werner; Thomson, Jeffrey L; Hough, Lindsay B; Fleck, Mark W

    2016-09-01

    Histamine is an important neurotransmitter that exerts its physiological actions through H1-4 metabotropic receptors in mammals. It also directly activates ionotropic GABAA receptor (GABAAR) β3 homooligomers and potentiates GABA responses in αβ heterooligomers in vitro, but the respective histamine binding sites in GABAARs are unknown. We hypothesized that histamine binds at the extracellular β+β- interface at a position homologous to the GABA binding site of heterooligomeric GABAARs. To test this, we individually mutated several residues at the putative ligand binding minus side of a rat GABAAR β3 wild type subunit and of a β3 subunit that was made insensitive to trace Zn(2+) inhibition [β3(H267A); called (Z)β3]. (Z)β3, (Z)β3(Y62L), (Z)β3(Q64A), (Z)β3(Q64E), α1(Z)β3, or α1(Z)β3(Y62L) receptors were studied in HEK293T cells using whole cell voltage clamp recording. β3, β3(Y62C), β3(Q64C), β3(N41C), β3(D43C), β3(A45C) or β3(M115C) receptors were examined in Xenopus oocytes using two-electrode voltage clamp. Histamine directly activated (Z)β3 and β3 homooligomers and potentiated GABA actions in α1(Z)β3 heterooligomers. Receptors containing (Z)β3(Y62L), β3(Y62C) and β3(D43C) showed markedly reduced histamine potency, but homo- and heterooligomers with (Z)β3(Q64E) exhibited increased potency. The GABAAR αβ(γ) competitive antagonist bicuculline elicited sub-maximal agonist currents through (Z)β3 homooligomers, the potency of which was strongly decreased by (Z)β3(Y62L). Mutations β3(N41C), β3(A45C) and β3(M115C) disturbed receptor expression or assembly. Computational docking into the crystal structure of homooligomeric β3 receptors resulted in a histamine pose highly consistent with the experimental findings, suggesting that histamine activates β3 receptors via a site homologous to the GABA site in αβγ receptors. PMID:27140694

  9. Kavain, the Major Constituent of the Anxiolytic Kava Extract, Potentiates GABAA Receptors: Functional Characteristics and Molecular Mechanism

    PubMed Central

    Chua, Han Chow; Christensen, Emilie T. H.; Hoestgaard-Jensen, Kirsten; Hartiadi, Leonny Y.; Ramzan, Iqbal; Jensen, Anders A.; Absalom, Nathan L.; Chebib, Mary

    2016-01-01

    Extracts of the pepper plant kava (Piper methysticum) are effective in alleviating anxiety in clinical trials. Despite the long-standing therapeutic interest in kava, the molecular target(s) of the pharmacologically active constituents, kavalactones have not been established. γ-Aminobutyric acid type A receptors (GABAARs) are assumed to be the in vivo molecular target of kavalactones based on data from binding assays, but evidence in support of a direct interaction between kavalactones and GABAARs is scarce and equivocal. In this study, we characterised the functional properties of the major anxiolytic kavalactone, kavain at human recombinant α1β2, β2γ2L, αxβ2γ2L (x = 1, 2, 3 and 5), α1βxγ2L (x = 1, 2 and 3) and α4β2δ GABAARs expressed in Xenopus oocytes using the two-electrode voltage clamp technique. We found that kavain positively modulated all receptors regardless of the subunit composition, but the degree of enhancement was greater at α4β2δ than at α1β2γ2L GABAARs. The modulatory effect of kavain was unaffected by flumazenil, indicating that kavain did not enhance GABAARs via the classical benzodiazepine binding site. The β3N265M point mutation which has been previously shown to profoundly decrease anaesthetic sensitivity, also diminished kavain-mediated potentiation. To our knowledge, this study is the first report of the functional characteristics of a single kavalactone at distinct GABAAR subtypes, and presents the first experimental evidence in support of a direct interaction between a kavalactone and GABAARs. PMID:27332705

