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Sample records for selective gabaa receptor

  1. Generation of Recombinant Antibodies to Rat GABAA Receptor Subunits by Affinity Selection on Synthetic Peptides

    PubMed Central

    Koduvayur, Sujatha P.; Gussin, Hélène A.; Parthasarathy, Rajni; Hao, Zengping; Kay, Brian K.; Pepperberg, David R.

    2014-01-01

    The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure. PMID:24586298

  2. 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

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

    PubMed

    Gallos, George; 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

    2015-05-01

    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 ([Ca(2+)]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 [Ca(2+)]i, store-operated Ca(2+) entry, and methacholine-induced Ca(2+) 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

  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. PMID:27120014

  5. Selective antagonism of the GABAA receptor by ciprofloxacin and biphenylacetic acid

    PubMed Central

    Green, M A; Halliwell, R F

    1997-01-01

    nerve. Ciprofloxacin also inhibited the GABA-evoked response with an IC50 of 334 μM (256–437) and BPAA (100 μM) potentiated these antagonist effects. However, the magnitude of the synergy was 48 times less than that seen in the vagus nerve. These data indicate that ciprofloxacin and BPAA are selective antagonists of GABAA receptors, an action that may contribute to their excitatory effects in vivo. Additionally, our data suggest that the molecular properties of GABAA receptors in different regions of the CNS influence the extent to which these drugs synergistically inhibit the GABAA receptor. PMID:9351519

  6. 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

  7. Selective antagonism of the GABA(A) receptor by ciprofloxacin and biphenylacetic acid.

    PubMed

    Green, M A; Halliwell, R F

    1997-10-01

    from that determined in the vagus nerve. Ciprofloxacin also inhibited the GABA-evoked response with an IC50 of 334 microM (256-437) and BPAA (100 microM) potentiated these antagonist effects. However, the magnitude of the synergy was 48 times less than that seen in the vagus nerve. 7. These data indicate that ciprofloxacin and BPAA are selective antagonists of GABA(A) receptors, an action that may contribute to their excitatory effects in vivo. Additionally, our data suggest that the molecular properties of GABA(A) receptors in different regions of the CNS influence the extent to which these drugs synergistically inhibit the GABA(A) receptor. PMID:9351519

  8. Photo-antagonism of the GABAA receptor.

    PubMed

    Mortensen, Martin; Iqbal, Favaad; Pandurangan, Arun P; Hannan, Saad; Huckvale, Rosemary; Topf, Maya; Baker, James R; Smart, Trevor G

    2014-01-01

    Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity. GABAA receptors and inhibitory synapses are vital components of brain function, yet much of our knowledge regarding receptor mobility and function at inhibitory synapses is derived indirectly from using recombinant receptors, antibody-tagged native receptors and pharmacological treatments. Here we describe the use of a set of research tools that can irreversibly bind to and affect the function of recombinant and neuronal GABAA receptors following ultraviolet photoactivation. These compounds are based on the competitive antagonist gabazine and incorporate a variety of photoactive groups. By using site-directed mutagenesis and ligand-docking studies, they reveal new areas of the GABA binding site at the interface between receptor β and α subunits. These compounds enable the selected inactivation of native GABAA receptor populations providing new insight into the function of inhibitory synapses and extrasynaptic receptors in controlling neuronal excitation. PMID:25072879

  9. Neurosteroids and GABA-A Receptor Function

    PubMed Central

    Wang, Mingde

    2011-01-01

    Neurosteroids represent a class of endogenous steroids that are synthesized in the brain, the adrenals, and the gonads and have potent and selective effects on the GABAA-receptor. 3α-hydroxy A-ring reduced metabolites of progesterone, deoxycorticosterone, and testosterone are positive modulators of GABAA-receptor in a non-genomic manner. Allopregnanolone (3α-OH-5α-pregnan-20-one), 5α-androstane-3α, 17α-diol (Adiol), and 3α5α-tetrahydrodeoxycorticosterone (3α5α-THDOC) enhance the GABA-mediated Cl- currents acting on a site (or sites) distinct from the GABA, benzodiazepine, barbiturate, and picrotoxin binding sites. 3α5α-P and 3α5α-THDOC potentiate synaptic GABAA-receptor function and activate δ-subunit containing extrasynaptic receptors that mediate tonic currents. On the contrary, 3β-OH pregnane steroids and pregnenolone sulfate (PS) are GABAA-receptor antagonists and induce activation-dependent inhibition of the receptor. The activities of neurosteroid are dependent on brain regions and types of neurons. In addition to the slow genomic action of the parent steroids, the non-genomic, and rapid actions of neurosteroids play a significant role in the GABAA-receptor function and shift in mood and memory function. This review describes molecular mechanisms underlying neurosteroid action on the GABAA-receptor, mood changes, and cognitive functions. PMID:22654809

  10. Selective Modulators of α5-Containing GABAA Receptors and their Therapeutic Significance.

    PubMed

    Soh, Ming Shiuan; Lynch, Joseph W

    2015-01-01

    GABAA receptors containing the α5 subunit (5GABAARs) are found mainly in the hippocampus where they mediate a tonic chloride leak current and contribute a slow component to GABAergic inhibitory synaptic currents. Their inhibitory effect on the excitability of hippocampal neurons at least partly explains why changes in the level of activity of 5GABAARs affect cognition, learning and memory. These receptors have been implicated as potential therapeutic targets for a range of clinical conditions including age-related dementia, stroke, schizophrenia, Down syndrome and anesthetic- induced amnesia. Accordingly, a range of pharmacological modulators that selectively target 5GABAARs, as either inhibitors or allosteric enhancers, have been developed. Although many of these compounds show therapeutic effects in animal models of the above clinical disorders, none has been marketed yet due to unsuccessful clinical trials and toxicity in humans. These experiments have also revealed paradoxical effects of 5GABAAR modulation (e.g., cognitive impairments can be reversed by both positive and negative modulation), suggesting that our knowledge of the physiological roles of 5GABAARs is incomplete. This review highlights the various positive and negative modulators for 5GABAARs that have been developed, key findings concerning their effects in behavioral studies, and their importance across a number of therapeutic fields. It also highlights some of the gaps in our knowledge of the physiological and pathological roles of α5GABAARs. PMID:25751008

  11. 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

  12. 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

  13. Valerenic acid derivatives as novel subunit-selective GABAA receptor ligands –in vitro and in vivo characterization

    PubMed Central

    Khom, S; Strommer, B; Ramharter, J; Schwarz, T; Schwarzer, C; Erker, T; Ecker, GF; Mulzer, J; Hering, S

    2010-01-01

    BACKGROUND AND PURPOSE Subunit-specific modulators of γ-aminobutyric acid (GABA) type A (GABAA) receptors can help to assess the physiological function of receptors with different subunit composition and also provide the basis for the development of new drugs. Valerenic acid (VA) was recently identified as a β2/3 subunit-specific modulator of GABAA receptors with anxiolytic potential. The aim of the present study was to generate VA derivatives as novel GABAA receptor modulators and to gain insight into the structure–activity relation of this molecule. EXPERIMENTAL APPROACH The carboxyl group of VA was substituted by an uncharged amide or amides with different chain length. Modulation of GABAA receptors composed of different subunit compositions by the VA derivatives was studied in Xenopus oocytes by means of the two-microelectrode voltage-clamp technique. Half-maximal stimulation of GABA-induced chloride currents (IGABA) through GABAA receptors (EC50) and efficacies (maximal stimulation of IGABA) were estimated. Anxiolytic activity of the VA derivatives was studied in mice, applying the elevated plus maze test. KEY RESULTS Valerenic acid amide (VA-A) displayed the highest efficacy (more than twofold greater IGABA enhancement than VA) and highest potency (EC50= 13.7 ± 2.3 µM) on α1β3 receptors. Higher efficacy and potency of VA-A were also observed on α1β2γ2s and α3β3γ2s receptors. Anxiolytic effects were most pronounced for VA-A. CONCLUSIONS AND IMPLICATIONS Valerenic acid derivatives with higher efficacy and affinity can be generated. Greater in vitro action of the amide derivative correlated with a more pronounced anxiolytic effect in vivo. The data give further confidence in targeting β3 subunit containing GABAA receptors for development of anxiolytics. PMID:20718740

  14. 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

  15. Attenuating GABAA Receptor Signaling in Dopamine Neurons Selectively Enhances Reward Learning and Alters Risk Preference in Mice

    PubMed Central

    Parker, Jones G.; Wanat, Matthew J.; Soden, Marta E.; Ahmad, Kinza; Zweifel, Larry S.; Bamford, Nigel S.; Palmiter, Richard D.

    2011-01-01

    Phasic dopamine transmission encodes the value of reward-predictive stimuli and influences both learning and decision-making. Altered dopamine signaling is associated with psychiatric conditions characterized by risky choices such as pathological gambling. These observations highlight the importance of understanding how dopamine neuron activity is modulated. While excitatory drive onto dopamine neurons is critical for generating phasic dopamine responses, emerging evidence suggests that inhibitory signaling also modulates these responses. To address the functional importance of inhibitory signaling in dopamine neurons, we generated mice lacking the β3 subunit of the GABAA receptor specifically in dopamine neurons (β3-KO mice) and examined their behavior in tasks that assessed appetitive learning, aversive learning, and risk preference. Dopamine neurons in midbrain slices from β3-KO mice exhibited attenuated GABA-evoked inhibitory post-synaptic currents. Furthermore, electrical stimulation of excitatory afferents to dopamine neurons elicited more dopamine release in the nucleus accumbens of β3-KO mice as measured by fast-scan cyclic voltammetry. β3-KO mice were more active than controls when given morphine, which correlated with potential compensatory upregulation of GABAergic tone onto dopamine neurons. β3-KO mice learned faster in two food-reinforced learning paradigms, but extinguished their learned behavior normally. Enhanced learning was specific for appetitive tasks, as aversive learning was unaffected in β3-KO mice. Finally, we found that β3-KO mice had enhanced risk preference in a probabilistic selection task that required mice to choose between a small certain reward and a larger uncertain reward. Collectively, these findings identify a selective role for GABAA signaling in dopamine neurons in appetitive learning and decision-making. PMID:22114279

  16. Specific targeting of the GABA-A receptor α5 subtype by a selective inverse agonist restores cognitive deficits in Down syndrome mice

    PubMed Central

    Braudeau, J; Delatour, B; Duchon, A; Pereira, P Lopes; Dauphinot, L; de Chaumont, F; Olivo-Marin, J-C; Dodd, RH; Hérault, Y; Potier, M-C

    2011-01-01

    An imbalance between inhibitory and excitatory neurotransmission has been proposed to contribute to altered brain function in individuals with Down syndrome (DS). Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system and accordingly treatment with GABA-A antagonists can efficiently restore cognitive functions of Ts65Dn mice, a genetic model for DS. However, GABA-A antagonists are also convulsant which preclude their use for therapeutic intervention in DS individuals. Here, we have evaluated safer strategies to release GABAergic inhibition using a GABA-A-benzodiazepine receptor inverse agonist selective for the α5-subtype (α5IA). We demonstrate that α5IA restores learning and memory functions of Ts65Dn mice in the novel-object recognition and in the Morris water maze tasks. Furthermore, we show that following behavioural stimulation, α5IA enhances learning-evoked immediate early gene products in specific brain regions involved in cognition. Importantly, acute and chronic treatments with α5IA do not induce any convulsant or anxiogenic effects that are associated with GABA-A antagonists or non-selective inverse agonists of the GABA-A-benzodiazepine receptors. Finally, chronic treatment with α5IA did not induce histological alterations in the brain, liver and kidney of mice. Our results suggest that non-convulsant α5-selective GABA-A inverse agonists could improve learning and memory deficits in DS individuals. PMID:21693554

  17. Selective pyramidal cell reduction of GABA(A) receptor α1 subunit messenger RNA expression in schizophrenia.

    PubMed

    Glausier, Jill R; Lewis, David A

    2011-09-01

    Levels of messenger RNA (mRNA) for the α1 subunit of the GABA(A) receptor, which is present in 60% of cortical GABA(A) receptors, have been reported to be lower in layer 3 of the prefrontal cortex (PFC) in subjects with schizophrenia. This subunit is expressed in both pyramidal cells and interneurons, and thus lower α1 subunit levels in each cell population would have opposite effects on net cortical excitation. We used dual-label in situ hybridization to quantify GABA(A) α1 subunit mRNA expression in calcium/calmodulin-dependent kinase II α (CaMKIIα)-containing pyramidal cells and glutamic acid decarboxylase 65 kDa (GAD65)-containing interneurons in layer 3 of the PFC from matched schizophrenia and healthy comparison subjects. In subjects with schizophrenia, mean GABA(A) α1 subunit mRNA expression was significantly 40% lower in pyramidal cells, but was not altered in interneurons. Lower α1 subunit mRNA expression in pyramidal cells was not attributable to potential confounding factors, and thus appeared to reflect the disease process of schizophrenia. These results suggest that pyramidal cell inhibition is reduced in schizophrenia, whereas inhibition of GABA neurons is maintained. The cell type specificity of these findings may reflect a compensatory response to enhance layer 3 pyramidal cell activity in the face of the diminished excitatory drive associated with the lower dendritic spine density on these neurons. PMID:21677653

  18. GABAA Receptors Containing the α2 Subunit Are Critical for Direction-Selective Inhibition in the Retina

    PubMed Central

    Auferkorte, Olivia Nicola; Baden, Tom; Kaushalya, Sanjeev Kumar; Zabouri, Nawal; Rudolph, Uwe; Haverkamp, Silke; Euler, Thomas

    2012-01-01

    Far from being a simple sensor, the retina actively participates in processing visual signals. One of the best understood aspects of this processing is the detection of motion direction. Direction-selective (DS) retinal circuits include several subtypes of ganglion cells (GCs) and inhibitory interneurons, such as starburst amacrine cells (SACs). Recent studies demonstrated a surprising complexity in the arrangement of synapses in the DS circuit, i.e. between SACs and DS ganglion cells. Thus, to fully understand retinal DS mechanisms, detailed knowledge of all synaptic elements involved, particularly the nature and localization of neurotransmitter receptors, is needed. Since inhibition from SACs onto DSGCs is crucial for generating retinal direction selectivity, we investigate here the nature of the GABA receptors mediating this interaction. We found that in the inner plexiform layer (IPL) of mouse and rabbit retina, GABAA receptor subunit α2 (GABAAR α2) aggregated in synaptic clusters along two bands overlapping the dendritic plexuses of both ON and OFF SACs. On distal dendrites of individually labeled SACs in rabbit, GABAAR α2 was aligned with the majority of varicosities, the cell's output structures, and found postsynaptically on DSGC dendrites, both in the ON and OFF portion of the IPL. In GABAAR α2 knock-out (KO) mice, light responses of retinal GCs recorded with two-photon calcium imaging revealed a significant impairment of DS responses compared to their wild-type littermates. We observed a dramatic drop in the proportion of cells exhibiting DS phenotype in both the ON and ON-OFF populations, which strongly supports our anatomical findings that α2-containing GABAARs are critical for mediating retinal DS inhibition. Our study reveals for the first time, to the best of our knowledge, the precise functional localization of a specific receptor subunit in the retinal DS circuit. PMID:22506070

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

    PubMed Central

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

    2005-01-01

    Classical benzodiazepines (BZs), such as diazepam, bind to GABAA receptors containing α1, α2, α3 or α5 subunits that are therefore described as diazepam-sensitive (DS) receptors. However, the corresponding binding site of GABAA receptors containing either an α4 or α6 subunit do not bind the classical BZs and are therefore diazepam-insensitive (DIS) receptors; a difference attributable to a single amino acid (histidine in α1, α2, α3 and α5 subunits and arginine in α4 and α6). Unlike classical BZs, the imidazobenzodiazepines Ro 15-4513 and bretazenil bind to both DS and DIS populations of GABAA receptors. In the present study, an in vivo assay was developed using lorazepam to fully occupy DS receptors such that [3H]Ro 15-4513 was then only able to bind to DIS receptors. When dosed i.v., [3H]Ro 15-4513 rapidly entered and was cleared from the brain, with approximately 70% of brain radioactivity being membrane-bound. Essentially all membrane binding to DS+DIS receptors could be displaced by unlabelled Ro 15-4513 or bretazenil, with respective ID50 values of 0.35 and 1.2 mg kg−1. A dose of 30 mg kg−1 lorazepam was used to block all DS receptors in a [3H]Ro 15-1788 in vivo binding assay. When predosed in a [3H]Ro 15-4513 binding assay, lorazepam blocked [3H]Ro 15-4513 binding to DS receptors, with the remaining binding to DIS receptors accounting for 5 and 23% of the total (DS plus DIS) receptors in the forebrain and cerebellum, respectively. The in vivo binding of [3H]Ro 15-4513 to DIS receptors in the presence of lorazepam was confirmed using α1H101R knock-in mice, in which α1-containing GABAA receptors are rendered diazepam insensitive by mutation of the histidine that confers diazepam sensitivity to arginine. In these mice, and in the presence of lorazepam, there was an increase of in vivo [3H]Ro 15-4513 binding in the forebrain and cerebellum from 4 and 15% to 36 and 59% of the total (i.e. DS plus DIS) [3H]Ro 15-4513 binding observed in the

  20. 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

  1. BDNF selectively regulates GABAA receptor transcription by activation of the JAK/STAT pathway.

    PubMed

    Lund, Ingrid V; Hu, Yinghui; Raol, YogendraSinh H; Benham, Rebecca S; Faris, Ramona; Russek, Shelley J; Brooks-Kayal, Amy R

    2008-01-01

    The gamma-aminobutyric acid (GABA) type A receptor (GABA(A)R) is the major inhibitory neurotransmitter receptor in the brain. Its multiple subunits show regional, developmental, and disease-related plasticity of expression; however, the regulatory networks controlling GABA(A)R subunit expression remain poorly understood. We report that the seizure-induced decrease in GABA(A)R alpha1 subunit expression associated with epilepsy is mediated by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway regulated by brain-derived neurotrophic factor (BDNF). BDNF- and seizure-dependent phosphorylation of STAT3 cause the adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB) family member ICER (inducible cAMP early repressor) to bind with phosphorylated CREB at the Gabra1:CRE site. JAK/STAT pathway inhibition prevents the seizure-induced decrease in GABA(A)R alpha1 abundance in vivo and, given that BDNF is known to increase the abundance of GABA(A)R alpha4 in a JAK/STAT-independent manner, indicates that BDNF acts through at least two distinct pathways to influence GABA(A)R-dependent synaptic inhibition. PMID:18922788

  2. An arylaminopyridazine derivative of gamma-aminobutyric acid (GABA) is a selective and competitive antagonist at the GABAA receptor site.

    PubMed Central

    Chambon, J P; Feltz, P; Heaulme, M; Restle, S; Schlichter, R; Biziere, K; Wermuth, C G

    1985-01-01

    In view of finding a new gamma-aminobutyric acid (GABA) receptor ligand we synthesized an arylaminopyridazine derivative of GABA, SR 95103 [2-(carboxy-3'-propyl)-3-amino-4-methyl-6-phenylpyridazinium chloride]. SR 95103 displaced [3H]GABA from rat brain membranes with an apparent Ki of 2.2 microM and a Hill number near 1.0. SR 95103 (1-100 microM) antagonized the GABA-mediated enhancement of [3H]diazepam binding in a concentration-dependent manner without affecting [3H]diazepam binding per se. SR 95103 competitively antagonized GABA-induced membrane depolarization in rat spinal ganglia. In all these experiments, the potency of SR 95103 was close to that of bicuculline. SR 95103 (100 microM) did not interact with a variety of central receptors--in particular the GABAB, the strychnine, and the glutamate receptors--did not inhibit Na+-dependent synaptosomal GABA uptake, and did not affect GABA-transaminase and glutamic acid decarboxylase activities. Intraperitoneally administered SR 95103 elicited clonicotonic seizures in mice (ED50 = 180 mg/kg). On the basis of these results it is postulated that St 95103 is a competitive antagonist of GABA at the GABAA receptor site. In addition to being an interesting lead structure for the search of GABA ligands, SR 95103 could also be a useful tool to investigate GABA receptor subtypes because it is freely soluble in water and chemically stable. Images PMID:2984669

  3. PET radioligands targeting the brain GABAA /benzodiazepine receptor complex.

    PubMed

    Andersson, Jan D; Halldin, Christer

    2013-01-01

    The development of positron emission tomography radioligands for the GABAA /benzodiazepine receptor complex (GABAA receptor) labeled with (11) C and (18) F is examined. The review covers labeling strategies as well as brief biological evaluations of radioligands. In addition, we assess the special considerations that must be taken during a development program for radioligands targeting the GABAA receptor and explore some of the challenges that lie ahead. PMID:24285326

  4. Sh-I-048A, an in vitro non-selective super-agonist at the benzodiazepine site of GABAA receptors: the approximated activation of receptor subtypes may explain behavioral effects.

    PubMed

    Obradović, Aleksandar Lj; Joksimović, Srđan; Poe, Michael M; Ramerstorfer, Joachim; Varagic, Zdravko; Namjoshi, Ojas; Batinić, Bojan; Radulović, Tamara; Marković, Bojan; Roth, Brian L; Sieghart, Werner; Cook, James M; Savić, Miroslav M

    2014-03-20

    Enormous progress in understanding the role of four populations of benzodiazepine-sensitive GABAA receptors was paralleled by the puzzling findings suggesting that substantial separation of behavioral effects may be accomplished by apparently non-selective modulators. We report on SH-I-048A, a newly synthesized chiral positive modulator of GABAA receptors characterized by exceptional subnanomolar affinity, high efficacy and non-selectivity. Its influence on behavior was assessed in Wistar rats and contrasted to that obtained with 2mg/kg diazepam. SH-I-048A reached micromolar concentrations in brain tissue, while the unbound fraction in brain homogenate was around 1.5%. The approximated electrophysiological responses, which estimated free concentrations of SH-I-048A or diazepam are able to elicit, suggested a similarity between the 10mg/kg dose of the novel ligand and 2mg/kg diazepam; however, SH-I-048A was relatively more active at α1- and α5-containing GABAA receptors. Behaviorally, SH-I-048A induced sedative, muscle relaxant and ataxic effects, reversed mechanical hyperalgesia 24h after injury, while it was devoid of clear anxiolytic actions and did not affect water-maze performance. While lack of clear anxiolytic actions may be connected with an enhanced potentiation at α1-containing GABAA receptors, the observed behavior in the rotarod, water maze and peripheral nerve injury tests was possibly affected by its prominent action at receptors containing the α5 subunit. The current results encourage further innovative approaches aimed at linking in vitro and in vivo data in order to help define fine-tuning mechanisms at four sensitive receptor populations that underlie subtle differences in behavioral profiles of benzodiazepine site ligands. PMID:24472579

  5. Puberty, steroids and GABAA receptor plasticity

    PubMed Central

    Smith, Sheryl S.; Aoki, Chiye; Shen, Hui

    2009-01-01

    Summary GABAA receptors (GABAR) mediate most inhibition in the CNS and are also a target for neuroactive steroids such as 3α,5[α]β-THP (3αOH-5[α]β-OH-pregnan-20-one or [allo]pregnanolone). Although these steroids robustly enhance current gated by α1β2δ GABAR, we have shown that 3α,5[α]β-THP effects at recombinant α4β2δ GABAR depend on the direction of Cl- flux, where the steroid increases outward flux, but decreases inward flux through the receptor. This polarity-dependent inhibition of α4β2δ GABAR resulted from an increase in the rate and extent of rapid desensitization of the receptor, recorded from recombinant receptors expressed in HEK-293 cells with whole cell voltage clamp techniques. This inhibitory effect of 3α,5[α]β-THP was not observed at other receptor subtypes, suggesting it was selective for α4β2δ GABAR. Furthermore, it was prevented by a selective mutation of basic residue arginine 353 in the intracellular loop of the receptor, suggesting that this might be a putative chloride modulatory site. Expression of α4βδ GABAR increases markedly at extrasynaptic sites at the onset of puberty in female mice. At this time, 3α,5[α]β–THP decreased the inhibitory tonic current, recorded with perforated patch techniques to maintain the physiological Cl- gradient. By decreasing this shunting inhibition, 3α,5[α]β–THP increased the excitability of CA1 hippocampal pyramidal cells at puberty: These effects of the steroid were opposite to those observed before puberty when 3α,5[α]β–THP reduced neuronal excitability as a pre-synaptic effect. Behaviorally, the excitatory effect of 3α,5[α]β–THP was reflected as an increase in anxiety at the onset of puberty in female mice. Taken together, these findings suggest that the emergence of α4β2δ GABAR at the onset of puberty reverses the effect of a stress steroid. These findings may be relevant for the mood swings and increased response to stressful events reported in adolescence

  6. 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

  7. GABAA receptor modulating steroid antagonists (GAMSA) are functional in vivo.

    PubMed

    Johansson, Maja; Strömberg, Jessica; Ragagnin, Gianna; Doverskog, Magnus; Bäckström, Torbjörn

    2016-06-01

    GABAA receptor modulating steroid antagonists (GAMSA) selectively inhibit neurosteroid-mediated enhancement of GABA-evoked currents at the GABAA receptor. 3α-hydroxy-neurosteroids, notably allopregnanolone and tetrahydrodeoxycorticosterone (THDOC), potentiate GABAA receptor-mediated currents. On the contrary, various 3β-hydroxy-steroids antagonize this positive neurosteroid-mediated modulation. Importantly, GAMSAs are specific antagonists of the positive neurosteroid-modulation of the receptor and do not inhibit GABA-evoked currents. Allopregnanolone and THDOC have both negative and positive actions. Allopregnanolone can impair encoding/consolidation and retrieval of memories. Chronic administration of a physiological allopregnanolone concentration reduces cognition in mice models of Alzheimer's disease. In humans an allopregnanolone challenge impairs episodic memory and in hepatic encephalopathy cognitive deficits are accompanied by increased brain ammonia and allopregnanolone. Hippocampal slices react in vitro to ammonia by allopregnanolone synthesis in CA1 neurons, which blocks long-term potentiation (LTP). Thus, allopregnanolone may impair learning and memory by interfering with hippocampal LTP. Contrary, pharmacological treatment with allopregnanolone can promote neurogenesis and positively influence learning and memory of trace eye-blink conditioning in mice. In rat the GAMSA UC1011 inhibits an allopregnanolone-induced learning impairment and the GAMSA GR3027 restores learning and motor coordination in rats with hepatic encephalopathy. In addition, the GAMSA isoallopregnanolone antagonizes allopregnanolone-induced anesthesia in rats, and in humans it antagonizes allopregnanolone-induced sedation and reductions in saccadic eye velocity. 17PA is also an effective GAMSA in vivo, as it antagonizes allopregnanolone-induced anesthesia and spinal analgesia in rats. In vitro the allopregnanolone/THDOC-increased GABA-mediated GABAA receptor activity is antagonized

  8. 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

  9. The Role of GABAA Receptors in the Development of Alcoholism

    PubMed Central

    Enoch, Mary-Anne

    2008-01-01

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

  10. Neurexins Physically and Functionally Interact with GABAA-receptors

    PubMed Central

    Zhang, Chen; Atasoy, Deniz; Araç, Demet; Yang, Xiaofei; Fucillo, Marc V.; Robison, Alfred J.; Ko, Jaewon; Brunger, Axel T.; Südhof, Thomas C.