  10. Identification of a GABAA receptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog.

    PubMed

    Li, Guo-Dong; Chiara, David C; Sawyer, Gregory W; Husain, S Shaukat; Olsen, Richard W; Cohen, Jonathan B

    2006-11-01

    General anesthetics, including etomidate, act by binding to and enhancing the function of GABA type A receptors (GABA(A)Rs), which mediate inhibitory neurotransmission in the brain. Here, we used a radiolabeled, photoreactive etomidate analog ([(3)H]azietomidate), which retains anesthetic potency in vivo and enhances GABA(A)R function in vitro, to identify directly, for the first time, amino acids that contribute to a GABA(A)R anesthetic binding site. For GABA(A)Rs purified by affinity chromatography from detergent extracts of bovine cortex, [(3)H]azietomidate photoincorporation was increased by GABA and inhibited by etomidate in a concentration-dependent manner (IC(50) = 30 microm). Protein microsequencing of fragments isolated from proteolytic digests established photolabeling of two residues: one within the alphaM1 transmembrane helix at alpha1Met-236 (and/or the homologous methionines in alpha2,3,5), not previously implicated in etomidate function, and one within the betaM3 transmembrane helix at beta3Met-286 (and/or the homologous methionines in beta1,2), an etomidate sensitivity determinant. The pharmacological specificity of labeling indicates that these methionines contribute to a single binding pocket for etomidate located in the transmembrane domain at the interface between beta and alpha subunits, in what is predicted by structural models based on homology with the nicotinic acetylcholine receptor to be a water-filled pocket approximately 50 A below the GABA binding site. The localization of the etomidate binding site to an intersubunit, not an intrasubunit, binding pocket is a novel conclusion that suggests more generally that the localization of drug binding sites to subunit interfaces may be a feature not only for GABA and benzodiazepines but also for etomidate and other intravenous and volatile anesthetics. PMID:17093081

  11. The GABAA receptor agonist muscimol induces an age- and region-dependent form of long-term depression in the mouse striatum.

    PubMed

    Zhang, Xiaoqun; Yao, Ning; Chergui, Karima

    2016-09-01

    Several forms of long-term depression (LTD) of glutamatergic synaptic transmission have been identified in the dorsal striatum and in the nucleus accumbens (NAc). Such experience-dependent synaptic plasticity might play important roles in reward-related learning. The GABAA receptor agonist muscimol was recently found to trigger a long-lasting depression of glutamatergic synaptic transmission in the NAc of adolescent mice, but the mechanisms that underlie this novel form of LTD were not studied. Here we examined the effect of muscimol applied in the perfusion solution on the amplitude of field excitatory postsynaptic potentials/population spikes (fEPSP/PSs) in mouse brain slices. We found that muscimol depressed the fEPSP/PS in the NAc of adolescent mice but not adult mice, through both postsynaptic and presynaptic mechanisms. Indeed, muscimol altered the fEPSP/PS paired-pulse ratio, depolarized the membrane of projection neurons, and decreased the frequency, but not amplitude, of spontaneous excitatory postsynaptic currents in the NAc of adolescent mice. The LTD induced by muscimol likely involved endocannabinoids, metabotropic glutamate receptors (mGluRs), but not TRPV1 receptors. Muscimol-LTD was occluded by prior induction of LTD through low-frequency stimulation (LFS) of the slice, demonstrating a common pathway in the induction of LFS-LTD and muscimol-LTD. We also found that muscimol induced a form of LTD in the dorsolateral striatum of adult but not adolescent mice. This LTD was mediated by endocannabinoids but did not involve mGluRs or TRPV1 receptors. These results identify a novel form of synaptic plasticity, and its mechanisms of induction, which is age and region dependent. These findings may contribute to a better understanding of the increased susceptibility of the adolescent brain to long-term synaptic changes in regions associated with reward mechanisms. PMID:27531838

  12. Memantine treatment reduces the expression of the K(+)/Cl(-) cotransporter KCC2 in the hippocampus and cerebral cortex, and attenuates behavioural responses mediated by GABA(A) receptor activation in mice.