    2011-01-01

    Neurexins are presynaptic cell-adhesion molecules that form trans-synaptic complexes with postsynaptic neuroligins. When overexpressed in non-neuronal cells, neurexins induce formation of postsynaptic specializations in co-cultured neurons, suggesting that neurexins are synaptogenic. However, we now find that when overexpressed in neurons, neurexins do not increase synapse density, but instead selectively suppressed GABAergic synaptic transmission without decreasing GABAergic synapse numbers. This suppression was mediated by all subtypes of neurexins tested, in a cell-autonomous and neuroligin-independent manner. Strikingly, addition of recombinant neurexin to cultured neurons at sub-micromolar concentrations induced the same suppression of GABAergic synaptic transmission as neurexin overexpression. Moreover, experiments with native brain proteins and with purified recombinant proteins revealed that neurexins directly and stoichiometrically bind to GABAA-receptors, suggesting that they decrease GABAergic synaptic responses by interacting with GABAA-receptors. Our findings suggest that besides their other well-documented interactions, presynaptic neurexins directly act on postsynaptic GABAA-receptors, which may contribute to regulate the excitatory/inhibitory balance in brain. PMID:20471353

  11. 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

  12. GABAA receptor target of tetramethylenedisulfotetramine.

    PubMed

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

    2014-06-10

    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 [(14)C]TETS (14 mCi/mmol, radiochemical purity >99%) by reacting sulfamide with H(14)CHO 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 [(14)C]TETS in neuroreceptor binding studies with rat brain membranes in comparison with the standard GABAAR radioligand 4'-ethynyl-4-n-[(3)H]propylbicycloorthobenzoate ([(3)H]EBOB) (46 Ci/mmol), illustrating the use of AMS for characterizing the binding sites of high-affinity (14)C radioligands. Fourteen noncompetitive antagonists of widely diverse chemotypes assayed at 1 or 10 µM inhibited [(14)C]TETS and [(3)H]EBOB binding to a similar extent (r(2) = 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 [(14)C]TETS and [(3)H]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

  13. 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

  14. Mutant GABA(A) receptor subunits in genetic (idiopathic) epilepsy.

    PubMed

    Hirose, Shinichi

    2014-01-01

    The γ-aminobutyric acid receptor type A (GABAA receptor) is a ligand-gated chloride channel that mediates major inhibitory functions in the central nervous system. GABAA receptors function mainly as pentamers containing α, β, and either γ or δ subunits. A number of antiepileptic drugs have agonistic effects on GABAA receptors. Hence, dysfunctions of GABAA receptors have been postulated to play important roles in the etiology of epilepsy. In fact, mutations or genetic variations of the genes encoding the α1, α6, β2, β3, γ2, or δ subunits (GABRA1, GABRA6, GABRB2, GABRB3, GABRG2, and GABRD, respectively) have been associated with human epilepsy, both with and without febrile seizures. Epilepsy resulting from mutations is commonly one of following, genetic (idiopathic) generalized epilepsy (e.g., juvenile myoclonic epilepsy), childhood absence epilepsy, genetic epilepsy with febrile seizures, or Dravet syndrome. Recently, mutations of GABRA1, GABRB2, and GABRB3 were associated with infantile spasms and Lennox-Gastaut syndrome. These mutations compromise hyperpolarization through GABAA receptors, which is believed to cause seizures. Interestingly, most of the insufficiencies are not caused by receptor gating abnormalities, but by complex mechanisms, including endoplasmic reticulum (ER)-associated degradation, nonsense-mediated mRNA decay, intracellular trafficking defects, and ER stress. Thus, GABAA receptor subunit mutations are now thought to participate in the pathomechanisms of epilepsy, and an improved understanding of these mutations should facilitate our understanding of epilepsy and the development of new therapies. PMID:25194483

  15. 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

  16. Identification and characterization of GABA(A) receptor autoantibodies in autoimmune encephalitis.

    PubMed

    Ohkawa, Toshika; Satake, Shin'Ichiro; Yokoi, Norihiko; Miyazaki, Yu; Ohshita, Tomohiko; Sobue, Gen; Takashima, Hiroshi; Watanabe, Osamu; Fukata, Yuko; Fukata, Masaki

    2014-06-11

    Autoimmune forms of encephalitis have been associated with autoantibodies against synaptic cell surface antigens such as NMDA- and AMPA-type glutamate receptors, GABA(B) receptor, and LGI1. However, it remains unclear how many synaptic autoantigens are yet to be defined. Using immunoproteomics, we identified autoantibodies against the GABA(A) receptor in human sera from two patients diagnosed with encephalitis who presented with cognitive impairment and multifocal brain MRI abnormalities. Both patients had antibodies directed against the extracellular epitope of the β3 subunit of the GABA(A) receptor. The β3-subunit-containing GABA(A) receptor was a major target of the patients' serum antibodies in rat hippocampal neurons because the serum reactivity to the neuronal surface was greatly decreased by 80% when the β3 subunit was knocked down. Our developed multiplex ELISA testing showed that both patients had similar levels of GABA(A) receptor antibodies, one patient also had a low level of LGI1 antibodies, and the other also had CASPR2 antibodies. Application of the patients' serum at the time of symptom presentation of encephalitis to rat hippocampal neuron cultures specifically decreased both synaptic and surface GABA(A) receptors. Furthermore, treatment of neurons with the patients' serum selectively reduced miniature IPSC amplitude and frequency without affecting miniature EPSCs. These results strongly suggest that the patients' GABA(A) receptor antibodies play a central role in the patients' symptoms. Therefore, this study establishes anti-GABA(A) receptor encephalitis and expands the pathogenic roles of GABA(A) receptor autoantibodies. PMID:24920620

  17. Tandem Subunits Effectively Constrain GABAA Receptor Stoichiometry and Recapitulate Receptor Kinetics But Are Insensitive to GABAA Receptor-Associated Protein

    PubMed Central

    Boileau, Andrew J.; Pearce, Robert A.; Czajkowski, Cynthia

    2008-01-01

    GABAergic synapses likely contain multiple GABAA receptor subtypes, making postsynaptic currents difficult to dissect. However, even in heterologous expression systems, analysis of receptors composed of α, β, and γ subunits can be confounded by receptors expressed from α and β subunits alone. To produce recombinant GABAA receptors containing fixed subunit stoichiometry, we coexpressed individual subunits with a “tandem” α1 subunit linked to a β2 subunit. Cotransfection of the γ2 subunit with αβ-tandem subunits in human embryonic kidney 293 cells produced currents that were similar in their macroscopic kinetics, single-channel amplitudes, and pharmacology to overexpression of the γ subunit with nonlinked α1 and β2 subunits. Similarly, expression of α subunits together with αβ-tandem subunits produced receptors having physiological and pharmacological characteristics that closely matched cotransfection of α with β subunits. In this first description of tandem GABAA subunits measured with patch-clamp and rapid agonist application techniques, we conclude that incorporation of αβ-tandem subunits can be used to fix stoichiometry and to establish the intrinsic kinetic properties of α1β2 and α1β2γ2 receptors. We used this method to test whether the accessory protein GABAA receptor-associated protein (GABARAP) alters GABAA receptor properties directly or influences subunit composition. In recombinant receptors with fixed stoichiometry, coexpression of GABARAP-enhanced green fluorescent protein (EGFP) fusion protein had no effect on desensitization, deactivation, or diazepam potentiation of GABA-mediated currents. However, in α1β2γ2S transfections in which stoichiometry was not fixed, GABARAP-EGFP altered desensitization, deactivation, and diazepam potentiation of GABA-mediated currents. The data suggest that GABARAP does not alter receptor kinetics directly but by facilitating surface expression of αβγ receptors. PMID:16339017

  18. Diverse voltage-sensitive dyes modulate GABAA receptor function

    PubMed Central

    Mennerick, Steven; Chisari, Mariangela; Shu, Hong-Jin; Taylor, Amanda; Vasek, Michael; Eisenman, Lawrence N.; Zorumski, Charles F.

    2010-01-01

    Voltage-sensitive dyes (VSDs) are important tools for assessing network and single-cell excitability, but an untested premise in most cases is that the dyes do not interfere with the parameters (membrane potential, excitability) that they are designed to measure. We found that popular members of several different families of voltage-sensitive dyes modulate GABAA receptor with maximum efficacy and potency similar to clinically used GABAA receptor modulators. Di-4-ANEPPS and DiBAC4(3) potentiated GABA function with micromolar and high nanomolar potency respectively and yielded strong maximum effects similar to barbiturates and neurosteroids. Newer blue oxonols had biphasic effects on GABAA receptor function at nanomolar and micromolar concentrations, with maximum potentiation comparable to that of saturating benzodiazepine effects. ANNINE 6 and ANNINE 6plus had no detectable effect on GABAA receptor function. Even dyes with no activity on GABAA receptors at baseline induced photodynamic enhancement of GABAA receptors. The basal effects of dyes were sufficient to prolong IPSCs and to dampen network activity in multielectrode array recordings. Therefore, the dual effects of voltage-sensitive dyes on GABAergic inhibition require caution in dye use for studies of excitability and network activity. PMID:20181584

  19. Mutations in GABAA receptor subunits associated with genetic epilepsies.

    PubMed

    Macdonald, Robert L; Kang, Jing-Qiong; Gallagher, Martin J

    2010-06-01

    Mutations in inhibitory GABAA receptor subunit genes (GABRA1, GABRB3, GABRG2 and GABRD) have been associated with genetic epilepsy syndromes including childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME), pure febrile seizures (FS), generalized epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome (DS)/severe myoclonic epilepsy in infancy (SMEI). These mutations are found in both translated and untranslated gene regions and have been shown to affect the GABAA receptors by altering receptor function and/or by impairing receptor biogenesis by multiple mechanisms including reducing subunit mRNA transcription or stability, impairing subunit folding, stability, or oligomerization and by inhibiting receptor trafficking. PMID:20308251

  20. 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

  1. Zolpidem is a potent stoichiometry-selective modulator of α1β3 GABAA receptors: evidence of a novel benzodiazepine site in the α1-α1 interface.

    PubMed

    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 Zn(2+). 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

  2. 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

  3. 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. PMID:27439531

  4. Effect of synthetic steroids on GABAA receptor binding in rat brain.

    PubMed

    Rey, M; Veleiro, A S; Ghini, A A; Kruse, M S; Burton, G; Coirini, H

    2015-04-01

    Neuroactive steroids, like allopregnanolone (A) and pregnanolone (P), bind to specifics sites on the GABAA receptor complex and modulate receptor function. They are capable to inhibit or stimulate the binding of GABAA receptor-specific ligands, like t-butyl-bicyclophosphorothionate, flunitrazepam and muscimol. We have previously characterized a set of oxygen-bridged synthetic steroids (SS) analogs to A or P using synaptosomes. Considering that the subunit composition of the GABAA receptor throughout the central nervous system affects the magnitude of the modulation of the GABAA receptor by NAS, we evaluated the action of two selected SS, in brain sections containing the cerebral cortex (CC) and hippocampus (HC) using quantitative receptor autoradiography. Both SS affected the binding of the three ligands in a similar way to A and P, with some differences on certain CC layers according to the ligand used. One of the SS, the 3α-hydroxy-6,19-epoxypregn-4-ene-20-one (compound 5), behaved similarly to the natural neuroactive steroids. However, significant differences with compound 5 were observed on the HC CA2 region, making it steroid suitable for a specific action. Those differences may be related to structural conformation of the SS and the subunits' composition present on the receptor complex. PMID:25617652

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. γ-Aminobutyric acid type A (GABA(A)) receptor subtype inverse agonists as therapeutic agents in cognition.

    PubMed

    Gabriella, Guerrini; Giovanna, Ciciani

    2010-01-01

    The gabaergic system has been identified as a relevant regulator of cognitive and emotional processing. In fact, the discovery that negative allosteric regulators (or inverse agonists) at GABA(A) (γ-aminobutyric acid) α5 subtype receptors improve learning and memory tasks, has further validated this concept. The localization of these extrasynaptic subtype receptors, mainly in the hippocampus, has suggested that they play a key role in the three stages of memory: acquisition, consolidation, and retrieval. The "α5 inverse agonist" binds to an allosteric site at GABA(A) receptor, provoking a reduction of chlorine current, but to elicit this effect, the necessary condition is the binding of agonist neurotransmitter (γ-amino butyric acid) at its orthosteric site. In this case, the GABA(A) receptor is not a "constitutively active receptor" and, however, the presence of spontaneous opening channels for native GABA(A) receptors is rare. Here, we present various classes of nonselective and α5 selective GABA(A) receptor ligands, and the in vitro and in vivo tests to elucidate their affinity and activity. The study of the GABA(A) α5 inverse agonists is one of the important tools, although not the only one, for the development of clinical strategies for treatment of Alzheimer disease and mild cognitive impairment. PMID:21050918

  11. Sex differences in effects of mild chronic stress on seizure risk and GABAA receptors in rats.

    PubMed

    Chadda, Ritu; Devaud, Leslie L

    2004-07-01

    Social stress is a common occurrence in our society that can negatively impact health. Therefore, we wanted to study the effects of a mild stressor designed to model social stress on seizure susceptibility and GABAA receptors in male and female rats. The mild chronic stress of individual housing consistently decreased bicuculline (but not pentylenetetrazol, PTZ) seizure thresholds by 10-15% in both sexes. Housing conditions did not alter the anticonvulsant activity of diazepam or ethanol, although the anticonvulsant effect of ethanol was significantly greater against PTZ-induced seizures. Experiments testing the addition of an acute restraint stress unmasked sex differences in seizure induction. The acute stress also selectively decreased the potency of GABA to modulate GABAA receptor-mediated chloride uptake in group-housed females. There were additional sex differences by housing condition for GABAA receptor-gated chloride uptake but no differences in [3H]flunitrazepam binding. We also found significant effects of sex and housing on ethanol-induced increases in corticosterone (CORT) levels. In summary, there were complex and sex-selective effects of mild chronic stress on seizure induction and GABAA receptors. Gaining a better understanding of mechanisms underlying interactions between sex and stress has important implications for addressing health concerns about stress in men and women. PMID:15251258

  12. The GABAA antagonist DPP-4-PIOL selectively antagonises tonic over phasic GABAergic currents in dentate gyrus granule cells.

    PubMed

    Boddum, Kim; Frølund, Bente; Kristiansen, Uffe

    2014-11-01

    GABAA receptors mediate two different types of inhibitory currents: phasic inhibitory currents when rapid and brief presynaptic GABA release activates postsynaptic GABAA receptors and tonic inhibitory currents generated by low extrasynaptic GABA levels, persistently activating extrasynaptic GABAA receptors. The two inhibitory current types are mediated by different subpopulations of GABAA receptors with diverse pharmacological profiles. Selective antagonism of tonic currents is of special interest as excessive tonic inhibition post-stroke has severe pathological consequences. Here we demonstrate that phasic and tonic GABAA receptor currents can be selectively inhibited by the antagonists SR 95531 and the 4-PIOL derivative, 4-(3,3-diphenylpropyl)-5-(4-piperidyl)-3-isoxazolol hydrobromide (DPP-4-PIOL), respectively. In dentate gyrus granule cells, SR 95531 was found approximately 4 times as potent inhibiting phasic currents compared to tonic currents (IC50 values: 101 vs. 427 nM). Conversely, DPP-4-PIOL was estimated to be more than 20 times as potent inhibiting tonic current compared to phasic current (IC50 values: 0.87 vs. 21.3 nM). Consequently, we were able to impose a pronounced reduction in tonic GABA mediated current (>70 %) by concentrations of DPP-4-PIOL, at which no significant effect on the phasic current was seen. Our findings demonstrate that selective inhibition of GABA mediated tonic current is possible, when targeting a subpopulation of GABAA receptors located extrasynaptically using the antagonist, DPP-4-PIOL. PMID:25103229

  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. Involvement of GABA(A) receptors in myoclonus.

    PubMed

    Matsumoto, R R; Truong, D D; Nguyen, K D; Dang, A T; Hoang, T T; Vo, P Q; Sandroni, P

    2000-01-01

    Alterations in multiple neurochemical systems have been reported in animal and human studies of posthypoxic myoclonus. It is impossible, however, to establish causative relationships between the observed changes and the myoclonic movements from these studies. Therefore, to establish causative links between neurochemical changes and myoclonus, ligands that target neurotransmitter systems that are altered in posthypoxic myoclonus were microinjected into the lateral ventricles of normal rats to identify the changes that can produce myoclonus. Of the ligands that were tested, only the GABA(A) antagonists produced myoclonus after intracerebroventricular administration, suggesting the importance of disinhibition of GABAergic systems in myoclonus. To further examine the role of GABA in myoclonus, GABAergic antagonists were microinjected into the nucleus reticularis of the thalamus (NRT), an area of the brain in which extensive pathologic changes are seen in posthypoxic animals. GABA(A), but not GABA(B), antagonists produced myoclonus after microinjection into the NRT. Earlier investigators have further reported the ability of GABA(A) antagonists to produce myoclonus after microinjection into the caudate. The data therefore suggest that disruption of activity at GABA(A) receptors at any one of a number of levels in the neural axis can produce myoclonus. PMID:10755272

  15. 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.

  16. 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

  17. GABAA receptor subtypes: the "one glass of wine" receptors.

    PubMed

    Olsen, Richard W; Hanchar, Harry J; Meera, Pratap; Wallner, Martin

    2007-05-01

    This review discusses evidence for and apparent controversy about, gamma-aminobutyric acid type A (GABAA) receptor (GABAAR) subtypes that mediate alcohol effects experienced during social drinking. GABAARs that contain the beta3 and delta subunits were shown to be enhanced by alcohol concentrations that mirror the concentration dependence of alcohol responses in humans. A mutation (alpha6R100Q) previously found in alcohol nontolerant rats in the cerebellar GABAAR alpha6 subunit is sufficient for increased alcohol-induced ataxia in rats homozygous for this mutation (alpha6-100QQ) and further increases alcohol sensitivity of tonic GABA currents (mediated by alpha6betadelta receptors) in cerebellar granule cells of alpha6-100QQ rats and in recombinant alpha6R100Qbeta3delta receptors. This provided the first direct evidence that these types of receptors mediate behavioral effects of ethanol. Furthermore, the behavioral alcohol antagonist Ro15-4513 specifically reverses ethanol enhancement on alpha4/6beta3delta receptors. Unexpectedly, native and recombinant alpha4/6beta3delta receptors bind the behavioral alcohol antagonist Ro15-4513 with high affinity and this binding is competitive with EtOH, suggesting a specific and mutually exclusive (competitive) ethanol/Ro15-4513 site, which explains the puzzling activity of Ro15-4513 as a behavioral alcohol antagonist. Our conclusion from these findings is that alcohol/Ro15-4513-sensitive GABAAR subtypes are important alcohol targets and that alcohol at relevant concentrations is more specific than previously thought. In this review, we discuss technical difficulties in expressing recombinant delta subunit-containing receptors in oocytes and mammalian cells that may have contributed to negative results and confusion. Not only because we have reproduced detailed positive results numerous times, and we and many others have built extensively on basic findings, but also because we explain and combine many previously puzzling

  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. Identification of a novel protein complex containing ASIC1a and GABAA receptors and their interregulation.

    PubMed

    Zhao, Dongbo; Ning, Nannan; Lei, Zhen; Sun, Hua; Wei, Chuanfei; Chen, Dawei; Li, Jingxin

    2014-01-01

    Acid-sensing ion channels (ASICs) belong to the family of the epithelial sodium channel/degenerin (ENaC/DEG) and are activated by extracellular protons. They are widely distributed within both the central and peripheral nervous systems. ASICs were modified by the activation of γ-aminobutyric acid receptors (GABAA), a ligand-gated chloride channels, in hippocampal neurons. In contrast, the activity of GABAA receptors were also modulated by extracellular pH. However so far, the mechanisms underlying this intermodulation remain obscure. We hypothesized that these two receptors-GABAA receptors and ASICs channels might form a novel protein complex and functionally interact with each other. In the study reported here, we found that ASICs were modified by the activation of GABAA receptors either in HEK293 cells following transient co-transfection of GABAA and ASIC1a or in primary cultured dorsal root ganglia (DRG) neurons. Conversely, activation of ASIC1a also modifies the GABAA receptor-channel kinetics. Immunoassays showed that both GABAA and ASIC1a proteins were co-immunoprecipitated mutually either in HEK293 cells co-transfected with GABAA and ASIC1a or in primary cultured DRG neurons. Our results indicate that putative GABAA and ASIC1a channels functionally interact with each other, possibly via an inter-molecular association by forming a novel protein complex. PMID:24923912

  20. Removal of GABAA Receptor γ2 Subunits from Parvalbumin Neurons Causes Wide-Ranging Behavioral Alterations

    PubMed Central

    Leppä, Elli; Linden, Anni-Maija; Vekovischeva, Olga Y.; Swinny, Jerome D.; Rantanen, Ville; Toppila, Esko; Höger, Harald; Sieghart, Werner; Wulff, Peer; Wisden, William; Korpi, Esa R.

    2011-01-01

    We investigated the behavioral significance of fast synaptic inhibition by αβγ2-type GABAA receptors on parvalbumin (Pv) cells. The GABAA receptor γ2 subunit gene was selectively inactivated in Pv-positive neurons by Cre/loxP recombination. The resulting Pv-Δγ2 mice were relatively healthy in the first postnatal weeks; but then as Cre started to be expressed, the mice progressively developed wide-ranging phenotypic alterations including low body weight, motor deficits and tremor, decreased anxiety levels, decreased pain sensitivity and deficient prepulse inhibition of the acoustic startle reflex and impaired spatial learning. Nevertheless, the deletion was not lethal, and mice did not show increased mortality even after one year. Autoradiography with t-butylbicyclophosphoro[35S]thionate suggested an increased amount of GABAA receptors with only α and β subunits in central nervous system regions that contained high levels of parvalbumin neurons. Using BAC-transgenesis, we reduced some of the Pv-Δγ2 phenotype by selectively re-expressing the wild-type γ2 subunit back into some Pv cells (reticular thalamic neurons and cerebellar Pv-positive neurons). This produced less severe impairments of motor skills and spatial learning compared with Pv-Δγ2 mice, but all other deficits remained. Our results reveal the widespread significance of fast GABAergic inhibition onto Pv-positive neurons for diverse behavioral modalities, such as motor coordination, sensorimotor integration, emotional behavior and nociception. PMID:21912668

  1. 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

  2. Zolpidem withdrawal induced uncoupling of GABA(A) receptors in vitro associated with altered GABA(A) receptor subunit mRNA expression.

    PubMed

    Jembrek, Maja Jazvinšćak; Vlainić, Josipa; Šuran, Jelena

    2015-01-01

    Hypnotic zolpidem produces its effects via the benzodiazepine binding site in α1-containing GABAA receptors. The aim of the study was to assess the influence of duration of zolpidem treatment and its withdrawal, as well as the role of alpha1-containing GABAA receptors in the development of physical dependence and tolerance. Namely, recombinant receptors can be used to characterize mechanisms involved in different processes in the brain and to delineate the contribution of specific receptor subtypes. To address the influence of chronic zolpidem treatment we exposed HEK293 cells stably expressing alpha1beta2gamma2S recombinant GABAA receptors for seven consecutive days, while withdrawal periods lasted for 24, 48, 72 and 96 hours. Using radioligand binding studies we determined that chronic zolpidem treatment did not induce changes in either GABAA receptor number or in the expression of subunit mRNAs. We observed the enhancement of binding sites and upregulated expression of subunit mRNAs only following 96-hour withdrawal. Moreover, zolpidem treatment and its withdrawal (All time points) induced functional uncoupling between GABA and benzodiazepine binding sites in the GABAA receptor complex. Accordingly, it might be assumed that zolpidem withdrawal-induced uncoupling of GABAA receptors is associated with altered GABAA receptor subunit mRNA expression. The results presented here provide an insight into molecular and cellular mechanisms probably underlying adaptive changes of GABAA receptor function in response to chronic usage and withdrawal of zolpidem and perhaps the observed molecular changes could be linked to the tolerance and dependence produced upon prolonged treatment with other GABAergic drugs. PMID:26232993

  3. Ionic Mechanisms of Neuronal Excitation by Inhibitory GABA_A Receptors

    NASA Astrophysics Data System (ADS)

    Staley, Kevin J.; Soldo, Brandi L.; Proctor, William R.

    1995-08-01

    Gamma-aminobutyric acid A (GABA_A) receptors are the principal mediators of synaptic inhibition, and yet when intensely activated, dendritic GABA_A receptors excite rather than inhibit neurons. The membrane depolarization mediated by GABA_A receptors is a result of the differential, activity-dependent collapse of the opposing concentration gradients of chloride and bicarbonate, the anions that permeate the GABA_A ionophore. Because this depolarization diminishes the voltage-dependent block of the N-methyl-D-aspartate (NMDA) receptor by magnesium, the activity-dependent depolarization mediated by GABA is sufficient to account for frequency modulation of synaptic NMDA receptor activation. Anionic gradient shifts may represent a mechanism whereby the rate and coherence of synaptic activity determine whether dendritic GABA_A receptor activation is excitatory or inhibitory.

  4. 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

  5. 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

  6. Hypothalamic oxytocin attenuates CRF expression via GABA(A) receptors in rats.

    PubMed

    Bülbül, Mehmet; Babygirija, Reji; Cerjak, Diana; Yoshimoto, Sazu; Ludwig, Kirk; Takahashi, Toku

    2011-04-28

    Centrally released oxytocin (OXT) has anxiolytic and anti-stress effects. Delayed gastric emptying (GE) induced by acute restraint stress (ARS) for 90 min is completely restored following 5 consecutive days of chronic homotypic restraint stress (CHS), via up-regulating hypothalamic OXT expression in rats. However, the mechanism behind the restoration of delayed GE following CHS remains unclear. Gamma-aminobutyric acid (GABA)-projecting neurons in the paraventricular nucleus (PVN) have been shown to inhibit corticotropin releasing factor (CRF) synthesis via GABA(A) receptors. We hypothesized that GABA(A) receptors are involved in mediating the inhibitory effect of OXT on CRF expression in the PVN, which in turn restores delayed GE following CHS. OXT (0.5 μg) and selective GABA(A) receptor antagonist, bicuculline methiodide (BMI) (100 ng), were administered intracerebroventricularly (icv). Solid GE was measured under non-stressed (NS), ARS and CHS conditions. Expression of CRF mRNA in the PVN was evaluated by real time RT-PCR. Neither OXT nor BMI changed GE and CRF mRNA expression under NS conditions. Delayed GE and increased CRF mRNA expression induced by ARS were restored by icv-injection of OXT. The effects of OXT on delayed GE and increased CRF mRNA expression in ARS were abolished by icv-injection of BMI. Following CHS, delayed GE was completely restored in saline (icv)-injected rats, whereas daily injection of BMI (icv) attenuated the restoration of delayed GE. Daily injection of BMI (icv) significantly increased CRF mRNA expression following CHS. It is suggested that central OXT inhibits ARS-induced CRF mRNA expression via GABA(A) receptors in the PVN. GABAergic system is also involved in OXT-mediated adaptation response of delayed GE under CHS conditions. PMID:21382355

  7. GABAA receptor complex function in frontal cortex membranes from control and neurological patients.

    PubMed

    Lloyd, G K; Lowenthal, A; Javoy-Agid, F; Constantidinis, J

    1991-05-01

    The functional integrity of the GABAA receptor-benzodiazepine (BZ) recognition site-Cl- ionophore complex was assessed by means of [35S]TBPS (t-butylbicyclophosphorothionate) binding to frontal cortex membranes prepared from frozen postmortem brain tissue taken from control (n = 4), Alzheimer (n = 7), Parkinson (n = 3) and Huntington's chorea (n = 2) patients. Specific [35S]TBPS binding was similar in control, Parkinson's disease and Huntington's chorea brains, but was significantly reduced (78% control, P less than 0.01) in frontal cortex membranes from Alzheimer's patients. The linkage between the BZ recognition sites and the GABAA receptor-linked Cl- ionophore was functionally intact in these membranes as BZ site agonists (zolpidem, alpidem, flunitrazepam and clonazepam) enhanced [35S]TBPS binding under the conditions used (well-washed membranes in the presence of 1.0 M NaCl). Zolpidem (BZ1 selective) exhibited a biphasic enhancement in control membranes whereas the other compounds induced a bell-shaped concentration-response curve. The enhancement of [35S]TBPS binding by alpidem, flunitrazepam and clonazepam was greater in frontal cortex membranes from Alzheimer's patients than in controls whereas it tended to be reduced in membranes from the brains of Huntington's chorea patients. These studies demonstrate the functional integrity of the GABAA receptor macromolecular complex and also the usefulness of [35S]TBPS binding in the study of human postmortem tissue. PMID:1654259

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

    PubMed

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

    2016-05-01

    The GABAA receptor is implicated in the pathophysiology of schizophrenia and regulated by PKA signalling. Current antipsychotics bind with D2-like receptors, but not the GABAA receptor. The cAMP-responsive element-binding protein 1 (CREB1) is also associated with PKA signalling and may be related to the positive symptoms of schizophrenia. This study investigated the effects of antipsychotics in modulating D2-mediated PKA signalling and its downstream GABAA receptors and CREB1. Rats were treated orally with aripiprazole (0.75 mg/kg, ter in die (t.i.d.)), bifeprunox (0.8 mg/kg, t.i.d.), haloperidol (0.1 mg/kg, t.i.d.) or vehicle for 1 week. The levels of PKA-Cα and p-PKA in the prefrontal cortex (PFC), nucleus accumbens (NAc) and caudate putamen (CPu) were detected by Western blots. The mRNA levels of Gabrb1, Gabrb2, Gabrb3 and Creb1, and their protein expression were measured by qRT-PCR and Western blots, respectively. Aripiprazole elevated the levels of p-PKA and the ratio of p-PKA/PKA in the NAc, but not the PFC and CPu. Correlated with this elevated PKA signalling, aripiprazole elevated the mRNA and protein expression of the GABAA (β-1) receptor and CREB1 in the NAc. While haloperidol elevated the levels of p-PKA and the ratio of p-PKA/PKA in both NAc and CPu, it only tended to increase the expression of the GABAA (β-1) receptor and CREB1 in the NAc, but not the CPu. Bifeprunox had no effects on PKA signalling in these brain regions. These results suggest that aripiprazole has selective effects on upregulating the GABAA (β-1) receptor and CREB1 in the NAc, probably via activating PKA signalling. PMID:26894264

  9. 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

  10. 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

  11. Neurosteroid Agonist at GABAA Receptor Induces Persistent Neuroplasticity in VTA Dopamine Neurons

    PubMed Central

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

    2014-01-01

    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

  12. 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

  13. Brain regional distribution of GABA(A) receptors exhibiting atypical GABA agonism: roles of receptor subunits.

    PubMed

    Halonen, Lauri M; Sinkkonen, Saku T; Chandra, Dev; Homanics, Gregg E; Korpi, Esa R

    2009-11-01

    The major inhibitory neurotransmitter in the brain, gamma-aminobutyric acid (GABA), has only partial efficacy at certain subtypes of GABA(A) receptors. To characterize these minor receptor populations in rat and mouse brains, we used autoradiographic imaging of t-butylbicyclophosphoro[(35)S]thionate ([(35)S]TBPS) binding to GABA(A) receptors in brain sections and compared the displacing capacities of 10mM GABA and 1mM 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a competitive GABA-site agonist. Brains from GABA(A) receptor alpha1, alpha4, delta, and alpha4+delta subunit knockout (KO) mouse lines were used to understand the contribution of these particular receptor subunits to "GABA-insensitive" (GIS) [(35)S]TBPS binding. THIP displaced more [(35)S]TBPS binding than GABA in several brain regions, indicating that THIP also inhibited GIS-binding. In these regions, GABA prevented the effect of THIP on GIS-binding. GIS-binding was increased in the cerebellar granule cell layer of delta KO and alpha4+delta KO mice, being only slightly diminished in that of alpha1 KO mice. In the thalamus and some other forebrain regions of wild-type mice, a significant amount of GIS-binding was detected. This GIS-binding was higher in alpha4 KO mice. However, it was fully abolished in alpha1 KO mice, indicating that the alpha1 subunit was obligatory for the GIS-binding in the forebrain. Our results suggest that native GABA(A) receptors in brain sections showing reduced displacing capacity of [(35)S]TBPS binding by GABA (partial agonism) minimally require the assembly of alpha1 and beta subunits in the forebrain and of alpha6 and beta subunits in the cerebellar granule cell layer. These receptors may function as extrasynaptic GABA(A) receptors. PMID:19397945

  14. Brain regional distribution of GABAA receptors exhibiting atypical GABA agonism: roles of receptor subunits

    PubMed Central

    Halonen, Lauri M.; Sinkkonen, Saku T.; Chandra, Dev; Homanics, Gregg E.; Korpi, Esa R.

    2009-01-01

    The major inhibitory neurotransmitter in the brain, γ-aminobutyric acid (GABA), has only partial efficacy at certain subtypes of GABAA receptors. To characterize these minor receptor populations in rat and mouse brains, we used autoradiographic imaging of t-butylbicyclophosphoro[35S]thionate ([35S]TBPS) binding to GABAA receptors in brain sections and compared the displacing capacities of 10 mM GABA and 1 mM 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a competitive GABA-site agonist. Brains from GABAA receptor α1, α4, δ, and α4 + δ subunit knockout (KO) mouse lines were used to understand the contribution of these particular receptor subunits to “GABA-insensitive” (GIS) [35S]TBPS binding. THIP displaced more [35S]TBPS binding than GABA in several brain regions, indicating that THIP also inhibited GIS-binding. In these regions, GABA prevented the effect of THIP on GIS-binding. GIS-binding was increased in the cerebellar granule cell layer of δ KO and α4 + δ KO mice, being only slightly diminished in that of α1 KO mice. In the thalamus and some other forebrain regions of wild-type mice, a significant amount of GIS-binding was detected. This GIS-binding was higher in α4 KO mice. However, it was fully abolished in α1 KO mice, indicating that the α1 subunit was obligatory for the GIS-binding in the forebrain. Our results suggest that native GABAA receptors in brain sections showing reduced displacing capacity of [35S]TBPS binding by GABA (partial agonism) minimally require the assembly of α1 and β subunits in the forebrain and of α6 and β subunits in the cerebellar granule cell layer. These receptors may function as extrasynaptic GABAA receptors. PMID:19397945

  15. 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...

  16. Souroubea sympetala (Marcgraviaceae): a medicinal plant that exerts anxiolysis through interaction with the GABAA benzodiazepine receptor.

    PubMed

    Mullally, Martha; Cayer, Christian; Kramp, Kari; Otárola Rojas, Marco; Sanchez Vindas, Pablo; Garcia, Mario; Poveda Alvarez, Luis; Durst, Tony; Merali, Zul; Trudeau, Vance L; Arnason, John T

    2014-09-01

    The mode of action of the anxiolytic medicinal plant Souroubea sympetala was investigated to test the hypothesis that extracts and the active principle act at the pharmacologically important GABAA-benzodiazepine (GABAA-BZD) receptor. Leaf extracts prepared by ethyl acetate extraction or supercritical extraction, previously determined to have 5.54 mg/g and 6.78 mg/g of the active principle, betulinic acid, respectively, reduced behavioural parameters associated with anxiety in a rat model. When animals were pretreated with the GABAA-BZD receptor antagonist flumazenil, followed by the plant extracts, or a more soluble derivative of the active principle, the methyl ester of betulinic acid (MeBA), flumazenil eliminated the anxiety-reducing effect of plant extracts and MeBA, demonstrating that S. sympetala acts via an agonist action on the GABAA-BZD receptor. An in vitro GABAA-BZD competitive receptor binding assay also demonstrated that S. sympetala extracts have an affinity for the GABAA-BZD receptor, with an EC50 value of 123 μg/mL (EtOAc leaf extract) and 154 μg/mL (supercritical CO2 extract). These experiments indicate that S. sympetala acts at the GABAA-BZD receptor to elicit anxiolysis. PMID:25140794

  17. Identification of the sites for CaMK-II-dependent phosphorylation of GABA(A) receptors.

    PubMed

    Houston, Catriona M; Lee, Henry H C; Hosie, Alastair M; Moss, Stephen J; Smart, Trevor G

    2007-06-15

    Phosphorylation can affect both the function and trafficking of GABA(A) receptors with significant consequences for neuronal excitability. Serine/threonine kinases can phosphorylate the intracellular loops between M3-4 of GABA(A) receptor beta and gamma subunits thereby modulating receptor function in heterologous expression systems and in neurons (1, 2). Specifically, CaMK-II has been demonstrated to phosphorylate the M3-4 loop of GABA(A) receptor subunits expressed as GST fusion proteins (3, 4). It also increases the amplitude of GABA(A) receptor-mediated currents in a number of neuronal cell types (5-7). To identify which substrate sites CaMK-II might phosphorylate and the consequent functional effects, we expressed recombinant GABA(A) receptors in NG108-15 cells, which have previously been shown to support CaMK-II modulation of GABA(A) receptors containing the beta3 subunit (8). We now demonstrate that CaMK-II mediates its effects on alpha1beta3 receptors via phosphorylation of Ser(383) within the M3-4 domain of the beta subunit. Ablation of beta3 subunit phosphorylation sites for CaMK-II revealed that for alphabetagamma receptors, CaMK-II has a residual effect on GABA currents that is not mediated by previously identified sites of CaMK-II phosphorylation. This residual effect is abolished by mutation of tyrosine phosphorylation sites, Tyr(365) and Tyr(367), on the gamma2S subunit, and by the tyrosine kinase inhibitor genistein. These results suggested that CaMK-II is capable of directly phosphorylating GABA(A) receptors and activating endogenous tyrosine kinases to phosphorylate the gamma2 subunit in NG108-15 cells. These findings were confirmed in a neuronal environment by expressing recombinant GABA(A) receptors in cerebellar granule neurons. PMID:17442679

  18. 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

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

    PubMed

    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

  20. pH-dependent inhibition of native GABAA receptors by HEPES

    PubMed Central

    Hugel, S; Kadiri, N; Rodeau, JL; Gaillard, S; Schlichter, R

    2012-01-01

    BACKGROUND AND PURPOSE Artificial buffers such as HEPES are extensively used to control extracellular pH (pHe) to investigate the effect of H+ ions on GABAA receptor function. EXPERIMENTAL APPROACH In neurones cultured from spinal cord dorsal horn (DH), dorsal root ganglia (DRG) and cerebellar granule cells (GC) of neonatal rats, we studied the effect of pHe on currents induced by GABAA receptor agonists, controlling pHe with HCO3- or different concentrations of HEPES. KEY RESULTS Changing HEPES concentration from 1 to 20 mM at constant pHe strongly inhibited the currents induced by submaximal GABA applications, but not those induced by glycine or glutamate, on DH, DRG or GC neurones, increasing twofold the EC50 for GABA in DH neurones and GC. Submaximal GABAA receptor-mediated currents were also inhibited by piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 3-(N-morpholino)propanesulfonic acid, tris(hydroxymethyl)aminomethane or imidazole. PIPES and HEPES, both piperazine derivatives, similarly inhibited GABAA receptors, whereas the other buffers had weaker effects and 2-(N-morpholino)ethanesulfonic acid had no effect. HEPES-induced inhibition of submaximal GABAA receptor-mediated currents was unaffected by diethylpyrocarbonate, a histidine-modifying reagent. HEPES-induced inhibition of GABAA receptors was independent of membrane potential, HCO3- and intracellular Cl- concentration and was not modified by flumazenil, which blocks the benzodiazepine binding site. However, it strongly depended on pHe. CONCLUSIONS AND IMPLICATIONS Inhibition of GABAA receptors by HEPES depended on pHe, leading to an apparent H+-induced inhibition of DH GABAA receptors, unrelated to the pH sensitivity of these receptors in both low and physiological buffering conditions, suggesting that protonated HEPES caused this inhibition. PMID:22452286

  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. 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

  3. Regulation of Action Potential Waveforms by Axonal GABAA Receptors in Cortical Pyramidal Neurons

    PubMed Central

    Xia, Yang; Zhao, Yuan; Yang, Mingpo; Zeng, Shaoqun; Shu, Yousheng

    2014-01-01

    GABAA receptors distributed in somatodendritic compartments play critical roles in regulating neuronal activities, including spike timing and firing pattern; however, the properties and functions of GABAA receptors at the axon are still poorly understood. By recording from the cut end (bleb) of the main axon trunk of layer –5 pyramidal neurons in prefrontal cortical slices, we found that currents evoked by GABA iontophoresis could be blocked by picrotoxin, indicating the expression of GABAA receptors in axons. Stationary noise analysis revealed that single-channel properties of axonal GABAA receptors were similar to those of somatic receptors. Perforated patch recording with gramicidin revealed that the reversal potential of the GABA response was more negative than the resting membrane potential at the axon trunk, suggesting that GABA may hyperpolarize the axonal membrane potential. Further experiments demonstrated that the activation of axonal GABAA receptors regulated the amplitude and duration of action potentials (APs) and decreased the AP-induced Ca2+ transients at the axon. Together, our results indicate that the waveform of axonal APs and the downstream Ca2+ signals are modulated by axonal GABAA receptors. PMID:24971996

  4. Stimulation of TM3 Leydig cell proliferation via GABAA receptors: A new role for testicular GABA

    PubMed Central

    Geigerseder, Christof; Doepner, Richard FG; Thalhammer, Andrea; Krieger, Annette; Mayerhofer, Artur

    2004-01-01

    The neurotransmitter gamma-aminobutyric acid (GABA) and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD), as well as GABAA and GABAB receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABAA receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABAA receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABAA receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA) we demonstrated that GABA or the GABAA agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABAA antagonist bicuculline, implying a role for GABAA receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABAA receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment. PMID:15040802

  5. Molecular Mechanisms of Antiseizure Drug Activity at GABAA Receptors

    PubMed Central

    Greenfield, L. John

    2013-01-01

    The GABAA receptor (GABAAR) is a major target of antiseizure drugs (ASDs). A variety of agents that act at GABAARs s are used to terminate or prevent seizures. Many act at distinct receptor sites determined by the subunit composition of the holoreceptor. For the benzodiazepines, barbiturates, and loreclezole, actions at the GABAAR are the primary or only known mechanism of antiseizure action. For topiramate, felbamate, retigabine, losigamone and stiripentol, GABAAR modulation is one of several possible antiseizure mechanisms. Allopregnanolone, a progesterone metabolite that enhances GABAAR function, led to the development of ganaxolone. Other agents modulate GABAergic “tone” by regulating the synthesis, transport or breakdown of GABA. GABAAR efficacy is also affected by the transmembrane chloride gradient, which changes during development and in chronic epilepsy. This may provide an additional target for “GABAergic” ASDs. GABAAR subunit changes occur both acutely during status epilepticus and in chronic epilepsy, which alter both intrinsic GABAAR function and the response to GABAAR-acting ASDs. Manipulation of subunit expression patterns or novel ASDs targeting the altered receptors may provide a novel approach for seizure prevention. PMID:23683707

  6. 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

  7. 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

  8. Extrasynaptic α6 subunit-containing GABAA receptors modulate excitability in turtle spinal motoneurons.

    PubMed

    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

  9. Ethanol activation of protein kinase A regulates GABAA α1 receptor function and trafficking in cultured cerebral cortical neurons.

    PubMed

    Carlson, Stephen L; Kumar, Sandeep; Werner, David F; Comerford, Christopher E; Morrow, A Leslie

    2013-05-01

    Ethanol exposure produces alterations in GABAergic signaling that are associated with dependence and withdrawal. Previously, we demonstrated that ethanol-induced protein kinase C (PKC) γ signaling selectively contributes to changes in GABAA α1 synaptic receptor activity and surface expression. Here, we demonstrate that protein kinase A (PKA) exerts opposing effects on GABAA receptor adaptations during brief ethanol exposure. Cerebral cortical neurons from day 0-1 rat pups were tested after 18 days in culture. Receptor trafficking was assessed by Western blot analysis, and functional changes were measured using whole-cell patch-clamp recordings of evoked and miniature inhibitory postsynaptic current (mIPSC) responses. One-hour ethanol exposure increased membrane-associated PKC and PKA, but steady-state GABAA α1 subunit levels were maintained. Activation of PKA by Sp-adenosine 3',5'-cyclic monophosphothioate triethylamine alone increased GABAA α1 subunit surface expression and zolpidem potentiation of GABA responses, whereas coexposure of ethanol with the PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine decreased α1 subunit expression and zolpidem responses. Exposure to the PKC inhibitor calphostin-C with ethanol mimicked the effect of direct PKA activation. The effects of PKA modulation on mIPSC decay τ were consistent with its effects on GABA currents evoked in the presence of zolpidem. Overall, the results suggest that PKA acts in opposition to PKC on α1-containing GABAA receptors, mediating the GABAergic effects of ethanol exposure, and may provide an important target for the treatment of alcohol dependence/withdrawal. PMID:23408117

  10. A network of autism linked genes stabilizes two pools of synaptic GABA(A) receptors.

    PubMed

    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 GABA(A) receptors are stabilized by distinct synaptic scaffolds at C. elegans neuromuscular junctions. Immobilized GABA(A) receptors are stabilized by binding to FRM-3/EPB4.1 and LIN-2A/CASK. Diffusing GABA(A) 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. PMID:26575289

  11. 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

  12. 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

  13. Ethanol reduces GABAA alpha1 subunit receptor surface expression by a protein kinase Cgamma-dependent mechanism in cultured cerebral cortical neurons.