    PubMed

    Molinaro, Gemma; Battaglia, Giuseppe; Riozzi, Barbara; Di Menna, Luisa; Rampello, Liborio; Bruno, Valeria; Nicoletti, Ferdinando

    2009-04-10

    A 7-day treatment with memantine (25 mg/kg, i.p.), a drug that is currently prescribed for the treatment of Alzheimer's disease, increased the levels of brain-derived neurotrophic factor (BDNF) and reduced the expression of the neuron-specific K(+)/Cl(-) co-transporter, KCC2, in the hippocampus and cerebral cortex of mice. Knowing that KCC2 maintains low intracellular Cl(-) concentrations, which drive Cl(-) influx in response to GABA(A) receptor activation, we monitored the behavioural response to the GABA(A) receptor enhancer, diazepam, in mice pre-treated for 7 days with saline or 25 mg/kg of memantine. Memantine treatment substantially attenuated motor impairment induced by an acute challenge with diazepam (6 mg/kg, i.p.), as assessed by the rotarod test and the horizontal wire test. We suggest that a prolonged treatment with memantine induces changes in the activity of GABA(A) receptors that might contribute to the therapeutic and/or toxic effects of the drug.

  13. Picrotoxin inhibition mechanism of a gamma-aminobutyric acid A receptor investigated by a laser-pulse photolysis technique.

    PubMed

    Ramakrishnan, Latha; Hess, George P

    2005-06-14

    The gamma-aminobutyric acid(A) (GABA(A)) receptor, a major inhibitory neurotransmitter receptor, belongs to a family of membrane-bound proteins that regulate signal transmission between approximately 10(12) cells of the nervous system. It plays a major role in many neurological disorders, including epilepsy. It is the target of many pharmacological agents, including the convulsant picrotoxin. Here, we present the mechanism of inhibition by picrotoxin of the rat alpha1beta2gamma2L GABA(A) receptor investigated using rapid kinetic techniques in combination with whole-cell current recordings. The following new results were obtained by using transient kinetic techniques, the cell-flow method and the laser-pulse photolysis (LaPP) technique with a microsecond to millisecond time resolution. (i) The apparent dissociation constant of picrotoxin for the open-channel form of the receptor was approximately 5 times higher than that of the closed-channel form. (ii) Picrotoxin increased the channel-closing rate constant (k(cl)) approximately 4-fold, while the rate constant for channel opening (k(op)) remained essentially unaffected. (iii) The mechanism indicates that picrotoxin binds to an allosteric site of the receptor with higher affinity for the closed-channel form than for the open-channel form and thereby inhibits the receptor by decreasing 4-fold its channel-opening equilibrium constant [Phi(I)(-)(1) = k(op(I))/k(cl(I))]. (iv) The mechanism further indicates that compounds that bind with equal affinity to the picrotoxin-binding site on the open-channel form of the receptor and the closed-channel form will not affect the channel-opening equilibrium and can, therefore, displace picrotoxin and prevent inhibition of the GABA(A) receptor by picrotoxin. Such compounds may be therapeutically useful in counteracting the effects of compounds and diseases that unfavorably affect the channel-opening equilibrium of the receptor channel.

  14. Anxiolytic effects of the GABA(A) receptor partial agonist, L-838,417: impact of age, test context familiarity, and stress.