    PubMed

    Kumar, Sandeep; Suryanarayanan, Asha; Boyd, Kevin N; Comerford, Chris E; Lai, Marvin A; Ren, Qinglu; Morrow, A Leslie

    2010-05-01

    Prolonged ethanol exposure causes central nervous system hyperexcitability that involves a loss of GABAergic inhibition. We previously demonstrated that long-term ethanol exposure enhances the internalization of synaptic GABA(A) receptors composed of alpha1beta2/3gamma2 subunits. However, the mechanisms of ethanol-mediated internalization are unknown. This study explored the effect of ethanol on surface expression of GABA(A) alpha1 subunit-containing receptors in cultured cerebral cortical neurons and the role of protein kinase C (PKC) beta, gamma, and epsilon isoforms in their trafficking. Cultured neurons were prepared from rat pups on postnatal day 1 and maintained for 18 days. Cells were exposed to ethanol, and surface receptors were isolated by biotinylation and P2 fractionation, whereas functional analysis was conducted by whole-cell patch-clamp recording of GABA- and zolpidem-evoked responses. Ethanol exposure for 4 h decreased biotinylated surface expression of GABA(A) receptor alpha1 subunits and reduced zolpidem (100 nM) enhancement of GABA-evoked currents. The PKC activator phorbol-12,13-dibutyrate mimicked the effect of ethanol, and the selective PKC inhibitor calphostin C prevented ethanol-induced internalization of these receptors. Ethanol exposure for 4 h also increased the colocalization and coimmunoprecipitation of PKCgamma with alpha1 subunits, whereas PKCbeta/alpha1 association and PKCepsilon/alpha1 colocalization were not altered by ethanol exposure. Selective PKCgamma inhibition by transfection of selective PKCgamma small interfering RNAs blocked ethanol-induced internalization of GABA(A) receptor alpha1 subunits, whereas PKCbeta inhibition using pseudo-PKCbeta had no effect. These findings suggest that ethanol exposure selectively alters PKCgamma translocation to GABA(A) receptors and PKCgamma regulates GABA(A) alpha1 receptor trafficking after ethanol exposure. PMID:20159950

  14. Development of ligands at γ-aminobutyrric acid type A (GABAA) receptor subtype as new agents for pain relief.

    PubMed

    Guerrini, Gabriella; Ciciani, Giovanna; Bruni, Fabrizio; Selleri, Silvia; Martini, Claudia; Daniele, Simona; Ghelardini, Carla; Di Cesare Mannelli, Lorenzo; Costanzo, Annarella

    2011-12-15

    The identification of compounds with selective anxiolytic-like effects, exerted through the benzodiazepine site on γ-aminobutyric acid type A (GABA(A)) receptors, and that show pronounced antihyperalgesia in several pain models, has oriented research towards the development of new agents for the relief of pain. Starting from our previously reported ligands at the benzodiazepine site on GABA(A) receptors showing selective anxiolytic-like effects, we have designed new compounds with the aim of identifying those devoid of the typical side effects of the classical benzodiazepines. Our preliminary results indicate that compounds 4, 10(±) and 11 have a very promising antihyperalgesic profile in different animal pain models (peripheral mono-neuropathy, STZ-induced hyperalgesia). In particular 11 exhibits high potency since it exerted its protective effect starting from the dose of 3mg/kg po, after single injection. PMID:22094278

  15. Benzodiazepine-site pharmacology on GABAA receptors in histaminergic neurons

    PubMed Central

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

    2013-01-01

    Background and Purpose The histaminergic tuberomamillary nucleus (TMN) of the posterior hypothalamus controls the cognitive aspects of vigilance which is reduced by common sedatives and anxiolytics. The receptors targeted by these drugs in histaminergic neurons are unknown. TMN neurons express nine different subunits of the GABAA receptor (GABAAR) with three α- (α1, α2 and α5) and two γ- (γ1, γ 2) subunits, which confer different pharmacologies of the benzodiazepine-binding site. Experimental Approach We investigated the actions of zolpidem, midazolam, diazepam, chlordiazepoxide, flumazenil (Ro15-1788) and methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) in TMN neurons using mouse genetics, electrophysiological and molecular biological methods. Key Results We find the sensitivity of GABAAR to zolpidem, midazolam and DMCM significantly reduced in TMN neurons from γ2F77I mice, but modulatory activities of diazepam, chlordiazepoxide and flumazenil not affected. Potencies and efficacies of these compounds are in line with the dominance of α2- and α1-subunit containing receptors associated with γ2- or γ1-subunits. Functional expression of the γ1-subunit is supported by siRNA-based knock-down experiments in γ2F77I mice. Conclusions and Implications GABAAR of TMN neurons respond to a variety of common sedatives with a high affinity binding site (γ2F77I) involved. The γ1-subunit likely contributes to the action of common sedatives in TMN neurons. This study is relevant for understanding the role of neuronal histamine and benzodiazepines in disorders of sleep and metabolism. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2013.170.issue-1 PMID:23799902

  16. Insights into structure-activity relationship of GABAA receptor modulating coumarins and furanocoumarins.

    PubMed

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

    2011-10-01

    The coumarins imperatorin and osthole are known to exert anticonvulsant activity. We have therefore analyzed the modulation of GABA-induced chloride currents (I(GABA)) by a selection of 18 coumarin derivatives on recombinant α(1)β(2)γ(2S) GABA(A) receptors expressed in Xenopus laevis oocytes by means of the two-microelectrode voltage clamp technique. Osthole (EC(50)=14 ± 1 μM) and oxypeucedanin (EC(50)=25 ± 8 μM) displayed the highest efficiency with I(GABA) potentiation of 116 ± 4 % and 547 ± 56 %, respectively. I(GABA) enhancement by osthole and oxypeucedanin was not inhibited by flumazenil (1 μM) indicating an interaction with a binding site distinct from the benzodiazepine binding site. In general, prenyl residues are essential for the positive modulatory activity, while longer side chains or bulkier residues (e.g. geranyl residues) diminish I(GABA) modulation. Generation of a binary classification tree revealed the importance of polarisability, which is sufficient to distinguish actives from inactives. A 4-point pharmacophore model based on oxypeucedanin - comprising three hydrophobic and one aromatic feature - identified 6 out of 7 actives as hits. In summary, (oxy-)prenylated coumarin derivatives from natural origin represent new GABA(A) receptor modulators. PMID:21749864

  17. 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

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

    PubMed

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

    2009-05-01

    The research described here investigates the role played by inhibitory processes in the discriminations made by the nervous system of humans and animals between familiar and unfamiliar and significant and nonsignificant events. This research compared the effects of two inhibitory mediators of gamma-aminobutyric acid (GABA): 1) phenibut, a nonselective agonist of ionotropic GABA(A) and metabotropic GABA(B) receptors and 2) gaboxadol a selective agonist of ionotropic GABA(A) receptors on the process of developing active defensive and inhibitory conditioned reflexes in alert non-immobilized rabbits. It was found that phenibut, but not gaboxadol, accelerates the development of defensive reflexes at an early stage of conditioning. Both phenibut and gaboxadol facilitate the development of conditioned inhibition, but the effect of gaboxadol occurs at later stages of conditioning and is less stable than that of phenibut. The earlier and more stable effects of phenibut, as compared to gaboxadol, on storage in memory of the inhibitory significance of a stimulus may occur because GABA(B) receptors play the dominant role in the development of internal inhibition during an early stage of conditioning. On the other hand this may occur because the participation of both GABA(A) and GABA(B) receptors are essential to the process. We discuss the polyfunctionality of GABA receptors as a function of their structure and the positions of the relevant neurons in the brain as this factor can affect regulation of various types of psychological processes. PMID:19476215

  19. 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. PMID:24244421

  20. γ-Hydroxybutyric Acid (GHB) Is Not an Agonist of Extrasynaptic GABAA Receptors

    PubMed Central

    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. PMID:24244421

  1. 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

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

    PubMed

    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

  3. 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

  4. GABAA Receptor Subtypes: the ‘One Glass of Wine’ Receptors

    PubMed Central

    Olsen, Richard W.; Hanchar, Harry J.; Meera, Pratap; Wallner, Martin

    2010-01-01

    This review discusses evidence for and apparent controversy about, GABAA receptor subtypes that mediate alcohol effects experienced during social drinking. GABAA receptors that contain the β3 and δ subunits were shown to be enhanced by alcohol concentrations that mirror the concentration-dependence of alcohol responses in humans. A mutation (α6R100Q) previously found in alcohol non-tolerant (ANT) rats in the cerebellar GABAA receptor α6 subunit is sufficient for increased alcohol-induced ataxia in rats homozygous for this mutation (α6-100QQ) and further increases alcohol-sensitivity of tonic GABA currents (mediated by α6βδ receptors) in cerebellar granule cells of α6-100QQ rats and in recombinant α6R100Qβ3δ receptors. This provided the first direct evidence that these types of receptors mediate behavioral effects of ethanol. Furthermore the behavioral alcohol antagonist Ro15-4513 specifically reverses ethanol enhancement on α4/6β3δ receptors. Unexpectedly, native and recombinant α4/6β3δ receptors bind the behavioral alcohol antagonist Ro15-4513 with high affinity and this binding is competitive with EtOH, suggesting a specific and mutually exclusive (competitive) ethanol/Ro15-4513 site which explains the puzzling activity of Ro15-4513 as a behavioral alcohol antagonist. Our conclusion from these findings is that alcohol/Ro15-4513-sensitive GABAA receptor subtypes are important alcohol targets and that alcohol at relevant concentrations is more specific than previously thought. In this review we discuss technical difficulties in expressing recombinant δ subunit-containing receptors in oocytes and mammalian cells, that may have contributed to negative results and confusion. Not only because we have reproduced detailed positive results numerous times, and we and many others have built extensively on basic findings, but also because we explain and combine many previously puzzling results into a coherent and highly plausible paradigm on how alcohol

  5. Amygdala-specific reduction of alpha1-GABAA receptors disrupts the anticonvulsant, locomotor, and sedative, but not anxiolytic, effects of benzodiazepines in mice.

    PubMed

    Heldt, Scott A; Ressler, Kerry J

    2010-05-26

    The heterogeneity and distribution of GABA(A) 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 GABA(A) receptor subtypes in particular brain regions is less clear. Such detailed knowledge of regional alpha1-GABA(A) receptor function will advance our understanding of the neural circuitry underlying the role of GABA(A) receptors and the effects of GABA(A)-modulating drugs on behavior. By combining inducible, site-specific alpha1 subunit deletion, using a lentivirus expressing Cre-recombinase in mice with the alpha1 subunit gene flanked by loxP sites, we examine baseline and pharmacological effects of deletion of amygdala alpha1-GABA(A) receptors. We find that amygdala-specific reduction of alpha1 receptor subunits does not affect mRNA or protein levels of amygdala alpha2 or alpha3 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 BZ-site ligands, diazepam and zolpidem, which is relatively alpha1-subunit selective. These data, using inducible, region and subunit-specific deletion, combined with pharmacogenetic approaches, demonstrate that amygdala expression of the alpha1-GABA(A) receptor subunit is required for normal BZ effects on sedation, locomotion, and seizure inhibition, but not for anxiolysis. PMID:20505082

  6. Quantitation of GABAA receptor inhibition required for quinolone-induced convulsions in mice.

    PubMed

    Tsutomi, Y; Matsubayashi, K; Akahane, K

    1994-11-01

    We quantified the amount of inhibition of gamma-aminobutyric acid (GABA)A receptor binding required for the onset of convulsions induced by ciprofloxacin in combination with biphenylacetic acid (BPAA) in mice. In fasting mice iv ciprofloxacin given 30 min after oral BPAA (50 mg/kg) induced convulsions at doses of 40 mg/kg or above. In contrast, ofloxacin caused no convulsions even at 100 mg/kg, the highest dose tested. When mice received 40 mg/kg of ciprofloxacin or ofloxacin, maximal brain concentrations of each quinolone at 30 min were 0.37 or 1.97 micrograms/g, respectively. These brain concentrations of ciprofloxacin and ofloxacin were not affected by combination with BPAA. In the presence of ciprofloxacin and BPAA (at brain tissue concentrations which induced convulsions), the binding of 3H-muscimol to GABAA receptor sites was inhibited by approximately 30%. Using results from a similar binding study, an impracticable iv dose of ofloxacin (500 mg/kg) was estimated to be required to inhibit GABAA receptor binding by 30%, and therefore to induce similar convulsions to those seen with ciprofloxacin at a dose of 40 mg/kg. These results may indicate that epileptic convulsions occur when ciprofloxacin and BPAA interact with each other to antagonize at least 30% of GABAA receptor binding in mice, and provide evidence for a significant role of GABAA receptor inhibition in the occurrence of quinolone-induced convulsions. PMID:7706169

  7. GABAA Receptor Expression in the Forebrain of Ataxic Rolling Nagoya Mice.

    PubMed

    Nielsen, Elsebet Østergaard; Kaja, Simon

    2014-01-01

    The human CACNA1A gene encodes the pore-forming α1 subunit of CaV2.1 (P/Q-type) calcium channels and is the locus for several neurological disorders, including episodic ataxia type 2 (EA2), spinocerebellar ataxia type 6 (SCA6) and Familial Hemiplegic Migraine type 1 (FHM1). Several spontaneous mouse Cacna1a mutant strains exist, among them Rolling Nagoya (tg (rol)), carrying the R1262G point mutation in the mouse Cacna1a gene. tg (rol) mice display a phenotype of severe gait ataxia and motor dysfunction of the hind limbs. At the functional level, the R1262G mutation results in a positive shift of the activation voltage of the CaV2.1 channel and reduced current density. γ-Aminobutyric acid type A (GABAA) receptor subunit expression depends critically on neuronal calcium influx, and GABAA receptor dysfunction has previously been described for the cerebellum of tg (rol) and other ataxic Cacna1a mutant mice. Given the expression pattern of CaV2.1, it was hypothesized that calcium dysregulation in tg (rol) might affect GABAA receptor expression in the forebrain. Herein, functional GABAA receptors in the forebrain of tg (rol) mice were quantified and pharmacologically dissociated using [(3)H] radioligand binding. No gross changes to functional GABAA receptors were identified. Future cell type-specific analyses are required to identify possible cortical contributions to the psychomotor phenotype of tg (rol) mice. PMID:25309056

  8. Dysfunction of GABAA receptor glycolysis-dependent modulation in human partial epilepsy

    PubMed Central

    Laschet, Jacques J.; Kurcewicz, Irène; Minier, Frédéric; Trottier, Suzanne; Khallou-Laschet, Jamila; Louvel, Jacques; Gigout, Sylvain; Turak, Baris; Biraben, Arnaud; Scarabin, Jean-Marie; Devaux, Bertrand; Chauvel, Patrick; Pumain, René

    2007-01-01

    A reduction in GABAergic neurotransmission has been put forward as a pathophysiological mechanism for human epilepsy. However, in slices of human epileptogenic neocortex, GABAergic inhibition can be clearly demonstrated. In this article we present data showing an increase in the functional lability of GABAergic inhibition in epileptogenic tissue compared with nonepileptogenic human tissue. We have previously shown that the glycolytic enzyme GAPDH is the kinase involved in the glycolysis-dependent endogenous phosphorylation of the α1-subunit of GABAA receptor, a mechanism necessary for maintaining GABAA function. In human epileptogenic cortex obtained during curative surgery of patients with partial seizures, we demonstrate an intrinsic deficiency of GABAA receptor endogenous phosphorylation resulting in an increased lability of GABAergic currents in neurons isolated from this tissue when compared with neurons from nonepileptogenic human tissue. This feature was not related to a reduction in the number of GABAA receptor α1-subunits in the epileptogenic tissue as measured by [3H]flunitrazepam photoaffinity labeling. Maintaining the receptor in a phosphorylated state either by favoring the endogenous phosphorylation or by inhibiting a membrane-associated phosphatase is needed to sustain GABAA receptor responses in epileptogenic cortex. The increased functional lability induced by the deficiency in phosphorylation can account for transient GABAergic disinhibition favoring seizure initiation and propagation. These findings imply new therapeutic approaches and suggest a functional link to the regional cerebral glucose hypometabolism observed in patients with partial epilepsy, because the dysfunctional GABAergic mechanism depends on the locally produced glycolytic ATP. PMID:17360668

  9. 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

  10. 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.

  11. 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

  12. GABAA Receptor-Modulating Steroids in Relation to Women's Behavioral Health.

    PubMed

    Bäckström, Torbjörn; Bixo, Marie; Strömberg, Jessica

    2015-11-01

    In certain women, increased negative mood relates to the progesterone metabolite, allopregnanolone (allo), during the luteal phase of ovulatory menstrual cycles, the premenstrual dysphoric disorder (PMDD). In anovulatory cycles, no symptom or sex steroid increase occurs but symptoms return during progesterone/allo treatment. Allo is a potent GABAA receptor-modulating steroid and as such is expected to be calming and anxiolytic. A relation to negative mood is unexpected. However, this paradoxical effect can be induced by all GABAA receptor modulators in low concentrations whereas higher concentrations are calming. The severity of the mood symptoms relate to allo in an inverted U-shaped curve at endogenous luteal-phase serum concentrations. Allo's effects on the GABAA receptor can be antagonized by isoallopregnanolone (ISO), an antagonist to allo. ISO has also been used in a preliminary clinical trial on PMDD ameliorating symptoms with good effect in PMDD patients. PMID:26396092

  13. AMPA receptors serum-dependently mediate GABAA receptor alpha1 and alpha6 subunit down-regulation in cultured mouse cerebellar granule cells.

    PubMed

    Uusi-Oukari, Mikko; Kontturi, Leena-Stiina; Kallinen, Sampsa A; Salonen, Virpi

    2010-04-01

    Depolarization of cultured mouse cerebellar granule cells with potassium or kainate results in developmentally arrested state that includes down-regulation of GABA(A) receptor alpha1, alpha6 and beta2 subunit expression. These subunits are normally strongly expressed in cerebellar granule cells from second postnatal week throughout the adulthood. In the present study we demonstrate that selective activation of AMPA subtype of glutamate receptors down-regulates alpha1 and alpha6 subunit mRNA expression. Removal of AMPA agonist from culture medium restores expression of these subunits indicating reversibility of the down-regulation. In serum-free culture medium AMPA receptor activation did not down-regulate alpha1 or alpha6 subunit expression. Furthermore, the down-regulation was strongly attenuated when the cells were cultured in the presence of dialysed fetal calf serum. The results indicate that down-regulation of GABA(A) receptor alpha1 and alpha6 subunits by AMPA receptor activation is dependent on the presence of low molecular weight compounds present in fetal calf serum. In order to study mouse cerebellar granule cell maturation and/or regulation of GABA(A) receptor subunit expression in culture, the experiments should be performed in the absence of fetal calf serum. PMID:20170697

  14. 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

  15. GABA(A) receptor downregulation in brains of subjects with autism.

    PubMed

    Fatemi, S Hossein; Reutiman, Teri J; Folsom, Timothy D; Thuras, Paul D

    2009-02-01

    Gamma-aminobutyric acid A (GABA(A)) receptors are ligand-gated ion channels responsible for mediation of fast inhibitory action of GABA in the brain. Preliminary reports have demonstrated altered expression of GABA receptors in the brains of subjects with autism suggesting GABA/glutamate system dysregulation. We investigated the expression of four GABA(A) receptor subunits and observed significant reductions in GABRA1, GABRA2, GABRA3, and GABRB3 in parietal cortex (Brodmann's Area 40 (BA40)), while GABRA1 and GABRB3 were significantly altered in cerebellum, and GABRA1 was significantly altered in superior frontal cortex (BA9). The presence of seizure disorder did not have a significant impact on GABA(A) receptor subunit expression in the three brain areas. Our results demonstrate that GABA(A) receptors are reduced in three brain regions that have previously been implicated in the pathogenesis of autism, suggesting widespread GABAergic dysfunction in the brains of subjects with autism. PMID:18821008

  16. 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

  17. α(5)GABA(A) receptors mediate primary afferent fiber tonic excitability in the turtle spinal cord.

    PubMed

    Loeza-Alcocer, Emanuel; Canto-Bustos, Martha; Aguilar, Justo; González-Ramírez, Ricardo; Felix, Ricardo; Delgado-Lezama, Rodolfo

    2013-11-01

    γ-Amino butyric acid (GABA) plays a key role in the regulation of central nervous system by activating synaptic and extrasynaptic GABAA receptors. It is acknowledged that extrasynaptic GABAA receptors located in the soma, dendrites, and axons may be activated tonically by low extracellular GABA concentrations. The activation of these receptors produces a persistent conductance that can hyperpolarize or depolarize nerve cells depending on the Cl(-) equilibrium potential. In an in vitro preparation of the turtle spinal cord we show that extrasynaptic α5GABAA receptors mediate the tonic state of excitability of primary afferents independently of the phasic primary afferent depolarization mediated by synaptic GABAA receptors. Blockade of α5GABAA receptors with the inverse agonist L-655,708 depressed the dorsal root reflex (DRR) without affecting the phasic increase in excitability of primary afferents. Using RT-PCR and Western blotting, we corroborated the presence of the mRNA and the α5GABAA protein in the dorsal root ganglia of the turtle spinal cord. The receptors were localized in primary afferents in dorsal root, dorsal root ganglia, and peripheral nerve terminals using immunoconfocal microscopy. Considering the implications of the DRR in neurogenic inflammation, α5GABAA receptors may serve as potential pharmacological targets for the treatment of pain. PMID:23966669

  18. 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

  19. GABAergic Afferents activate both GABAA and GABAB receptors in mouse substantia nigra dopaminergic neurons in vivo

    PubMed Central

    Brazhnik, Elena; Shah, Fulva; Tepper, James M.

    2008-01-01

    Most in vivo electrophysiological studies of substantia nigra have employed rats. With the recent proliferation of the use of mice for in vitro neurophysiological studies due to the availability of various genetically modified strains to identify the roles of various channels and proteins in neuronal function, it is crucial to obtain data on in vivo responses in mice to verify that the in vitro results reflect functioning of systems comparable to those that have been well studied in rat. Inhibitory responses of rat nigral dopaminergic neurons by stimulation of afferents from striatum, globus pallidus or pars reticulata have been shown to be mediated predominantly or exclusively by GABAA receptors. This is puzzling given the substantial expression of GABAB receptors and the ubiquitous appearance of GABAB synaptic responses in rat dopaminergic neurons in vitro. In the present study we studied electrically evoked GABAergic inhibition in nigral dopaminergic neurons in C57BL/6J mice. Stimulation of the three major GABAergic inputs elicited stronger and longer lasting inhibitory responses than those seen in rats. The early inhibition was GABAA mediated, whereas the later component, absent in rats, was GABAB mediated and selectively enhanced by GABA uptake inhibition. Striatal-evoked inhibition exhibited a slower onset and a weaker initial component compared to inhibition from globus pallidus or substantia nigra pars reticulata. These results are discussed with respect to differences in the size and neuronal density of the rat and mouse brain, and the different sites of synaptic contact of the synapses from the three GABAergic afferents. PMID:18842898

  20. Abuse and Dependence Liability of Benzodiazepine-Type Drugs: GABAA Receptor Modulation and Beyond

    PubMed Central

    Licata, Stephanie C.; Rowlett, James K.

    2008-01-01

    Benzodiazepine-type drugs (benzodiazepines and the newer non-benzodiazepines) are similar to older sedative/hypnotic drugs, such as the barbiturates, in that they act at the GABAA receptor (9, 188). Unfortunately, benzodiazepine-type drugs also retain the liability for abuse and dependence associated with the earlier anxiolytics (133, 208). Action at GABAA receptors likely plays a key role in both the therapeutic as well as abuse-related effects of this important class of drugs. While the extent to which therapeutic efficacy and abuse potential can be dissociated is not yet understood fully, the biochemical processes underlying these behavioral effects are even less understood. A more comprehensive understanding of the etiology of benzodiazepine-type drug-induced abuse and dependence is likely to provide information that can inform drug development strategies to help design anxiolytics and hypnotics that have maximum clinical benefit with reduced abuse potential. Thus, this review will explore issues related to the abuse and dependence potential of benzodiazepine-type drugs and the role that GABAA receptors play in this phenomenon. Further, this review will discuss putative intracellular events that may occur as a result of the interaction between benzodiazepine-type drugs and GABAA receptors, and how those events may ultimately give rise to the abuse-related behaviors associated with these drugs. PMID:18295321

  1. Linkage disequilibrium between the beta frequency of the human EEG and a GABAA receptor gene locus

    PubMed Central

    Porjesz, Bernice; Almasy, Laura; Edenberg, Howard J.; Wang, Kongming; Chorlian, David B.; Foroud, Tatiana; Goate, Alison; Rice, John P.; O'Connor, Sean J.; Rohrbaugh, John; Kuperman, Samuel; Bauer, Lance O.; Crowe, Raymond R.; Schuckit, Marc A.; Hesselbrock, Victor; Conneally, P. Michael; Tischfield, Jay A.; Li, Ting-Kai; Reich, Theodore; Begleiter, Henri

    2002-01-01

    Human brain oscillations represent important features of information processing and are highly heritable. A common feature of beta oscillations (13–28 Hz) is the critical involvement of networks of inhibitory interneurons as pacemakers, gated by γ-aminobutyric acid type A (GABAA) action. Advances in molecular and statistical genetics permit examination of quantitative traits such as the beta frequency of the human electroencephalogram in conjunction with DNA markers. We report a significant linkage and linkage disequilibrium between beta frequency and a set of GABAA receptor genes. Uncovering the genes influencing brain oscillations provides a better understanding of the neural function involved in information processing. PMID:11891318

  2. 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

  3. A dual mechanism for impairment of GABAA receptor activity by NMDA receptor activation in rat cerebellum granule cells.

    PubMed

    Robello, M; Amico, C; Cupello, A

    1997-01-01

    The function of the GABAA receptor has been studied using the whole cell voltage clamp recording technique in rat cerebellum granule cells in culture. Activation of NMDA-type glutamate receptors causes a reduction in the effect of GABA. Full GABAA receptor activity was recovered after washing out NMDA and NMDA action was prevented in a Mg+2 containing medium. The NMDA effect was also absent when extracellular Ca+2 was replaced by Ba+2 and when 10 mM Bapta was present in the intracellular solution. Charge accumulations via voltage activated Ca+2 channels greater than the ones via NMDA receptors do not cause any reduction in GABAA receptor function, suggesting that Ca+2 influx through NMDA receptor channels is critical for the effect. The NMDA effect was reduced by including adenosine-5'-O-3-thiophosphate (ATP-gamma-S) in the internal solution and there was a reduction in the NMDA effect caused by deltamethrin, a calcineurin inhibitor. Part of the NMDA induced GABAA receptor impairment was prevented by prior treatment with L-arginine. Analogously, part of the NMDA effect was prevented by blockage of NO-synthase activity by N omega-nitro-L-arginine. A combination of NO-synthase and calcineurin inhibitors completely eliminated the NMDA action. An analogous result was obtained by combining the NO-synthase inhibitor with the addition of ATP-gamma-S to the pipette medium. The additivity of the prevention of the NMDA impairment of GABAA receptor by blocking the L-arginine/NO pathway and inhibiting calcineurin activity suggests an independent involvement of these two pathways in the interaction between NMDA and the GABAA receptor. On the one hand Ca+2 influx across NMDA channels activates calcineurin and dephosphorylates the GABAA receptor complex directly or dephosphorylates proteins critical for the function of the receptor. On the other hand, Ca+2 influx activates NO-synthase and induces nitric oxide production, which regulates such receptors via protein kinase G

  4. 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

  5. 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

  6. An Investigation of the Differential Effects of Ursane Triterpenoids from Centella asiatica, and Their Semisynthetic Analogues, on GABAA Receptors.

    PubMed

    Hamid, Kaiser; Ng, Irene; Tallapragada, Vikram J; Váradi, Linda; Hibbs, David E; Hanrahan, Jane; Groundwater, Paul W

    2016-09-01

    The ursane triterpenoids, asiatic acid 1 and madecassic acid 2, are the major pharmacological constituents of Centella asiatica, commonly known as Gotu Kola, which is used traditionally for the treatment of anxiety and for the improvement of cognition and memory. Using the two-electrode voltage-clamp technique, these triterpenes, and some semisynthetic derivatives, were found to exhibit selective negative modulation of different subtypes of the GABAA receptor expressed in Xenopus laevis oocytes. Despite differing by only one hydroxyl group, asiatic acid 1 was found to be a negative modulator of the GABA-induced current at α1 β2 γ2L, α2 β2 γ2L and α5 β3 γ2L GABAA receptors, while madecassic acid 2 was not. Asiatic acid 1 exhibited the greatest effect at α1 β2 γ2L (IC50 37.05 μm), followed by α5 β3 γ2L (IC50 64.05 μm) then α2 β2 γ2L (IC50 427.2 μm) receptors. Conversion of the carboxylic acid group of asiatic acid 1 to a carboxamide group (2α,3β,23-trihydroxy-urs-12-en-28-amide 5) resulted in enhanced inhibition at both the α1 β2 γ2L (IC50 14.07 μm) and α2 β2 γ2L receptor subtypes (IC50 28.41 μm). The results of this study, and the involvement of α5 -containing GABAA receptors in cognition and memory, suggest that asiatic acid 1 may be a lead compound for the enhancement of cognition and memory. PMID:27062315

  7. 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

  8. 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

  9. Flavonoids as GABAA receptor ligands: the whole story?

    PubMed Central

    Wasowski, Cristina; Marder, Mariel

    2012-01-01

    Benzodiazepines are the most widely prescribed class of psychoactive drugs in current therapeutic use, despite the important unwanted side effects that they produce, such as sedation, myorelaxation, ataxia, amnesia, and ethanol and barbiturate potentiation and tolerance. They exert their therapeutic effects via binding to the benzodiazepine binding site of gamma-aminobutyric acid (GABA) type A receptors, and allosterically modulating the chloride flux through the ion channel complex. First isolated from plants used as tranquilizers in folkloric medicine, some natural flavonoids have been shown to possess selective affinity for the benzodiazepine binding site with a broad spectrum of central nervous system effects. Since the initial search for alternative benzodiazepine ligands amongst the flavonoids, a list of successful synthetic derivatives has been generated with enhanced activities. This review provides an update on research developments that have established the activity of natural and synthetic flavonoids on GABA type A receptors. Flavonoids are prominent drugs in the treatment of mental disorders, and can also be used as tools to study modulatory sites at GABA type A receptors and to develop GABA type A selective agents further.

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

    PubMed Central

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

    2012-01-01

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

  11. Effects of Pharmacological Block of GABAA Receptors on Pallidal Neurons in Normal and Parkinsonian State

    PubMed Central

    Xue, Yan; Han, Xiao-Hua; Chen, Lei

    2009-01-01

    The globus pallidus plays a central integrative role in the basal ganglia circuitry. Morphological studies have revealed a high level of GABA and GABAA receptors in the globus pallidus. To further investigate the effects of endogenous GABAA neurotransmission in the globus pallidus of normal and parkinsonian rats, in vivo extracellular recording and behavioral tests were performed in the present studies. In normal rats, micro-pressure ejection of GABAA receptor antagonist gabazine (0.1 mM) increased the spontaneous firing rate of pallidal neurons by 28.3%. Furthermore, in 6-hydroxydopamine parkinsonian rats, gabazine increased the firing rate by 46.0% on the lesioned side, which was significantly greater than that on the unlesioned side (21.5%, P < 0.05), as well as that in normal rats (P < 0.05). In the behaving rats, unilateral microinjection of gabazine (0.1 mM) evoked consistent contralateral rotation in normal rats, and significantly potentiated the number of apomorphine-induced contralateral rotations in parkinsonian rats. The present electrophysiological and behavioral findings may provide a rational for further investigations into the potential of pallidal endogenous GABAA neurotransmission in the treatment of Parkinson's disease. PMID:20204138

  12. Molecular basis for the high THIP/gaboxadol sensitivity of extrasynaptic GABA(A) receptors.

    PubMed

    Meera, Pratap; Wallner, Martin; Otis, Thomas S

    2011-10-01

    Extrasynaptic GABA(A) receptors (eGABARs) allow ambient GABA to tonically regulate neuronal excitability and are implicated as targets for ethanol and anesthetics. These receptors are thought to be heteropentameric proteins made up of two α subunits-either α4 or α6-two β2 or β3 subunits, and one δ subunit. The GABA analog 4,5,6,7-tetrahydroisoxazolo (5,4-c)pyridin-3(-ol) (THIP) has been proposed as a selective ligand for eGABARs. Behavioral and in vitro studies suggest that eGABARs have nanomolar affinity for THIP; however, all published studies on recombinant versions of eGABARs report micromolar affinities. Here, we examine THIP sensitivity of native eGABARs on cerebellar neurons and on reconstituted GABARs in heterologous systems. Concentration-response data for THIP, obtained from cerebellar granule cells and molecular layer interneurons in wild-type and δ subunit knockout slices, confirm that submicromolar THIP sensitivity requires δ subunits. In recombinant experiments, we find that δ subunit coexpression leads to receptors activated by nanomolar THIP concentrations (EC(50) of 30-50 nM for α4β3δ and α6β3δ), a sensitivity almost 1,000-fold higher than receptors formed by α4/6 and β3 subunits. In contrast, γ2 subunit expression significantly reduces THIP sensitivity. Even when δ subunit cDNA or cRNA was supplied in excess, high- and low-sensitivity THIP responses were often apparent, indicative of variable mixtures of low-affinity αβ and high-affinity αβδ receptors. We conclude that δ subunit incorporation into GABARs leads to a dramatic increase in THIP sensitivity, a defining feature that accounts for the unique behavioral and neurophysiological properties of THIP. PMID:21795619

  13. Molecular basis for the high THIP/gaboxadol sensitivity of extrasynaptic GABAA receptors

    PubMed Central

    Meera, Pratap; Wallner, Martin

    2011-01-01

    Extrasynaptic GABAA receptors (eGABARs) allow ambient GABA to tonically regulate neuronal excitability and are implicated as targets for ethanol and anesthetics. These receptors are thought to be heteropentameric proteins made up of two α subunits—either α4 or α6—two β2 or β3 subunits, and one δ subunit. The GABA analog 4,5,6,7-tetrahydroisoxazolo (5,4-c)pyridin-3(-ol) (THIP) has been proposed as a selective ligand for eGABARs. Behavioral and in vitro studies suggest that eGABARs have nanomolar affinity for THIP; however, all published studies on recombinant versions of eGABARs report micromolar affinities. Here, we examine THIP sensitivity of native eGABARs on cerebellar neurons and on reconstituted GABARs in heterologous systems. Concentration-response data for THIP, obtained from cerebellar granule cells and molecular layer interneurons in wild-type and δ subunit knockout slices, confirm that submicromolar THIP sensitivity requires δ subunits. In recombinant experiments, we find that δ subunit coexpression leads to receptors activated by nanomolar THIP concentrations (EC50 of 30–50 nM for α4β3δ and α6β3δ), a sensitivity almost 1,000-fold higher than receptors formed by α4/6 and β3 subunits. In contrast, γ2 subunit expression significantly reduces THIP sensitivity. Even when δ subunit cDNA or cRNA was supplied in excess, high- and low-sensitivity THIP responses were often apparent, indicative of variable mixtures of low-affinity αβ and high-affinity αβδ receptors. We conclude that δ subunit incorporation into GABARs leads to a dramatic increase in THIP sensitivity, a defining feature that accounts for the unique behavioral and neurophysiological properties of THIP. PMID:21795619

  14. Antiseizure Activity of Midazolam in Mice Lacking δ-Subunit Extrasynaptic GABA(A) Receptors.

    PubMed

    Reddy, Sandesh D; Younus, Iyan; Clossen, Bryan L; Reddy, Doodipala Samba

    2015-06-01

    Midazolam is a benzodiazepine anticonvulsant with rapid onset and short duration of action. Midazolam is the current drug of choice for acute seizures and status epilepticus, including those caused by organophosphate nerve agents. The antiseizure activity of midazolam is thought to result from its allosteric potentiation of synaptic GABA(A) receptors in the brain. However, there are indications that benzodiazepines promote neurosteroid synthesis via the 18-kDa cholesterol transporter protein (TSPO). Therefore, we investigated the role of neurosteroids and their extrasynaptic GABA(A) receptor targets in the antiseizure activity of midazolam. Here, we used δ-subunit knockout (DKO) mice bearing a targeted deletion of the extrasynaptic receptors to investigate the contribution of the extrasynaptic receptors to the antiseizure activity of midazolam using the 6-Hz and hippocampus kindling seizure models. In both models, midazolam produced rapid and dose-dependent protection against seizures (ED50, 0.4 mg/kg). Moreover, the antiseizure potency of midazolam was undiminished in DKO mice compared with control mice. Pretreatment with PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide], a TSPO blocker, or finasteride, a 5α-reductase neurosteroid inhibitor, did not affect the antiseizure effect of midazolam. The antiseizure activity of midazolam was significantly reversed by pretreatment with flumazenil, a benzodiazepine antagonist. Plasma and brain levels of the neurosteroid allopregnanolone were not significantly greater in midazolam-treated animals. These studies therefore provide strong evidence that neurosteroids and extrasynaptic GABA(A) receptors are not involved in the antiseizure activity of midazolam, which mainly occurs through synaptic GABA(A) receptors via direct binding to benzodiazepine sites. This study reaffirms midazolam's use for controlling acute seizures and status epilepticus. PMID:25784648

  15. Differential effects of diazepam treatment and withdrawal on recombinant GABAA receptor expression and functional coupling.

    PubMed

    Svob Strac, Dubravka; Vlainić, Josipa; Jazvinsćak Jembrek, Maja; Pericić, Danka

    2008-12-30

    Prolonged exposure to benzodiazepines, drugs known to produce tolerance and dependence and also to be abused, leads to adaptive changes in GABA(A) receptors. To further explore the mechanisms responsible for these phenomena, we studied the effects of prolonged diazepam treatment on the recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptors, stably expressed in human embryonic kidney (HEK) 293 cells. The results demonstrating that long-term (48 and 72 h) exposure of cells to a high concentration of diazepam (50 microM) enhanced the maximum number (B(max)) of [(3)H]flunitrazepam, [(3)H]muscimol and [(3)H]t-butylbicycloorthobenzoate ([(3)H]TBOB) binding sites, without changing their affinity (K(d)), suggested the up-regulation of GABA(A) receptors. As demonstrated by cell counting and WST-1 proliferation assay, the observed increase in receptor expression was not a consequence of stimulated growth of cells exposed to diazepam. Semi-quantitative RT-PCR and Western blot analysis, showing elevated levels of alpha(1) subunit mRNA as well as beta(2) and gamma(2) subunit proteins, respectively, suggested that prolonged high dose diazepam treatment induced de novo receptor synthesis by acting at both transcriptional and translational levels. The finding that the number of GABA(A) receptor binding sites returned to control value 24 h following diazepam withdrawal, makes this process less likely to account for the development of benzodiazepine tolerance and dependence. On the other hand, the results demonstrating that observed functional uncoupling between GABA and benzodiazepine binding sites persisted after the termination of diazepam treatment supported the hypothesis of its possible role in these phenomena. PMID:18955034

  16. Enhanced GABAergic inhibition in the premammillary nucleus of photorefractory turkey hens via GABAA receptor upregulation.

    PubMed

    Kosonsiriluk, Sunantha; Chaiworakul, Voravasa; Mauro, Laura J; El Halawani, Mohamed E

    2016-05-01

    The premammillary nucleus (PMM) of the turkey mediobasal hypothalamus, where dopamine-melatonin (DA-Mel) neurons are localized, is a site for photoreception and photoperiodic time measurement, which is essential for the initiation of avian reproductive seasonality. In addition, this area could also be responsible for the onset and maintenance of photorefractoriness at the end of the breeding season due to the enhanced inhibitory effect of γ-aminobutyric acid (GABA). GABA is an inhibitory neurotransmitter in the central nervous system which interferes with the photosexual response in the turkey, a seasonally breeding bird. Here, we further characterized the GABAA receptor subunits in the PMM DA-Mel neurons related to reproductive seasonality and the onset of photorefractoriness. GABAA receptor subunits and GABA synthesis enzymes in the PMM of photosensitive and photorefractory turkey hens were identified using real-time qRT-PCR. The upregulation of GABAA receptor α1-3, β2-3, γ1-3, ρ1-3, δ, and θ mRNA expression were observed in the PMM of photorefractory birds when compared to those of photosensitive ones while there is no change observed in the GABA synthesis enzymes, glutamate decarboxylase 1 and 2. Those upregulated GABAA receptor subunits were further examined using immunohistochemical staining and they appeared to be co-localized within the PMM DA-Mel neurons. The upregulation of GABAA receptor subunits observed in the PMM of photorefractory birds coincides with a lack of responsiveness to a light stimulus provided during the photosensitive phase. This is supported by the absence of c-fos induction and TH upregulation in the PMM and a subsequence inhibition of c-fos and GnRH-I expression in the nucleus commissurae pallii. The augmented GABAA receptor subunits expression may mediate an enhancement of inhibitory GABAergic neurotransmission and the subsequent interference with the photosexual response. This could contribute to the state of

  17. 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

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

    PubMed

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

    2016-01-01

    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

  19. 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

  20. Differential effects of GABAA receptor antagonists in the control of respiratory neuronal discharge patterns.

    PubMed

    Dogas, Z; Krolo, M; Stuth, E A; Tonkovic-Capin, M; Hopp, F A; McCrimmon, D R; Zuperku, E J

    1998-11-01

    To ascertain the role of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in shaping and controlling the phasic discharge patterns of medullary respiratory premotor neurons, localized pressure applications of the competitive GABAA receptor antagonist bicuculline (BIC) and the noncompetitive GABAA receptor antagonist picrotoxin (PIC) were studied. Multibarrel micropipettes were used in halothane anesthetized, paralyzed, ventilated, vagotomized dogs to record single unit activity from inspiratory and expiratory neurons in the caudal ventral respiratory group and to picoeject GABAA receptor antagonists. The moving time average of phrenic nerve activity was used to determine respiratory phase durations and to synchronize cycle-triggered histograms of discharge patterns. Picoejection of BIC and PIC had qualitatively different effects on the discharge patterns of respiratory neurons. BIC caused an increase in the discharge rate during the neuron's active phase without inducing activity during the neuron's normally silent phase. The resulting discharge patterns were amplified replicas (x2-3) of the underlying preejection phasic patterns. In contrast, picoejection of PIC did not increase the peak discharge rate during the neuron's active phase but induced a tonic level of activity during the neuron's normally silent phase. The maximum effective BIC dose (15 +/- 1.8 pmol/min) was considerably smaller than that for PIC (280 +/- 53 pmol/min). These findings suggest that GABAA receptors with differential pharmacology mediate distinct functions within the same neuron, 1) gain modulation that is BIC sensitive but PIC insensitive and 2) silent-phase inhibition blocked by PIC. These studies also suggest that the choice of an antagonist is an important consideration in the determination of GABA receptor function within the respiratory motor control system. PMID:9819249

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

    PubMed

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

    2016-06-01

    GABAA receptors meet all of the pharmacological requirements necessary to be considered important targets for the action of general anesthetic agents in the mammalian brain. In the following patch-clamp study, the relative modulatory effects of 2,6-dimethylcyclohexanol diastereomers were investigated on human GABAA (α1β3γ2s) receptor currents stably expressed in human embryonic kidney cells. Cis,cis-, trans,trans-, and cis,trans-isomers were isolated from commercially available 2,6-dimethylcyclohexanol and were tested for positive modulation of submaximal GABA responses. For example, the addition of 30 μM cis,cis-isomer resulted in an approximately 2- to 3-fold enhancement of the EC20 GABA current. Coapplications of 30 μM 2,6-dimethylcyclohexanol isomers produced a range of positive enhancements of control GABA responses with a rank order for positive modulation: cis,cis > trans,trans ≥ mixture of isomers > > cis,trans-isomer. In molecular modeling studies, the three cyclohexanol isomers bound with the highest binding energies to a pocket within transmembrane helices M1 and M2 of the β3 subunit through hydrogen-bonding interactions with a glutamine at the 224 position and a tyrosine at the 220 position. The energies for binding to and hydrogen-bond lengths within this pocket corresponded with the relative potencies of the agents for positive modulation of GABAA receptor currents (cis,cis > trans,trans > cis,trans-2,6-dimethylcyclohexanol). In conclusion, the stereochemical configuration within the dimethylcyclohexanols is an important molecular feature in conferring positive modulation of GABAA receptor activity and for binding to the receptor, a consideration that needs to be taken into account when designing novel anesthetics with enhanced therapeutic indices. PMID:27029583

  2. Prefrontal GABA(A) receptor alpha-subunit expression in normal postnatal human development and schizophrenia.

    PubMed

    Duncan, Carlotta E; Webster, Maree J; Rothmond, Debora A; Bahn, Sabine; Elashoff, Michael; Shannon Weickert, Cynthia

    2010-07-01

    Cortical GABA deficits that are consistently reported in schizophrenia may reflect an etiology of failed normal postnatal neurotransmitter maturation. Previous studies have found prefrontal cortical GABA(A) receptor alpha subunit alterations in schizophrenia, yet their relationship to normal developmental expression profiles in the human cortex has not been determined. The aim of this study was to quantify GABA(A) receptor alpha-subunit mRNA expression patterns in human dorsolateral prefrontal cortex (DLPFC) during normal postnatal development and in schizophrenia cases compared to controls. Transcript levels of GABA(A) receptor alpha subunits were measured using microarray and qPCR analysis of 60 normal individuals aged 6weeks to 49years and in 37 patients with schizophrenia/schizoaffective disorder and 37 matched controls. We detected robust opposing changes in cortical GABA(A) receptor alpha1 and alpha5 subunits during the first few years of postnatal development, with a 60% decrease in alpha5 mRNA expression and a doubling of alpha1 mRNA expression with increasing age. In our Australian schizophrenia cohort we detected decreased GAD67 mRNA expression (p=0.0012) and decreased alpha5 mRNA expression (p=0.038) in the DLPFC with no significant change of other alpha subunits. Our findings confirm that GABA deficits (reduced GAD67) are a consistent feature of schizophrenia postmortem brain studies. Our study does not confirm alterations in cortical alpha1 or alpha2 mRNA levels in the schizophrenic DLPFC, as seen in previous studies, but instead we report a novel down-regulation of alpha5 subunit mRNA suggesting that post-synaptic alterations of inhibitory receptors are an important feature of schizophrenia but may vary between cohorts. PMID:20100621

  3. GABAA-receptor modification in taurine transporter knockout mice causes striatal disinhibition

    PubMed Central

    Sergeeva, O A; Fleischer, W; Chepkova, A N; Warskulat, U; Häussinger, D; Siebler, M; Haas, H L

    2007-01-01

    The Striatum is involved in the regulation of movements and motor skills. We have shown previously, that the osmolyte and neuromodulator taurine plays a role in striatal plasticity. We demonstrate now that hereditary taurine deficiency in taurine-transporter knock-out (TAUT KO) mice results in disinhibition of striatal network activity, which can be corrected by taurine supplementation. Modification of GABAA but not glycine receptors (taurine is a ligand for both receptor types) underlies this disinhibition. Whole-cell recordings from acutely isolated as well as cultured striatal neurons revealed a decreased agonist sensitivity of the GABAA receptor in TAUT KO neurons in the absence of changes in the maximal GABA-evoked current amplitude. The striatal GABA level in TAUT KO mice was unchanged. The amplitude enhancement of spontaneous IPSCs by zolpidem was stronger in TAUT KO than in wild-type (WT) animals. Tonic inhibition was absent in striatal neurons under control conditions but was detected after incubation with the GABA-transaminase inhibitor vigabatrin: bicuculline induced a larger shift of baseline current in WT as compared to TAUT KO neurons. Lack of taurine leads to reduced sensitivity of synaptic and extrasynaptic GABAA receptors and consequently to disinhibition. These findings help in understanding neuropathologies accompanied by the loss of endogenous taurine, for instance in hepatic encephalopathy. PMID:17962336

  4. Modulation of GABAA receptor desensitization uncouples sleep onset and maintenance in Drosophila

    PubMed Central

    Agosto, Jose; Choi, James C; Parisky, Katherine M; Stilwell, Geoffrey; Rosbash, Michael; Griffith, Leslie C

    2009-01-01

    Many lines of evidence indicate that GABA and GABAA receptors make important contributions to human sleep regulation. Pharmacological manipulation of these receptors has differential effects on sleep onset and sleep maintenance insomnia. Here we show that sleep is regulated by GABA in Drosophila and that a mutant GABAA receptor, RdlA302S, specifically decreases sleep latency. The drug carbamazepine (CBZ) has the opposite effect on sleep; it increases sleep latency as well as decreasing sleep. Behavioral and physiological experiments indicated that RdlA302S mutant flies are resistant to the effects of CBZ on sleep latency and that mutant RDLA302S channels are resistant to the effects of CBZ on desensitization, respectively. These results suggest that this biophysical property of the channel, specifically channel desensitization, underlies the regulation of sleep latency in flies. These experiments uncouple the regulation of sleep latency from that of sleep duration and suggest that the kinetics of GABAA receptor signaling dictate sleep latency. PMID:18223647

  5. GABA-A Receptor Inhibition of Local Calcium Signaling in Spines and Dendrites

    PubMed Central

    Marlin, Joseph J.