    PubMed

    Morales, Melissa; Varlinskaya, Elena I; Spear, Linda P

    2013-08-01

    The partial α2,3,5 GABA(A) receptor agonist, L-838,417 has been reported to have anxiolytic effects in adult rodents. Although maturational differences exist for the GABA(A) receptor subunits, the anxiolytic effects of L-838,417 have not been tested in younger animals. The goal of the present experiments was to determine whether L-838,417 reverses anxiety-like behavior induced by either an unfamiliar environment (Experiment 1) or repeated restraint stress (Experiment 2) differentially in adolescent and adult, male and female Sprague-Dawley rats using a modified social interaction test. In Experiment 1, rats were injected with 0, 0.5, 1.0, 2.0, or 4.0 mg/kg L-838,417, i.p. and tested 30 min later in an unfamiliar test context for 10 min. In Experiment 2, rats were exposed to restraint stress (90 min daily for 5 days). Immediately after the last restraint session, animals were injected with L-838,417 and placed alone for 30 min in the test apparatus to familiarize them to this context prior to the 10 min social interaction test. In Experiment 1, L-838,417 produced anxiolytic effects in adults at 1.0 mg/kg, as indexed by a transformation of social avoidance into preference and an increase in social investigation. In adolescents, a dose of 2.0 mg/kg eliminated social avoidance, but had no anxiolytic effects on social investigation. Testing under familiar circumstances (Experiment 2) after repeated restraint stress eliminated age differences in sensitivity to L-838,417, with 0.5 mg/kg reversing the anxiogenic effects of prior stress regardless of age, but with doses ≥ 1 mg/kg decreasing social investigation, an effect possibly due in part to locomotor-impairing effects of this compound. Although locomotor activity was suppressed in both experiments, higher doses of L-838,417 were necessary to suppress locomotor activity in Experiment 1. Thus, anxiolytic effects of L-838,417 were found to be context-, age-, and stress-dependent.

  15. PX-RICS-deficient mice mimic autism spectrum disorder in Jacobsen syndrome through impaired GABAA receptor trafficking.

    PubMed

    Nakamura, Tsutomu; Arima-Yoshida, Fumiko; Sakaue, Fumika; Nasu-Nishimura, Yukiko; Takeda, Yasuko; Matsuura, Ken; Akshoomoff, Natacha; Mattson, Sarah N; Grossfeld, Paul D; Manabe, Toshiya; Akiyama, Tetsu

    2016-01-01

    Jacobsen syndrome (JBS) is a rare congenital disorder caused by a terminal deletion of the long arm of chromosome 11. A subset of patients exhibit social behavioural problems that meet the diagnostic criteria for autism spectrum disorder (ASD); however, the underlying molecular pathogenesis remains poorly understood. PX-RICS is located in the chromosomal region commonly deleted in JBS patients with autistic-like behaviour. Here we report that PX-RICS-deficient mice exhibit ASD-like social behaviours and ASD-related comorbidities. PX-RICS-deficient neurons show reduced surface γ-aminobutyric acid type A receptor (GABAAR) levels and impaired GABAAR-mediated synaptic transmission. PX-RICS, GABARAP and 14-3-3ζ/θ form an adaptor complex that interconnects GABAAR and dynein/dynactin, thereby facilitating GABAAR surface expression. ASD-like behavioural abnormalities in PX-RICS-deficient mice are ameliorated by enhancing inhibitory synaptic transmission with a GABAAR agonist. Our findings demonstrate a critical role of PX-RICS in cognition and suggest a causal link between PX-RICS deletion and ASD-like behaviour in JBS patients. PMID:26979507

  16. PX-RICS-deficient mice mimic autism spectrum disorder in Jacobsen syndrome through impaired GABAA receptor trafficking.

    PubMed

    Nakamura, Tsutomu; Arima-Yoshida, Fumiko; Sakaue, Fumika; Nasu-Nishimura, Yukiko; Takeda, Yasuko; Matsuura, Ken; Akshoomoff, Natacha; Mattson, Sarah N; Grossfeld, Paul D; Manabe, Toshiya; Akiyama, Tetsu

    2016-01-01

    Jacobsen syndrome (JBS) is a rare congenital disorder caused by a terminal deletion of the long arm of chromosome 11. A subset of patients exhibit social behavioural problems that meet the diagnostic criteria for autism spectrum disorder (ASD); however, the underlying molecular pathogenesis remains poorly understood. PX-RICS is located in the chromosomal region commonly deleted in JBS patients with autistic-like behaviour. Here we report that PX-RICS-deficient mice exhibit ASD-like social behaviours and ASD-related comorbidities. PX-RICS-deficient neurons show reduced surface γ-aminobutyric acid type A receptor (GABAAR) levels and impaired GABAAR-mediated synaptic transmission. PX-RICS, GABARAP and 14-3-3ζ/θ form an adaptor complex that interconnects GABAAR and dynein/dynactin, thereby facilitating GABAAR surface expression. ASD