    2014-01-01

    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 Ca2+ 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 Ca2+ 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 Ca2+ signals via GABA-A receptors at both spines and dendrites. These findings reveal how multiple interneurons regulate local Ca2+ signaling in pyramidal neurons, with implications for cortical function and disease. PMID:25429132

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

    PubMed Central

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

    2011-01-01

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

  7. Adenosine A1 Receptor Suppresses Tonic GABAA Receptor Currents in Hippocampal Pyramidal Cells and in a Defined Subpopulation of Interneurons.

    PubMed

    Rombo, Diogo M; Dias, Raquel B; Duarte, Sofia T; Ribeiro, Joaquim A; Lamsa, Karri P; Sebastião, Ana M

    2016-03-01

    Adenosine is an endogenous neuromodulator that decreases excitability of hippocampal circuits activating membrane-bound metabotropic A1 receptor (A1R). The presynaptic inhibitory action of adenosine A1R in glutamatergic synapses is well documented, but its influence on inhibitory GABAergic transmission is poorly known. We report that GABAA receptor (GABAAR)-mediated tonic, but not phasic, transmission is suppressed by A1R in hippocampal neurons. Adenosine A1R activation strongly inhibits GABAAR agonist (muscimol)-evoked currents in Cornu Ammonis 1 (CA1) pyramidal neurons and in a specific subpopulation of interneurons expressing axonal cannabinoid receptor type 1. In addition, A1R suppresses tonic GABAAR currents measured in the presence of elevated ambient GABA as well as in naïve slices. The inhibition of GABAergic currents involves both protein kinase A (PKA) and protein kinase C (PKC) signaling pathways and decreases GABAAR δ-subunit expression. On the contrary, no A1R-mediated modulation was detected in phasic inhibitory postsynaptic currents evoked either by afferent electrical stimulation or by spontaneous quantal release. The results show that A1R modulates extrasynaptic rather than synaptic GABAAR-mediated signaling, and that this modulation selectively occurs in hippocampal pyramidal neurons and in a specific subpopulation of inhibitory interneurons. We conclude that modulation of tonic GABAAR signaling by adenosine A1R in specific neuron types may regulate neuronal gain and excitability in the hippocampus. PMID:25452570

  8. 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

  9. A Reinforcing Circuit Action of Extrasynaptic GABAA Receptor Modulators on Cerebellar Granule Cell Inhibition

    PubMed Central

    Santhakumar, Vijayalakshmi; Otis, Thomas S.

    2013-01-01

    GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants. PMID:23977374

  10. A reinforcing circuit action of extrasynaptic GABAA receptor modulators on cerebellar granule cell inhibition.

    PubMed

    Santhakumar, Vijayalakshmi; Meera, Pratap; Karakossian, Movses H; Otis, Thomas S

    2013-01-01

    GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants. PMID:23977374

  11. 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. PMID:8224749

  12. A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABAA receptor chloride channels.

    PubMed

    Kruger, Wade; Gilbert, Daniel; Hawthorne, Rebecca; Hryciw, Deanne H; Frings, Stephan; Poronnik, Philip; Lynch, Joseph W

    2005-06-01

    There is a significant clinical need to identify novel ligands with high selectivity and potency for GABA(A), GABA(C) and glycine receptor Cl- channels. Two recently developed, yellow fluorescent protein variants (YFP-I152L and YFP-V163S) are highly sensitive to quench by small anions and are thus suited to reporting anionic influx into cells. The aim of this study was to establish the optimal conditions for using these constructs for high-throughput screening of GABA(A), GABA(C) and glycine receptors transiently expressed in HEK293 cells. We found that a 70% fluorescence reduction was achieved by quenching YFP-I152L with a 10 s influx of I- ions, driven by an external I- concentration of at least 50 mM. The fluorescence quench was rapid, with a mean time constant of 3 s. These responses were similar for all anion receptor types studied. We also show the assay is sufficiently sensitive to measure agonist and antagonist concentration-responses using either imaging- or photomultiplier-based detection systems. The robustness, sensitivity and low cost of this assay render it suited for high-throughput screening of transiently expressed anionic ligand-gated channels. PMID:15862914

  13. From synapse to behavior: rapid modulation of defined neuronal types with engineered GABAA receptors

    PubMed Central

    Wulff, Peer; Goetz, Thomas; Leppä, Elli; Linden, Anni-Maija; Renzi, Massimiliano; Swinny, Jerome D; Vekovischeva, Olga Y; Sieghart, Werner; Somogyi, Peter; Korpi, Esa R; Farrant, Mark; Wisden, William

    2007-01-01

    In mammals, identifying the contribution of specific neurons or networks to behavior is a key challenge. Here we describe an approach that facilitates this process by enabling the rapid modulation of synaptic inhibition in defined cell populations. Binding of zolpidem, a systemically active allosteric modulator that enhances the function of the GABAA receptor, requires a phenylalanine residue (Phe77) in the γ2 subunit. Mice in which this residue is changed to isoleucine are insensitive to zolpidem. By Cre recombinase-induced swapping of the γ2 subunit (that is, exchanging Ile77 for Phe77), zolpidem sensitivity can be restored to GABAA receptors in chosen cell types. We demonstrate the power of this method in the cerebellum, where zolpidem rapidly induces significant motor deficits when Purkinje cells are made uniquely sensitive to its action. This combined molecular and pharmacological technique has demonstrable advantages over targeted cell ablation and will be invaluable for investigating many neuronal circuits. PMID:17572671

  14. From synapse to behavior: rapid modulation of defined neuronal types with engineered GABAA receptors.

    PubMed

    Wulff, Peer; Goetz, Thomas; Leppä, Elli; Linden, Anni-Maija; Renzi, Massimiliano; Swinny, Jerome D; Vekovischeva, Olga Y; Sieghart, Werner; Somogyi, Peter; Korpi, Esa R; Farrant, Mark; Wisden, William

    2007-07-01

    In mammals, identifying the contribution of specific neurons or networks to behavior is a key challenge. Here we describe an approach that facilitates this process by enabling the rapid modulation of synaptic inhibition in defined cell populations. Binding of zolpidem, a systemically active allosteric modulator that enhances the function of the GABAA receptor, requires a phenylalanine residue (Phe77) in the gamma2 subunit. Mice in which this residue is changed to isoleucine are insensitive to zolpidem. By Cre recombinase-induced swapping of the gamma2 subunit (that is, exchanging Ile77 for Phe77), zolpidem sensitivity can be restored to GABAA receptors in chosen cell types. We demonstrate the power of this method in the cerebellum, where zolpidem rapidly induces significant motor deficits when Purkinje cells are made uniquely sensitive to its action. This combined molecular and pharmacological technique has demonstrable advantages over targeted cell ablation and will be invaluable for investigating many neuronal circuits. PMID:17572671

  15. Prediction of receptor properties and binding affinity of ligands to benzodiazepine/GABAA receptors using artificial neural networks.

    PubMed

    Maddalena, D J; Johnston, G A

    1995-02-17

    To date the use of artificial neural networks (ANNs) in quantitative structure-activity relationship (QSAR) studies has been primarily concerned in comparing the predictive accuracy of the technique using known data sets where the data set parameters had been preselected and optimized for use with other statistical methods. Little effort has been directed at optimizing the input parameters for use with ANNs or exploring other potential strengths of ANNs. In this study, back-propagation ANNs and multilinear regression (MLR) were used to examine the QSAR between substituent constants and random noise at six positions on 57 1,4-benzodiazepin-2-ones (1,4-BZs) and their binding affinities (log IC50) for benzodiazepine GABAA receptor preparations. By using selective pruning and cross-validation techniques, it was found possible to use ANNs to indicate an optimum set of 10 input parameters from a choice of 48 which were then used to train back-propagation ANNs that best predicted the receptor binding affinity with a high correlation between known and predicted data sets. Using the optimum set of input parameters, three-layer ANNs performed no better than the two-layer ANNs which gave marginally better results than MLR. Using the trained ANNs to examine the individual parameters showed that increases in the lipophilicity and F polar value at position 7, F polar value at position 2', and dipole at position 1 on the molecule all enhanced receptor binding affinity of 1,4-BZ ligands. Increases in molar refractivity and resonance parameters at position 1, molar refractivity at positions 6' and 2', Hammet meta constant at position 3', and Hammet para constant at position 8 on the molecule all caused decreases in receptor binding affinity. By considering the optimal ANNs as pharmacophore models representing the internal physicochemical structure of the receptor site, it was found that they could be used to critically examine the properties of the receptor site. PMID:7861419

  16. A Comprehensive Optogenetic Pharmacology Toolkit for In Vivo Control of GABA(A) Receptors and Synaptic Inhibition.

    PubMed

    Lin, Wan-Chen; Tsai, Ming-Chi; Davenport, Christopher M; Smith, Caleb M; Veit, Julia; Wilson, Neil M; Adesnik, Hillel; Kramer, Richard H

    2015-12-01

    Exogenously expressed opsins are valuable tools for optogenetic control of neurons in circuits. A deeper understanding of neural function can be gained by bringing control to endogenous neurotransmitter receptors that mediate synaptic transmission. Here we introduce a comprehensive optogenetic toolkit for controlling GABA(A) receptor-mediated inhibition in the brain. We developed a series of photoswitch ligands and the complementary genetically modified GABA(A) receptor subunits. By conjugating the two components, we generated light-sensitive versions of the entire GABA(A) receptor family. We validated these light-sensitive receptors for applications across a broad range of spatial scales, from subcellular receptor mapping to in vivo photo-control of visual responses in the cerebral cortex. Finally, we generated a knockin mouse in which the "photoswitch-ready" version of a GABA(A) receptor subunit genomically replaces its wild-type counterpart, ensuring normal receptor expression. This optogenetic pharmacology toolkit allows scalable interrogation of endogenous GABA(A) receptor function with high spatial, temporal, and biochemical precision. PMID:26606997

  17. Association of nonsense mutation in GABRG2 with abnormal trafficking of GABAA receptors in severe epilepsy.

    PubMed

    Ishii, Atsushi; Kanaumi, Takeshi; Sohda, Miwa; Misumi, Yoshio; Zhang, Bo; Kakinuma, Naoto; Haga, Yoshiko; Watanabe, Kazuyoshi; Takeda, Sen; Okada, Motohiro; Ueno, Shinya; Kaneko, Sunao; Takashima, Sachio; Hirose, Shinichi

    2014-03-01

    Mutations in GABRG2, which encodes the γ2 subunit of GABAA receptors, can cause both genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. Most GABRG2 truncating mutations associated with Dravet syndrome result in premature termination codons (PTCs) and are stably translated into mutant proteins with potential dominant-negative effects. This study involved search for mutations in candidate genes for Dravet syndrome, namely SCN1A, 2A, 1B, 2B, GABRA1, B2, and G2. A heterozygous nonsense mutation (c.118C>T, p.Q40X) in GABRG2 was identified in dizygotic twin girls with Dravet syndrome and their apparently healthy father. Electrophysiological studies with the reconstituted GABAA receptors in HEK cells showed reduced GABA-induced currents when mutated γ2 DNA was cotransfected with wild-type α1 and β2 subunits. In this case, immunohistochemistry using antibodies to the α1 and γ2 subunits of GABAA receptor showed granular staining in the soma. In addition, microinjection of mutated γ2 subunit cDNA into HEK cells severely inhibited intracellular trafficking of GABAA receptor subunits α1 and β2, and retention of these proteins in the endoplasmic reticulum. The mutated γ2 subunit-expressing neurons also showed impaired axonal transport of the α1 and β2 subunits. Our findings suggested that different phenotypes of epilepsy, e.g., GEFS+ and Dravet syndrome (which share similar abnormalities in causative genes) are likely due to impaired axonal transport associated with the dominant-negative effects of GABRG2. PMID:24480790

  18. 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

  19. γ-Aminobutyric Acid Type A (GABAA) Receptor Activation Modulates Tau Phosphorylation*

    PubMed Central

    Nykänen, Niko-Petteri; Kysenius, Kai; Sakha, Prasanna; Tammela, Päivi; Huttunen, Henri J.

    2012-01-01

    Abnormal phosphorylation and aggregation of the microtubule-associated protein Tau are hallmarks of various neurodegenerative diseases, such as Alzheimer disease. Molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. We have developed a novel live cell reporter system based on protein-fragment complementation assay to study dynamic changes in Tau phosphorylation status. In this assay, fusion proteins of Tau and Pin1 (peptidyl-prolyl cis-trans-isomerase 1) carrying complementary fragments of a luciferase protein serve as a sensor of altered protein-protein interaction between Tau and Pin1, a critical regulator of Tau dephosphorylation at several disease-associated proline-directed phosphorylation sites. Using this system, we identified several structurally distinct GABAA receptor modulators as novel regulators of Tau phosphorylation in a chemical library screen. GABAA receptor activation promoted specific phosphorylation of Tau at the AT8 epitope (Ser-199/Ser-202/Thr-205) in cultures of mature cortical neurons. Increased Tau phosphorylation by GABAA receptor activity was associated with reduced Tau binding to protein phosphatase 2A and was dependent on Cdk5 but not GSK3β kinase activity. PMID:22235112

  20. 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. PMID:25675485

  1. 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

  2. GABAA and GABAC receptors in adult porcine cones: evidence from a photoreceptor–glia co-culture model

    PubMed Central

    Picaud, Serge; Pattnaik, Bikash; Hicks, David; Forster, Valérie; Fontaine, Valérie; Sahel, José; Dreyfus, Henri

    1998-01-01

    Edge contrast enhancement is an integrated visual function based on the complex centre-surround organization of the cone photoreceptor light response. While centre responses result from direct light activation, surround responses are thought to result from lateral inhibition mediated by horizontal cells. This feedback signal has been attributed to GABAA receptors which have been found in lower vertebrate cones. In order to study the GABA response of adult mammalian photoreceptors, we designed a culture system consisting of isolated photoreceptors seeded on a layer of retinal glial cells. Mature rods and cones required the presence of Müller glial cells to survive and develop neurites; they degenerated in the absence of glial cells. Cone photoreceptors generated large GABA responses whereas rod photoreceptors did not respond to GABA applications. Cone GABA responses consisted of two distinct components, one suppressed by the GABAA receptor blockers bicuculline and SR95531, and the second by the GABAC receptor antagonists TPMPA and imidazole-4-acetic acid (I4AA). Pentobarbital greatly increased the GABAA receptor component whereas it did not affect, or even reduced, the GABAC receptor component. During long GABA applications, GABAA receptor currents desensitized by 78 %, contrasting with the sustained GABAC response. Expression of GABAC receptors in cone photoreceptors was confirmed by anti-ρ-subunit immunolabelling of porcine retinal sections. These results indicate that both GABAA and GABAC receptors may participate in the feedback synapse from horizontal cells to cone photoreceptors in the mammalian retina. PMID:9782157

  3. Anxiolytic-Like Effects of Chrysanthemum indicum Aqueous Extract in Mice: Possible Involvement of GABAA Receptors and 5-HT1A Receptors.

    PubMed

    Hong, Sa-Ik; Kwon, Seung-Hwan; Kim, Min-Jung; Ma, Shi-Xun; Kwon, Je-Won; Choi, Seung-Min; Choi, Soo-Im; Kim, Sun-Yeou; Lee, Seok-Yong; Jang, Choon-Gon

    2012-07-01

    Chrysanthemum indicum Linne is an ancient herbal medicine used to treat bone and muscle deterioration, ocular infl ammation, headache, and anxiety in Korea, China, and Japan. Furthermore, tea derived from Chrysanthemum indicum Linne has been used to treat anxiety by facilitating relaxation and curing insomnia. However, no reports exist on the anxiolytic-like effects of Chrysanthemum indicum Linne water extract (CWE) in mice. In the present study, we investigated the anxiolytic-like effects of CWE using the elevated plus-maze (EPM) test in mice. CWE, at a dose of 500 mg/kg (p.o.), signifi cantly increased the time spent in the open arms of the EPM compared to a vehicle-injected control group. Moreover, the effect of CWE (500 mg/kg) was blocked by bicuculline (a selective GABAA receptor antagonist) and WAY 100635 (a selective 5-HT1A receptor antagonist). Taken together, these fi ndings suggest that the anxiolytic-like effects of CWE might be mediated by the GABAA receptor and the 5-HT1A receptor. PMID:24009829

  4. Seizure-Related Regulation of GABAA Receptors in Spontaneously Epileptic Rats

    PubMed Central

    González, Marco I.; Grabenstatter, Heidi L.; del Rio, Christian Cea; Del Angel, Yasmin Cruz; Carlsen, Jessica; Laoprasert, Rick; White, Andrew M.; Huntsman, Molly M.; Brooks-Kayal, Amy

    2015-01-01

    In this study, we analyzed the impact that spontaneous seizures might have on the plasma membrane expression, composition and function of GABAA receptors (GABAARs). For this, tissue of chronically epileptic rats was collected within 3 hours of seizure occurrence (≤3 hours group) or at least 24 hours after seizure occurrence (≥24 hours group). A retrospective analysis of seizure frequency revealed that selecting animals on the bases of seizure proximity also grouped animals in terms of overall seizure burden with a higher seizure burden observed in the ≤3 hours group. A biochemical analysis showed that although animals with more frequent/recent seizures (≤3 hours group) had similar levels of GABAAR at the plasma membrane they showed deficits in inhibitory neurotransmission. In contrast, tissue obtained from animals experiencing infrequent seizures (≥24 hours group) had increased plasma membrane levels of GABAAR and showed no deficit in inhibitory function. Together, our findings offer an initial insight into the molecular changes that might help to explain how alterations in GABAAR function can be associated with differential seizure burden. Our findings also suggest that increased plasma membrane levels of GABAAR might act as a compensatory mechanism to more effectively maintain inhibitory function, repress hyperexcitability and reduce seizure burden. This study is an initial step towards a fuller characterization of the molecular events that trigger alterations in GABAergic neurotransmission during chronic epilepsy. PMID:25769812

  5. The expression of GABAA beta subunit isoforms in synaptic and extrasynaptic receptor populations of mouse dentate gyrus granule cells.

    PubMed

    Herd, Murray B; Haythornthwaite, Alison R; Rosahl, Thomas W; Wafford, Keith A; Homanics, Gregg E; Lambert, Jeremy J; Belelli, Delia

    2008-02-15

    The subunit composition of GABA(A) receptors influences their biophysical and pharmacological properties, dictates neuronal location and the interaction with associated proteins, and markedly influences the impact of intracellular biochemistry. The focus has been on alpha and gamma subunits, with little attention given to beta subunits. Dentate gyrus granule cells (DGGCs) express all three beta subunit isoforms and exhibit both synaptic and extrasynaptic receptors that mediate 'phasic' and 'tonic' transmission, respectively. To investigate the subcellular distribution of the beta subunits we have utilized the patch-clamp technique to compare the properties of 'tonic' and miniature inhibitory postsynaptic currents (mIPSCs) recorded from DGGCs of hippocampal slices of P20-26 wild-type (WT), beta(2)(-/-), beta(2N265S) (etomidate-insensitive), alpha(1)(-/-) and delta(-/-) mice. Deletion of either the beta(2) or the delta subunit produced a significant reduction of the tonic current and attenuated the increase of this current induced by the delta subunit-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). By contrast, mIPSCs were not influenced by deletion of these genes. Enhancement of the tonic current by the beta(2/3) subunit-selective agent etomidate was significantly reduced for DGGCs derived from beta(2N265S) mice, whereas this manipulation had no effect on the prolongation of mIPSCs produced by this anaesthetic. Collectively, these observations, together with previous studies on alpha(4)(-/-) mice, identify a population of extrasynaptic alpha(4)beta(2)delta receptors, whereas synaptic GABA(A) receptors appear to primarily incorporate the beta(3) subunit. A component of the tonic current is diazepam sensitive and is mediated by extrasynaptic receptors incorporating alpha(5) and gamma(2) subunits. Deletion of the beta(2) subunit had no effect on the diazepam-induced current and therefore these extrasynaptic receptors do not contain this

  6. Are extrasynaptic GABAA receptors important targets for sedative/hypnotic drugs?

    PubMed Central

    Houston, Catriona M; McGee, Thomas P; MacKenzie, Georgina; Troyano-Cuturi, Kevin; Rodriguez, Pablo Mateos; Kutsarova, Elena; Diamanti, Efthymia; Hosie, Alastair M; Franks, Nicholas P; Brickley, Stephen G

    2012-01-01

    High-affinity extrasynaptic GABAA receptors are persistently activated by the low ambient GABA levels that are known to be present in the extracellular space. The resulting tonic conductance generates a form of shunting inhibition that is capable of altering cellular and network behaviour. It has been suggested that this tonic inhibition will be enhanced by neurosteroids, anti-epileptics, and sedative/hypnotic drugs. However, we show that the ability of sedative/hypnotic drugs to enhance tonic inhibition in the mouse cerebellum will critically depend upon ambient GABA levels. For example, we show that the intravenous anaesthetic propofol only enhances tonic inhibition when ambient GABA levels are below 100 nM. More surprisingly, the actions of the sleep promoting drug THIP (4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol) are attenuated at ambient GABA levels of just 20 nM. In contrast, our data suggests that neurosteroid enhancement of tonic inhibition will be greater at high ambient GABA concentrations. We present a model that takes into account realistic estimates of ambient GABA levels and predicted extrasynaptic GABAA numbers when considering the ability of sedative/hypnotic drugs to enhance tonic inhibition. These issues will be important when considering drug strategies designed to target extrasynaptic GABAA receptors in the treatment of sleep disorders and other neurological conditions. PMID:22423109

  7. 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

  8. Tonic inhibition in spinal ventral horn interneurons mediated by α5 subunit-containing GABA(A) receptors.

    PubMed

    Castro, Alberto; Aguilar, Justo; González-Ramírez, Ricardo; Loeza-Alcocer, Emanuel; Canto-Bustos, Martha; Felix, Ricardo; Delgado-Lezama, Rodolfo

    2011-08-19

    GABA(A) receptors mediate synaptic and tonic inhibition in many neurons of the central nervous system. These receptors can be constructed from a range of different subunits deriving from seven identified families. Among these subunits, α(5) has been shown to mediate GABAergic tonic inhibitory currents in neurons from supraspinal nuclei. Likewise, immunohistochemical and in situ hybridization studies have shown the presence of the α(5) subunit in spinal cord neurons, though almost nothing is known about its function. In the present report, using slices of the adult turtle spinal cord as a model system we have recorded a tonic inhibitory current in ventral horn interneurons (VHIs) and determined the functional contribution of the α(5) subunit-containing GABA(A) receptors to this current. Patch clamp studies show that the GABAergic tonic inhibitory current in VHIs is not affected by the application of antagonists of the α(4/6) subunit-containing GABA(A) receptors, but is sensitive to L-655708, an antagonist of the GABA(A) receptors containing α(5) subunits. Last, by using RT-PCR and immunohistochemistry we confirmed the expression of the α(5) subunit in the turtle spinal cord. Together, these results suggest that GABA(A) receptors containing the α(5) subunit mediate the tonic inhibitory currents observed in VHIs. PMID:21798246

  9. Alcohol- and alcohol antagonist-sensitive human GABAA receptors: tracking δ subunit incorporation into functional receptors.

    PubMed

    Meera, Pratap; Olsen, Richard W; Otis, Thomas S; Wallner, Martin

    2010-11-01

    GABA(A) receptors (GABA(A)Rs) have long been a focus as targets for alcohol actions. Recent work suggests that tonic GABAergic inhibition mediated by extrasynaptic δ subunit-containing GABA(A)Rs is uniquely sensitive to ethanol and enhanced at concentrations relevant for human alcohol consumption. Ethanol enhancement of recombinant α4β3δ receptors is blocked by the behavioral alcohol antagonist 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (Ro15-4513), suggesting that EtOH/Ro15-4513-sensitive receptors mediate important behavioral alcohol actions. Here we confirm alcohol/alcohol antagonist sensitivity of α4β3δ receptors using human clones expressed in a human cell line and test the hypothesis that discrepant findings concerning the high alcohol sensitivity of these receptors are due to difficulties incorporating δ subunits into functional receptors. To track δ subunit incorporation, we used a functional tag, a single amino acid change (H68A) in a benzodiazepine binding residue in which a histidine in the δ subunit is replaced by an alanine residue found at the homologous position in γ subunits. We demonstrate that the δH68A substitution confers diazepam sensitivity to otherwise diazepam-insensitive α4β3δ receptors. The extent of enhancement of α4β3δH68A receptors by 1 μM diazepam, 30 mM EtOH, and 1 μM β-carboline-3-carboxy ethyl ester (but not 1 μM Zn(2+) block) is correlated in individual recordings, suggesting that δ subunit incorporation into recombinant GABA(A)Rs varies from cell to cell and that this variation accounts for the variable pharmacological profile. These data are consistent with the notion that δ subunit-incorporation is often incomplete in recombinant systems yet is necessary for high ethanol sensitivity, one of the features of native δ subunit-containing GABA(A)Rs. PMID:20699325

  10. Alcohol- and Alcohol Antagonist-Sensitive Human GABAA Receptors: Tracking δ Subunit Incorporation into Functional Receptors

    PubMed Central

    Meera, Pratap; Olsen, Richard W.; Otis, Thomas S.

    2010-01-01

    GABAA receptors (GABAARs) have long been a focus as targets for alcohol actions. Recent work suggests that tonic GABAergic inhibition mediated by extrasynaptic δ subunit-containing GABAARs is uniquely sensitive to ethanol and enhanced at concentrations relevant for human alcohol consumption. Ethanol enhancement of recombinant α4β3δ receptors is blocked by the behavioral alcohol antagonist 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (Ro15-4513), suggesting that EtOH/Ro15-4513-sensitive receptors mediate important behavioral alcohol actions. Here we confirm alcohol/alcohol antagonist sensitivity of α4β3δ receptors using human clones expressed in a human cell line and test the hypothesis that discrepant findings concerning the high alcohol sensitivity of these receptors are due to difficulties incorporating δ subunits into functional receptors. To track δ subunit incorporation, we used a functional tag, a single amino acid change (H68A) in a benzodiazepine binding residue in which a histidine in the δ subunit is replaced by an alanine residue found at the homologous position in γ subunits. We demonstrate that the δH68A substitution confers diazepam sensitivity to otherwise diazepam-insensitive α4β3δ receptors. The extent of enhancement of α4β3δH68A receptors by 1 μM diazepam, 30 mM EtOH, and 1 μM β-carboline-3-carboxy ethyl ester (but not 1 μM Zn2+ block) is correlated in individual recordings, suggesting that δ subunit incorporation into recombinant GABAARs varies from cell to cell and that this variation accounts for the variable pharmacological profile. These data are consistent with the notion that δ subunit-incorporation is often incomplete in recombinant systems yet is necessary for high ethanol sensitivity, one of the features of native δ subunit-containing GABAARs. PMID:20699325

  11. A cycloartane glycoside derived from Actaea racemosa L. modulates GABAA receptors and induces pronounced sedation in mice.

    PubMed

    Strommer, Barbara; Khom, Sophia; Kastenberger, Iris; Cicek, Serhat Sezai; Stuppner, Hermann; Schwarzer, Christoph; Hering, Steffen

    2014-11-01

    23-O-Acetylshengmanol 3-O-β-D-xylopyranoside (Ac-SM) isolated from Actaea racemosa L.-an herbal remedy for the treatment of mild menopausal disorders-has been recently identified as a novel efficacious modulator of GABAA receptors composed of α1-, β2-, and γ2S-subunits. In the present study, we analyzed a potential subunit-selective modulation of GABA-induced chloride currents (IGABA) at GABA concentrations eliciting 3-8% of the maximal GABA response (EC3-8) through nine GABAA receptor isoforms expressed in Xenopus laevis oocytes by Ac-SM with two-microelectrode voltage clamp and behavioral effects 30 minutes after intraperitoneal application in a mouse model. Efficacy of IGABA enhancement by Ac-SM displayed a mild α-subunit dependence with α2β2γ2S (maximal IGABA potentiation [Emax] = 1454 ± 97%) and α5β2γ2S (Emax = 1408 ± 87%) receptors being most efficaciously modulated, followed by slightly weaker IGABA enhancement through α1β2γ2S (Emax = 1187 ± 166%), α3β2γ2S (Emax = 1174 ± 218%), and α6β2γ2S (Emax = 1171 ± 274%) receptors and less pronounced effects on receptors composed of α4β2γ2S (Emax = 752 ± 53%) subunits, whereas potency was not affected by the subunit composition (EC50 values ranging from α1β2γ2S = 35.4 ± 12.3 µM to α5β2γ2S = 50.9 ± 11.8 µM). Replacing β2- with β1- or β3-subunits as well as omitting the γ2S-subunit affected neither efficacy nor potency of IGABA enhancement by Ac-SM. Ac-SM shifted the GABA concentration-response curve toward higher GABA sensitivity (about 3-fold) and significantly increased the maximal GABA response by 44 ± 13%, indicating a pharmacological profile distinct from a pure allosteric GABAA receptor modulator. In mice, Ac-SM significantly reduced anxiety-related behavior in the elevated plus maze test at a dose of 0.6 mg/kg, total ambulation in the open field test at doses ≥6 mg/kg, stress-induced hyperthermia at doses ≥0.6 mg/kg, and significantly elevated seizure threshold at

  12. 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

  13. Effects of subunit types of the recombinant GABAA receptor on the response to a neurosteroid.

    PubMed

    Zaman, S H; Shingai, R; Harvey, R J; Darlison, M G; Barnard, E A

    1992-04-10

    When vertebrate brain poly(A)+ RNA is expressed in Xenopus oocytes the response of the GABA receptors formed is found to be inhibited allosterically by a neurosteroid, pregnenolone sulphate (PS). This negative modulation was reproduced after expressing RNAs encoding bovine GABAA receptor subunits in the combinations alpha i + beta 1, or alpha i + beta 1 + gamma 2 (where i = 1, 2 or 3). The characteristics of this inhibition vary significantly with the type of the alpha subunit (alpha 1, alpha 2, or alpha 3) used. When the bovine gamma 2L alternate form of the gamma 2 subunit was replaced by the human gamma 2S subunit, the behaviour was unchanged: the human gamma 2S subunit used is a newly-cloned form, which encodes a polypeptide with two amino acid differences from the human gamma 2 subunit previously described. The results of co-application of PS and 3 alpha-hydroxy-5 alpha-pregnan-ol-20-one, a neurosteroid which is a positive modulator of the GABAA receptor, indicate that these act at different sites on the receptor. PS also increases the desensitisation of the receptor by GABA. This effect, also, is alpha-subunit-type dependent and occurs by an acceleration of the fast phase of desensitisation. PMID:1323476

  14. Midazolam inhibits long-term potentiation through modulation of GABAA receptors.

    PubMed

    Evans, M S; Viola-McCabe, K E

    1996-03-01

    Benzodiazepine drugs (BZ) are used for anxiety, insomnia, and seizures. They worsen memory, especially in large doses, but the mechanism of this action is uncertain. In micromolar concentrations, benzodiazepines have been shown to reduce long-term potentiation (LTP), which could be a cellular basis for their amnesic action. We have found that the LTP-inhibiting effects of BZ occur in the nanomolar concentrations attained in humans, and that this effect occurs through modulation of GABAA receptor function. We recorded extracellular synaptic input/output (I/O) curves for population spikes (PS) and EPSPs in rat hippocampal slices before and after induction of LTP. LTP increased maximal PS and EPSPs and shifted I/O curves for PS and EPSPs to the left, reflecting increased synaptic responsiveness after LTP. Curves relating EPSPs to PS were also shifted, so that after LTP larger PS were elicited for the same size EPSP (E-S potentiation). Midazolam (0.5 microM) markedly inhibited the left-shift in PS I/O curves due to E-S potentiation but did not significantly affect other parameters. 8-Phenyltheophylline (10 microM), an adenosine receptor antagonist, did not prevent midazolam inhibition of LTP. Bicuculline, a GABAA receptor antagonist, caused a dose-dependent antagonism of midazolam's LTP inhibition. Our results suggest that benzodiazepines reduce LTP primarily through reduction of E-S potentiation, and that this effect occurs through modulation of GABAA receptor function. This could in part account for the ability of benzodiazepines to disturb new memory formation. PMID:8783210

  15. The role of α1 and α5 subunit-containing GABAA receptors in motor impairment induced by benzodiazepines in rats.

    PubMed

    Milić, Marija; Divljaković, Jovana; Rallapalli, Sundari; van Linn, Michael L; Timić, Tamara; Cook, James M; Savić, Miroslav M

    2012-04-01

    Benzodiazepines negatively affect motor coordination and balance and produce myorelaxation. The aim of the present study was to examine the extent to which populations of γ-aminobutyric acid A (GABAA) receptors containing α1 and α5 subunits contribute to these motor-impairing effects in rats. We used the nonselective agonist diazepam and the α1-selective agonist zolpidem, as well as nonselective, α1-subunit and α5-subunit-selective antagonists flumazenil, βCCt, and XLi093, respectively. Ataxia and muscle relaxation were assessed by rotarod and grip strength tests performed 20 min after intraperitoneal treatment. Diazepam (2 mg/kg) induced significant ataxia and muscle relaxation, which were completely prevented by pretreatment with flumazenil (10 mg/kg) and βCCt (20 mg/kg). XLi093 antagonized the myorelaxant, but not the ataxic actions of diazepam. All three doses of zolpidem (1, 2, and 5 mg/kg) produced ataxia, but only the highest dose (5 mg/kg) significantly decreased the grip strength. These effects of zolpidem were reversed by βCCt at doses of 5 and 10 mg/kg, respectively. The present study demonstrates that α1 GABAA receptors mediate ataxia and indirectly contribute to myorelaxation in rats, whereas α5 GABAA receptors contribute significantly, although not dominantly, to muscle relaxation but not ataxia. PMID:22327019

  16. 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

  17. Synthesis of novel cognition enhancers with pyrazolo[5,1-c][1,2,4]benzotriazine core acting at γ-aminobutyric acid type A (GABA(A)) receptor.

    PubMed

    Guerrini, Gabriella; Ciciani, Giovanna; Costanzo, Annarella; Daniele, Simona; Martini, Claudia; Ghelardini, Carla; Di Cesare Mannelli, Lorenzo; Ciattini, Samuele

    2013-04-15

    Memory dysfunction associated with aging, neurodegenerative and psychiatric disorders represents an increasing medical need. Advances in research exploring the biological mechanisms underlying learning and memory have opened new potential approaches for development of memory-enhancing therapies addressed to selective neuronal targets. In this work, we synthesized some derivatives with a pyrazolo[5,1-c][1,2,4]benzotriazine core to identify ligands on GABAA receptors subtype (benzodiazepine site on GABAA-receptor) endowed with the potential of enhancing cognition activity without the side effects usually associated with non-selective GABAA modulators. In fact, there is much evidence that GABAA-R (γ-aminobutyric acid, type A receptor) subtype ligands have relevance in learning and memory. In vitro and in vivo tests have been performed. Pharmacological data indicate that compounds 7, 13, 14 and 22 act as dual functional modulators of GABAA-Rs (promnemonic and anxiolytic agents) while only compounds 3 and 10 stand out as selectively displaying good antiamnesic and procognitive activity (1 and 3 mg/kg, respectively). PMID:23490154

  18. Identification of novel positive allosteric modulators and null modulators at the GABAA receptor α+β− interface

    PubMed Central

    Varagic, Zdravko; Wimmer, Laurin; Schnürch, Michael; Mihovilovic, Marko D; Huang, Shengming; Rallapalli, Sundari; Cook, James M; Mirheydari, Pantea; Ecker, Gerhard F; Sieghart, Werner; Ernst, Margot

    2013-01-01

    Background and Purpose GABAA receptors are the major inhibitory neurotransmitter receptors in the mammalian brain and the target of many clinically important drugs interacting with different binding sites. Recently, we demonstrated that CGS 9895 (2-(4-methoxyphenyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one) acts as a null modulator (antagonist) at the high affinity benzodiazepine binding site, but in addition elicits a strong enhancement of GABA-induced currents via a novel drug binding site at the extracellular α+β− interface. Here, we investigated 32 structural analogues of CGS 9895 for their ability to mediate their effects via the α1+β3− interface of GABAA receptors. Experimental Approach GABAA receptors were expressed in Xenopus laevis oocytes and investigated by the two-electrode voltage clamp method. Key Results We not only identified compounds with higher efficacy/potency than CGS 9895 for stimulating GABA-induced currents via the α1+β3-binding site, but also discovered compounds acting as null modulators at this site. Most of the compounds also acted as null modulators via the benzodiazepine binding site of GABAA receptors. But some of the positive allosteric modulators or null modulators exclusively exerted their action via the α+β− binding site. Conclusion and Implications Pyrazoloquinolinones and pyrazolopyridinones represent the first prototype of drug candidates mediating benzodiazepine like modulatory effects via the α+β-interface of GABAA receptors. The discovery of null modulators acting as inhibitors of the plus modulators provides a highly useful tool for the discovery of additional classes of compounds that can modulate GABAA receptors via this site, which may lead to novel therapeutic principles. Linked Article This article is accompanied by Varagic et al., pp. 384–399 of this issue. To view this article visit http://dx.doi.org/10.1111/bph.12153 PMID:23472852

  19. 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

  20. Novel α1 and γ2 GABAA receptor subunit mutations in families with idiopathic generalized epilepsy.

    PubMed

    Lachance-Touchette, Pamela; Brown, Patricia; Meloche, Caroline; Kinirons, Peter; Lapointe, Line; Lacasse, Hélène; Lortie, Anne; Carmant, Lionel; Bedford, Fiona; Bowie, Derek; Cossette, Patrick

    2011-07-01

    Epilepsy is a heterogeneous neurological disease affecting approximately 50 million people worldwide. Genetic factors play an important role in both the onset and severity of the condition, with mutations in several ion-channel genes being implicated, including those encoding the GABA(A) receptor. Here, we evaluated the frequency of additional mutations in the GABA(A) receptor by direct sequencing of the complete open reading frame of the GABRA1 and GABRG2 genes from a cohort of French Canadian families with idiopathic generalized epilepsy (IGE). Using this approach, we have identified three novel mutations that were absent in over 400 control chromosomes. In GABRA1, two mutations were found, with the first being a 25-bp insertion that was associated with intron retention (i.e. K353delins18X) and the second corresponding to a single point mutation that replaced the aspartate 219 residue with an asparagine (i.e. D219N). Electrophysiological analysis revealed that K353delins18X and D219N altered GABA(A) receptor function by reducing the total surface expression of mature protein and/or by curtailing neurotransmitter effectiveness. Both defects would be expected to have a detrimental effect on inhibitory control of neuronal circuits. In contrast, the single point mutation identified in the GABRG2 gene, namely P83S, was indistinguishable from the wildtype subunit in terms of surface expression and functionality. This finding was all the more intriguing as the mutation exhibited a high degree of penetrance in three generations of one French Canadian family. Further experimentation will be required to understand how this mutation contributes to the occurrence of IGE in these individuals. PMID:21714819

  1. Developmental regulation of voltage-gated K+ channel and GABAA receptor expression in Bergmann glial cells.

    PubMed

    Müller, T; Fritschy, J M; Grosche, J; Pratt, G D; Möhler, H; Kettenmann, H

    1994-05-01

    Bergmann glial cells are closely associated with neurons: during development they provide guiding structures for migrating granule cells and in the adult cerebellum they display intimate interactions with Purkinje cells. In this study, we have addressed the question of whether such changes in neuronal-glial interactions during development are accompanied by variations in the membrane properties of Bergmann glial cells. We used a mouse cerebellum slice preparation to study membrane currents of the Bergmann glial cells at various stages of development in situ using the patch-clamp technique. The distinct morphology of Bergmann glial cells was revealed by Lucifer yellow injections during recording. While Bergmann glial cells in mice of postnatal day 20 (P20) to P30 have thick processes with arborized, irregularly shaped leaf-like appendages, the processes of cells from younger mice (P5-P7) are thinner and smoother. This morphological maturation is accompanied by a variation in voltage-gated currents. In cells from P5 to P7, delayed outward- and inward-rectifying K+ currents were recorded, while older Bergmann glial cells were characterized by, large, voltage- and time-independent K+ currents. In addition, application of GABA induces two effects, a rapid activation of a Cl- conductance and a longer-lasting decrease in the (resting) K+ conductance. Both effects were mediated by benzodiazepine-insensitive GABAA receptors. Responses in cells of P5-P7 mice were large as compared to the small or even undetectable responses in P20-P30 cells. These GABAA receptors were characterized immunohistochemically in mice and rat brain sections with five subunit-specific antibodies. Bergmann glial cells exhibit a distinct but transient immunoreactivity for the GABAA receptor alpha 2-, alpha 3-, and delta-subunits. Staining is maximal between P7 and P10 and decreases gradually thereafter. In contrast, antibodies to the alpha 1- and beta 2,3-subunits fail to decorate Bergmann glial cells

  2. Association between GABA(A) receptor subunit polymorphisms and autism spectrum disorder (ASD).

    PubMed

    Sesarini, Carla V; Costa, Lucas; Grañana, Nora; Coto, Miguel Garcia; Pallia, Roberto C; Argibay, Pablo F

    2015-09-30

    ASD might be associated with alterations in excitation/inhibition ratio and GABA(A) has been implicated since it mediates synaptic inhibition. Polymorphisms in GABA receptor (GABAR) were studied: significant differences in allele and genotype frequencies observed between cases and controls (rs1912960, GABRA4). Haplotype analysis: rs1912960 (GABRA4) and rs211037 (GABRG2) overrepresented in cases. Rs1912960 has been associated with ASD and rs211037 with epilepsy. GABRA4 is associated with autism in the Argentinean dataset independently or in combination with GABRG2. PMID:26239769

  3. Cocaine effects on mouse incentive-learning and human addiction are linked to α2 subunit-containing GABAA receptors

    PubMed Central

    Dixon, Claire I.; Morris, Hannah V.; Breen, Gerome; Desrivieres, Sylvane; Jugurnauth, Sarah; Steiner, Rebecca C.; Vallada, Homero; Guindalini, Camila; Laranjeira, Ronaldo; Messas, Guilherme; Rosahl, Thomas W.; Atack, John R.; Peden, Dianne R.; Belelli, Delia; Lambert, Jeremy J.; King, Sarah L.; Schumann, Gunter; Stephens, David N.

    2010-01-01

    Because GABAA receptors containing α2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltage-clamp recordings from NAcc medium spiny neurons of mice with α2 gene deletion showed reduced synaptic GABAA receptor-mediated responses. Behaviorally, the deletion abolished cocaine’s ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of α2-GABAA receptors (α2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 15-4513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In α2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated locomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of α2−GABAA receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction. PMID:20133874

  4. 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. PMID:23103412

  5. 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

  6. 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

  7. 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

  8. GABAA-receptor activation in the subthalamic nucleus compensates behavioral asymmetries in the hemiparkinsonian rat.

    PubMed

    Petri, David; Pum, Martin; Vesper, Jan; Huston, Joseph P; Schnitzler, Alfons

    2013-09-01

    The subthalamic nucleus (STN) has a pivotal role in the pathophysiology of Parkinson's disease (PD). Modulation of STN activity (by lesions, pharmacological or electrical stimulation) has been shown to improve motor parameters in PD patients and in animal models of PD. In an attempt to characterize the neurochemical bases for such antiparkinsonian action, we address specific neurotransmitter systems via local pharmacological manipulation of the STN in hemiparkinsonian rats. Here, we have focused on the GABAergic and glutamatergic receptors in the STN. In animals with unilateral 6-hydroxydopamine lesions of the nigro-striatal tract, we administered either the selective GABAA-agonist muscimol (0.5 μg and 1.0 μg), the non-competitive N-methyl-d-aspartate (NMDA)-antagonist MK-801 (dizocilpine; 2.5 μg), or vehicle (0.25 μl) into the STN. The effects of GABAergic and glutamatergic modulation of the STN on motor parameters were assessed by gauging rotational behavior and locomotion. Application of muscimol ipsilateral to the side of dopamine-depletion influenced turning behavior in a dose-dependent fashion, with the low dose re-adjusting turning behavior to a non-biased distribution, and the high dose evoking contraversive turning. The administration of MK-801 did not have such effects. These findings give evidence for the involvement of GABAergic activation in the STN in the compensation of motor asymmetries in the hemiparkinsonian rat, whereas N-methyl-d-aspartate (NMDA)-antagonism was ineffective in this model of PD. PMID:23727148

  9. 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. PMID:25298123

  10. 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

  11. Tetramethylenedisulfotetramine Alters Ca2+ Dynamics in Cultured Hippocampal Neurons: Mitigation by NMDA Receptor Blockade and GABAA Receptor-Positive Modulation

    PubMed Central

    Pessah, Isaac N.

    2012-01-01

    Tetramethylenedisulfotetramine (TETS) is a potent convulsant that is considered a chemical threat agent. We characterized TETS as an activator of spontaneous Ca2+ oscillations and electrical burst discharges in mouse hippocampal neuronal cultures at 13–17 days in vitro using FLIPR Fluo-4 fluorescence measurements and extracellular microelectrode array recording. Acute exposure to TETS (≥ 2µM) reversibly altered the pattern of spontaneous neuronal discharges, producing clustered burst firing and an overall increase in discharge frequency. TETS also dramatically affected Ca2+ dynamics causing an immediate but transient elevation of neuronal intracellular Ca2+ followed by decreased frequency of Ca2+ oscillations but greater peak amplitude. The effect on Ca2+ dynamics was similar to that elicited by picrotoxin and bicuculline, supporting the view that TETS acts by inhibiting type A gamma-aminobutyric acid (GABAA) receptor function. The effect of TETS on Ca2+ dynamics requires activation of N-methyl-d-aspartic acid (NMDA) receptors, because the changes induced by TETS were prevented by MK-801 block of NMDA receptors, but not nifedipine block of L-type Ca2+ channels. Pretreatment with the GABAA receptor-positive modulators diazepam and allopregnanolone partially mitigated TETS-induced changes in Ca2+ dynamics. Moreover, low, minimally effective concentrations of diazepam (0.1µM) and allopregnanolone (0.1µM), when administered together, were highly effective in suppressing TETS-induced alterations in Ca2+ dynamics, suggesting that the combination of positive modulators of synaptic and extrasynaptic GABAA receptors may have therapeutic potential. These rapid throughput in vitro assays may assist in the identification of single agents or combinations that have utility in the treatment of TETS intoxication. PMID:22889812

  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. 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. PMID:26898547

  14. SAHA Enhances Proteostasis of Epilepsy-Associated α1(A322D)β2γ2 GABAa Receptors

    PubMed Central

    Di, Xiao-Jing; Han, Dong-Yun; Wang, Ya-Juan; Chance, Mark R.; Mu, Ting-Wei

    2013-01-01

    SUMMARY GABAa receptors are the primary inhibitory ion channels in the mammalian central nervous system. The A322D mutation in the α1 subunit of GABAa receptors is known to result in its degradation and reduce its cell surface expression, leading to loss of GABAa receptor function in autosomal dominant juvenile myoclonic epilepsy. Here, we show that SAHA, a FDA-approved drug, increases the transcription of the α1(A322D) subunit, enhances its folding and trafficking post-translationally, increases its cell surface level, and restores the GABA-induced maximal current in HEK293 cells expressing α1(A322D)β2γ2 receptors to 10% of that for wild type receptors. To enhance the trafficking efficiency of the α1(A322D) subunit, SAHA increases the BiP protein level and the interaction between the α1(A322D) subunit and calnexin. SAHA is the first reported drug that enhances epilepsy-associated GABAa receptor proteostasis. PMID:24211135

  15. Distinct structural changes in the GABAA receptor elicited by pentobarbital and GABA.

    PubMed

    Muroi, Yukiko; Theusch, Cassandra M; Czajkowski, Cynthia; Jackson, Meyer B

    2009-01-01

    The barbiturate pentobarbital binds to gamma-aminobutyric acid type A (GABA(A)) receptors, and this interaction plays an important role in the anesthetic action of this drug. Depending on its concentration, pentobarbital can potentiate (approximately 10-100 microM), activate (approximately 100-800 microM), or block (approximately 1-10 mM) the channel, but the mechanisms underlying these three distinct actions are poorly understood. To investigate the drug-induced structural rearrangements in the GABA(A) receptor, we labeled cysteine mutant receptors expressed in Xenopus oocytes with the sulfhydryl-reactive, environmentally sensitive fluorescent probe tetramethylrhodamine-6-maleimide (TMRM). We then used combined voltage clamp and fluorometry to monitor pentobarbital-induced channel activity and local protein movements simultaneously in real time. High concentrations of pentobarbital induced a decrease in TMRM fluorescence (F(TMRM)) of labels tethered to two residues in the extracellular domain (alpha(1)L127C and beta(2)L125C) that have been shown previously to produce an increase in F(TMRM) in response to GABA. Label at beta(2)K274C in the extracellular end of the M2 transmembrane helix reported a small but significant F(TMRM) increase during application of low modulating pentobarbital concentrations, and it showed a much greater F(TMRM) increase at higher concentrations. In contrast, GABA decreased F(TMRM) at this site. These results indicate that GABA and pentobarbital induce different structural rearrangements in the receptor, and thus activate the receptor by different mechanisms. Labels at alpha(1)L127C and beta(2)K274C change their fluorescence by substantial amounts during channel blockade by pentobarbital. In contrast, picrotoxin blockade produces no change in F(TMRM) at these sites, and the pattern of F(TMRM) signals elicited by the antagonist SR95531 differs from that produced by other antagonists. Thus, with either channel block by antagonists or

  16. [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. PMID:21800540

  17. 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

  18. 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

  19. Lamina-specific alterations in cortical GABA(A) receptor subunit expression in schizophrenia.

    PubMed

    Beneyto, Monica; Abbott, Andrew; Hashimoto, Takanori; Lewis, David A

    2011-05-01

    Dysfunction of the dorsolateral prefrontal cortex (DLPFC) in schizophrenia is associated with lamina-specific alterations in particular subpopulations of interneurons. In pyramidal cells, postsynaptic γ-aminobutyric acid (GABA(A)) receptors containing different α subunits are inserted preferentially in distinct subcellular locations targeted by inputs from specific interneuron subpopulations. We used in situ hybridization to quantify the laminar expression of α1, α2, α3, and α5 subunit, and of β1-3 subunit, mRNAs in the DLFPC of schizophrenia, and matched normal comparison subjects. In subjects with schizophrenia, mean GABA(A) α1 mRNA expression was 17% lower in layers 3 and 4, α2 expression was 14% higher in layer 2, α5 expression was 15% lower in layer 4, and α3 expression did not differ relative to comparison subjects. The mRNA expression of β2, which preferentially assembles with α1 subunits, was also 20% lower in layers 3 and 4, whereas β1 and β3 mRNA levels were not altered in schizophrenia. These expression differences were not attributable to medication effects or other potential confounds. These findings suggest that GABA neurotransmission in the DLPFC is altered at the postsynaptic level in a receptor subunit- and layer-specific manner in subjects with schizophrenia and support the hypothesis that GABA neurotransmission in this illness is predominantly impaired in certain cortical microcircuits. PMID:20843900

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

    PubMed Central

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

    2010-01-01

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

  1. Antibodies to GABAA receptor α1 and γ2 subunits

    PubMed Central

    Pettingill, Philippa; Kramer, Holger B.; Coebergh, Jan Adriaan; Pettingill, Rosie; Maxwell, Susan; Nibber, Anjan; Malaspina, Andrea; Jacob, Anu; Irani, Sarosh R.; Buckley, Camilla; Beeson, David; Lang, Bethan; Waters, Patrick

    2015-01-01

    Objective: To search for antibodies against neuronal cell surface proteins. Methods: Using immunoprecipitation from neuronal cultures and tandem mass spectrometry, we identified antibodies against the α1 subunit of the γ-aminobutyric acid A receptor (GABAAR) in a patient whose immunoglobulin G (IgG) antibodies bound to hippocampal neurons. We searched 2,548 sera for antibodies binding to GABAAR α, β, and γ subunits on live HEK293 cells and identified the class, subclass, and GABAAR subunit specificities of the positive samples. Results: GABAAR-Abs were identified in 40 of 2,046 (2%) referred sera previously found negative for neuronal antibodies, in 5/502 (1%) previously positive for other neuronal surface antibodies, but not in 92 healthy individuals. The antibodies in 40% bound to either the α1 (9/45, 20%) or the γ2 subunits (9/45, 20%) and were of IgG1 (94%) or IgG3 (6%) subclass. The remaining 60% had lower antibody titers (p = 0.0005), which were mainly immunoglobulin M (IgM) (p = 0.0025), and showed no defined subunit specificity. Incubation of primary hippocampal neurons with GABAAR IgG1 sera reduced surface GABAAR membrane expression. The clinical features of 15 patients (GABAAR α1 n = 6, γ2 n = 5, undefined n = 4) included seizures (47%), memory impairment (47%), hallucinations (33%), or anxiety (20%). Most patients had not been given immunotherapies, but one with new-onset treatment-resistant catatonia made substantial improvement after plasma exchange. Conclusions: The GABAAR α1 and γ2 are new targets for antibodies in autoimmune neurologic disease. The full spectrum of clinical features, treatment responses, correlation with antibody specificity, and in particular the role of the IgM antibodies will need to be assessed in future studies. PMID:25636713

  2. Abnormal subcellular localization of GABAA receptor subunits in schizophrenia brain.

    PubMed

    Mueller, T M; Remedies, C E; Haroutunian, V; Meador-Woodruff, J H

    2015-01-01

    Inhibitory neurotransmission is primarily mediated by γ-aminobutyric acid (GABA) activating synaptic GABA type A receptors (GABA(A)R). In schizophrenia, presynaptic GABAergic signaling deficits are among the most replicated findings; however, postsynaptic GABAergic deficits are less well characterized. Our lab has previously demonstrated that although there is no difference in total protein expression of the α1-6, β1-3 or γ2 GABA(A)R subunits in the superior temporal gyrus (STG) in schizophrenia, the α1, β1 and β2 GABA(A)R subunits are abnormally N-glycosylated. N-glycosylation is a posttranslational modification that has important functional roles in protein folding, multimer assembly and forward trafficking. To investigate the impact that altered N-glycosylation has on the assembly and trafficking of GABA(A)Rs in schizophrenia, this study used western blot analysis to measure the expression of α1, α2, β1, β2 and γ2 GABA(A)R subunits in subcellular fractions enriched for endoplasmic reticulum (ER) and synapses (SYN) from STG of schizophrenia (N = 16) and comparison (N = 14) subjects and found evidence of abnormal localization of the β1 and β2 GABA(A)R subunits and subunit isoforms in schizophrenia. The β2 subunit is expressed as three isoforms at 52 kDa (β2(52 kDa)), 50 kDa (β2(50 kDa)) and 48 kDa (β2(48 kDa)). In the ER, we found increased total β2 GABA(A)R subunit (β2(ALL)) expression driven by increased β2(50 kDa), a decreased ratio of β(248 kDa):β2(ALL) and an increased ratio of β2(50 kDa):β2(48 kDa). Decreased ratios of β1:β2(ALL) and β1:β2(50 kDa) in both the ER and SYN fractions and an increased ratio of β2(52 kDa):β(248 kDa) at the synapse were also identified in schizophrenia. Taken together, these findings provide evidence that alterations of N-glycosylation may contribute to GABAergic signaling deficits in schizophrenia by disrupting the assembly and trafficking of GABA(A)Rs. PMID:26241350

  3. Abnormal subcellular localization of GABAA receptor subunits in schizophrenia brain

    PubMed Central

    Mueller, T M; Remedies, C E; Haroutunian, V; Meador-Woodruff, J H

    2015-01-01

    Inhibitory neurotransmission is primarily mediated by γ-aminobutyric acid (GABA) activating synaptic GABA type A receptors (GABAAR). In schizophrenia, presynaptic GABAergic signaling deficits are among the most replicated findings; however, postsynaptic GABAergic deficits are less well characterized. Our lab has previously demonstrated that although there is no difference in total protein expression of the α1–6, β1–3 or γ2 GABAAR subunits in the superior temporal gyrus (STG) in schizophrenia, the α1, β1 and β2 GABAAR subunits are abnormally N-glycosylated. N-glycosylation is a posttranslational modification that has important functional roles in protein folding, multimer assembly and forward trafficking. To investigate the impact that altered N-glycosylation has on the assembly and trafficking of GABAARs in schizophrenia, this study used western blot analysis to measure the expression of α1, α2, β1, β2 and γ2 GABAAR subunits in subcellular fractions enriched for endoplasmic reticulum (ER) and synapses (SYN) from STG of schizophrenia (N=16) and comparison (N=14) subjects and found evidence of abnormal localization of the β1 and β2 GABAAR subunits and subunit isoforms in schizophrenia. The β2 subunit is expressed as three isoforms at 52 kDa (β252 kDa), 50 kDa (β250 kDa) and 48 kDa (β248 kDa). In the ER, we found increased total β2 GABAAR subunit (β2ALL) expression driven by increased β250 kDa, a decreased ratio of β248 kDa:β2ALL and an increased ratio of β250 kDa:β248 kDa. Decreased ratios of β1:β2ALL and β1:β250 kDa in both the ER and SYN fractions and an increased ratio of β252 kDa:β248 kDa at the synapse were also identified in schizophrenia. Taken together, these findings provide evidence that alterations of N-glycosylation may contribute to GABAergic signaling deficits in schizophrenia by disrupting the assembly and trafficking of GABAARs. PMID:26241350

  4. SH-I-048A, AN IN VITRO NONSELECTIVE SUPER-AGONIST AT THE BENZODIAZEPINE SITE OF GABAA RECEPTORS: THE APPROXIMATED ACTIVATION OF RECEPTOR SUBTYPES MAY EXPLAIN BEHAVIORAL EFFECTS

    PubMed Central

    Obradović, Aleksandar Lj.; Joksimović, Srđan; Poe, Michael M.; Ramerstorfer, Joachim; Varagic, Zdravko; Namjoshi, Ojas; Batinić, Bojan; Radulović, Tamara; Marković, Bojan; Roth, Brian; Sieghart, Werner; Cook, James M.; Savić, Miroslav M.

    2014-01-01

    Enormous progress in understanding the role of four populations of benzodiazepine-sensitive GABAA receptors was paralleled by the puzzling findings suggesting that substantial separation of behavioral effects may be accomplished by apparently non-selective modulators. We report on SH-I-048A, a newly-synthesized chiral positive modulator of GABAA receptors characterized by exceptional subnanomolar affinity, high efficacy and non-selectivity. Its influence on behavior was assessed in Wistar rats and contrasted to that obtained with 2 mg/kg diazepam. SH-I-048A reached micromolar concentrations in brain tissue, while the unbound fraction in brain homogenate was around 1.5%. The approximated electrophysiological responses, which estimated free concentrations of SH-I-048A or diazepam are able to elicit, suggested a similarity between the 10 mg/kg dose of the novel ligand and 2 mg/kg diazepam; however, SH-I-048A was relatively more active at α1- and α5-containing GABAA receptors. Behaviorally, SH-I-048A induced sedative, muscle relaxant and ataxic effects, reversed mechanical hyperalgesia 24 hours after injury, while it was devoid of clear anxiolytic actions and did not affect water-maze performance. While lack of clear anxiolytic actions may be connected with an enhanced potentiation at α1-containing GABAA receptors, the observed behavior in the rotarod, water maze and peripheral nerve injury tests was possibly affected by its prominent action at receptors containing the α5 subunit. The current results encourage further innovative approaches aimed at linking in vitro and in vivo data in order to help define fine-tuning mechanisms at four sensitive receptor populations that underlie subtle differences in behavioral profiles of benzodiazepine site ligands. PMID:24472579

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

    PubMed

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

    2016-06-01

    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/kg), whereas

  6. 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

  7. GABAA Receptor in the Thalamic Specific Relay System Contributes to the Propofol-Induced Somatosensory Cortical Suppression in Rat

    PubMed Central

    Zhang, Yu; Wang, Chaoping; Zhang, Yi; Zhang, Lin; Yu, Tian

    2013-01-01

    Interaction with the gamma-aminobutyric-acid-type-A (GABAA) receptors is recognized as an important component of the mechanism of propofol, a sedative-hypnotic drug commonly used as anesthetic. However the contribution of GABAA receptors to the central nervous system suppression is still not well understood, especially in the thalamocortical network. In the present study, we investigated if intracerebral injection of bicuculline (a GABAA receptor antagonist) into the thalamus ventral posteromedial nucleus (VPM, a thalamus specific relay nuclei that innervated S1 mostly) could reverse propofol-induced cortical suppression, through recording the changes of both spontaneous and somatosensory neural activities in rat’s somatosensory cortex (S1). We found that after injection of bicuculline into VPM, significant increase of neural activities were observed in all bands of local field potentials (total band, 182±6%), while the amplitude of all components in somatosensory evoked potentials were also increased (negative, 121±9% and positive, 124±6%).These data support that the potentiation of GABAA receptor-mediated synaptic inhibition in a thalamic specific relay system seems to play a crucial role in propofol-induced cortical suppression in the somatosensory cortex of rats. PMID:24324778

  8. Bioactivity-guided isolation of GABA(A) receptor modulating constituents from the rhizomes of Actaea racemosa.

    PubMed

    Cicek, Serhat S; Khom, Sophia; Taferner, Barbara; Hering, Steffen; Stuppner, Hermann

    2010-12-27

    Black cohosh (Actaea racemosa) is a frequently used herbal remedy for the treatment of mild climacteric symptoms. In the present study, the modulation of γ-aminobutryic acid (GABA)-induced chloride currents (I(GABA)) through GABA type A (GABA(A)) receptors by black cohosh extracts and isolated compounds was investigated. GABA(A) receptors, consisting of α(1), β(2), and γ(2S) subunits, were expressed in Xenopus laevis oocytes, and potentiation of I(GABA) was measured using the two-microelectrode voltage clamp technique. In a bioactivity-guided isolation procedure the positive modulation of I(GABA) could be restricted to the plant terpenoid fractions, resulting in the isolation of 11 cycloartane glycosides, of which four significantly (p < 0.05) enhanced I(GABA). The most efficient effect was observed for 23-O-acetylshengmanol 3-O-β-d-xylopyranoside (4, 100 μM), enhancing I(GABA) by 1692 ± 201%, while actein (1), cimigenol 3-O-β-d-xylopyranoside (6), and 25-O-acetylcimigenol 3-O-α-l-arabinopyranoside (8) were significantly less active. In the absence of GABA, only 4 induced small (not exceeding 1% of I(GABA-max)) chloride inward currents through GABA(A) receptors. It is hypothesized that the established positive allosteric modulation of GABA(A) receptors may contribute to beneficial effects of black cohosh extracts in the treatment of climacteric symptoms. PMID:21082802

  9. HPLC-based activity profiling of Angelica pubescens roots for new positive GABAA receptor modulators in Xenopus oocytes.

    PubMed

    Zaugg, Janine; Eickmeier, Eva; Rueda, Diana C; Hering, Steffen; Hamburger, Matthias

    2011-04-01

    A petroleum ether extract of the traditional Chinese herbal drug Duhuo (roots of Angelica pubescens Maxim. f. biserrata Shan et Yuan), showed significant activity in a functional two-microelectrode voltage clamp assay with Xenopus oocytes which expressed recombinant γ-aminobutyric acid type A (GABA(A)) receptors of the subtype α(1)β(2)γ(2S). HPLC-based activity profiling of the active extract revealed six compounds responsible for the GABA(A) receptor modulating activity. They were identified by microprobe NMR and high resolution mass spectrometry as columbianetin acetate (1), imperatorin (3), cnidilin (4), osthol (5), and columbianedin (6). In concentration-dependent experiments, osthol and cnidilin showed the highest potentiation of the GABA induced chloride current (273.6%±39.4% and 204.5%±33.2%, respectively at 300 μM). Bisabolangelone (2) only showed minor activity at the GABA(A) receptor. The example demonstrates that HPLC-based activity profiling is a simple and efficient method to rapidly identify GABA(A) receptor modulators in a bioactive plant extract. PMID:21147202

  10. 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

  11. 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. PMID:23293594

  12. Direct activation of GABAA receptors by barbiturates in cultured rat hippocampal neurons.

    PubMed Central

    Rho, J M; Donevan, S D; Rogawski, M A

    1996-01-01

    1. The direct activation of the GABAA receptor by pentobarbitone (PB) and phenobarbitone (PHB) was characterized in cultured rat hippocampal neurons using whole-cell voltage clamp and single channel recording techniques. 2. In whole-cell recordings, PB and PHB produced a concentration-dependent activation of Cl- current (EC50 values, 0.33 and 3.0 mM, respectively). The response to the barbiturates was similar to that produced by GABA, although GABA was more potent (EC50, 5.5 microM). PB and PHB were substantially more potent in enhancing the response to 1 microM GABA (EC50 values, 94 microM and 0.89 mM, respectively). The maximal magnitude of the responses to PB was similar to that of the maximal response to GABA or GABA + PB. PHB appeared to be modestly less efficacious. 3. The mean deactivation time constant for whole-cell Cl- currents evoked by 1 mM PB + 1 microM GABA was significantly longer (480 +/- 34 ms) than for 1 mM PB (170 +/- 9 ms) or 1 microM GABA (180 +/- 14 ms) alone. 4. Whole-cell currents directly activated by 300 microM PB and 1 microM GABA were blocked by the GABA receptor antagonists bicuculline and picrotoxin. 5. Unitary GABAA receptor channel currents evoked by 300 microM PB had similar main conductance, mean open time and mean burst duration as those activated by 2 microM GABA alone. Single channel openings and bursts were of shorter mean duration when 100 and 300 microM PHB were used. 6. High concentrations of PB (1-3 mM) and PHB (3-10 mM) produced a rapid block of currents activated by the barbiturate alone or by the barbiturate in the presence of 1 microM GABA. The estimated IC50 values for block of PB- and PHB-potentiated GABA currents were 2.8 and 12.9 mM, respectively. 7. Single channel currents activated by high concentrations of PB and PHB alone or in the presence of GABA demonstrated flickering, probably reflecting fast channel block. 8. We conclude that the gating of the GABAA receptor channel by PHB and PB is functionally similar to

  13. 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

  14. Hippocampal Extracellular Signal-Regulated Kinase Signaling has a Role in Passive Avoidance Memory Retrieval Induced by GABAA Receptor Modulation in Mice

    PubMed Central

    Kim, Dong Hyun; Kim, Jong Min; Park, Se Jin; Lee, Seungheon; Shin, Chan Young; Cheong, Jae Hoon; Ryu, Jong Hoon

    2012-01-01

    Available evidence strongly suggests that the γ-aminobutyric acid type A (GABAA) receptor has a crucial role in memory retrieval. However, the signaling mechanisms underlying the role of GABAA receptor modulation in memory retrieval are unclear. We conducted one-trial passive avoidance task with pre-retention trial drug administration in the hippocampus to test the effects of GABAA receptor modulation on memory retrieval. We further tested the co-involvement of signaling molecules: extracellular signal-regulated kinase (ERK), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and cAMP responsive element-binding protein (CREB). First, we observed that the phosphorylation of hippocampal ERK was required for memory retrieval during the task. Accordingly, to investigate whether GABAA receptor activation or inhibition induces ERK phosphorylation during memory retrieval, drugs that target the GABAA receptor were administered into the hippocampus before the retention trial. Muscimol, a GABAA receptor agonist, and diazepam, an agonist to benzodiazepine-binding site of GABAA receptor, blocked retention trial-induced ERK phosphorylation and impaired memory retrieval. Furthermore, co-treatment with sub-effective dose of U0126, a mitogen-activated protein kinase inhibitor, blocked the upregulation of ERK phosphorylation and impaired memory retrieval, and bicuculline methiodide (BMI), a GABAA receptor antagonist, increased ERK phosphorylation induced by the retention trial and facilitated memory retrieval. Finally, the effects of BMI were blocked by the co-application of a sub-effective dose of U0126. These results suggest that GABAA receptor-mediated memory retrieval is closely related to ERK activity. PMID:22169949

  15. Differential cerebellar GABAA receptor expression in mice with mutations in CaV2.1 (P/Q-type) calcium channels.

    PubMed

    Kaja, S; Payne, A J; Nielsen, E Ø; Thompson, C L; van den Maagdenberg, A M J M; Koulen, P; Snutch, T P

    2015-09-24

    Ataxia is the predominant clinical manifestation of cerebellar dysfunction. Mutations in the human CACNA1A gene, encoding the pore-forming α1 subunit of CaV2.1 (P/Q-type) calcium channels, underlie several neurological disorders, including Episodic Ataxia type 2 and Familial Hemiplegic Migraine type 1 (FHM1). Several mouse mutants exist that harbor mutations in the orthologous Cacna1a gene. The spontaneous Cacna1a mutants Rolling Nagoya (tg(rol)), Tottering (tg) and Leaner (tg(ln)) mice exhibit behavioral motor phenotypes, including ataxia. Transgenic knock-in (KI) mouse strains with the human FHM1 R192Q and S218L missense mutations have been generated. R192Q KI mice are non-ataxic, whereas S218L KI mice display a complex behavioral phenotype that includes cerebellar ataxia. Given the dependence of γ-aminobutyric acid type A (GABAA) receptor subunit functioning on localized calcium currents, and the functional link between GABAergic inhibition and ataxia, we hypothesized that cerebellar GABAA receptor expression is differentially affected in Cacna1a mutants and contributes to the ataxic phenotype. Herein we quantified functional GABAA receptors and pharmacologically dissociated cerebellar GABAA receptors in several Cacna1a mutants. We did not identify differences in the expression of GABAA receptor subunits or in the number of functional GABAA receptors in the non-ataxic R192Q KI strain. In contrast, tg(rol) mice had a ∼15% decrease in the number of functional GABAA receptors, whereas S218L KI mice showed a ∼29% increase. Our data suggest that differential changes in cerebellar GABAA receptor expression profile may contribute to the neurological phenotype of cerebellar ataxia and that targeting GABAA receptors might represent a feasible complementary strategy to treat cerebellar ataxia. PMID:26208839

  16. GABAA Receptor Blockade Enhances Memory Consolidation by Increasing Hippocampal BDNF Levels

    PubMed Central

    Kim, Dong Hyun; Kim, Jong Min; Park, Se Jin; Cai, Mudan; Liu, Xiaotong; Lee, Seungheon; Shin, Chan Young; Ryu, Jong Hoon

    2012-01-01

    Memory consolidation is the process by which acquired information is converted to something concrete to be retrieved later. Here we examined a potential role for brain-derived neurotrophic factor (BDNF) in mediating the enhanced memory consolidation induced by the GABAA receptor antagonist, bicuculline methiodide. With the administration of an acquisition trial in naïve mice using a passive avoidance task, mature BDNF (mBDNF) levels were temporally changed in the hippocampal CA1 region, and the lowest levels were observed 9 h after the acquisition trial. In the passive avoidance task, bicuculline methiodide administration within 1 h of training but not after 3 h significantly increased latency time in the retention trial 24 h after the acquisition trial. Concomitantly, 1 h post-training administration of bicuculline methiodide, which enhanced memory consolidation, significantly increased mBDNF levels 9 h after training compared to those of the vehicle-treated control group. In addition, exogenous human recombinant BDNF (hrBDNF) administration 9 h after training into the hippocampal CA1 region facilitated memory consolidation confirming that the increase in mBDNF at around 9 h after training plays a key role in the enhancement of memory consolidation. Moreover, the increases in latency time and immediate early gene expressions by bicuculline methiodide or hrBDNF were significantly blocked by anisomycin, a protein synthesis inhibitor, K252a, a tyrosine receptor kinase (Trk) inhibitor, or anti-TrkB IgG. These findings suggest that the increase in the level of mBDNF and its function during a restricted time window after training are required for the enhancement of memory consolidation by GABAA receptor blockade. PMID:21900885

  17. 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

  18. 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. PMID:26857960

  19. 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

  20. Modulation of human GABAA receptor function: a novel mode of action of drugs of abuse.

    PubMed

    Hondebrink, L; Meulenbelt, J; van Kleef, R G D M; van den Berg, M; Westerink, R H S

    2011-12-01

    Drugs of abuse are known to mainly affect the dopaminergic and serotonergic system, although behavioral studies indicated that the GABA-ergic system also plays a role. We therefore investigated the acute effects of several commonly used drugs of abuse (methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA) and meta-chlorophenylpiperazine (mCPP)) on the function of the human α(1)β(2)γ(2) GABA(A) receptor (hGABA(A)-R), expressed in Xenopus oocytes, using the two-electrode voltage-clamp technique. Although none of the tested drugs acted as full agonist on the hGABA(A)-R, some drugs induced differential modulation of hGABA(A)-R function, depending on the degree of receptor occupancy. Methamphetamine did not affect the GABA-evoked current at high receptor occupancy, but induced a minor inhibition at low receptor occupancy. Its metabolite amphetamine slightly potentiated the GABA-evoked current. MDMA and its metabolite MDA both inhibited the current at low receptor occupancy. However, MDMA did not affect the current at high occupancy, whereas MDA induced a potentiation. mCPP induced a strong inhibition (max. ∼ 80%) at low receptor occupancy, but ∼ 25% potentiation at high receptor occupancy. Competitive binding to one of the GABA-binding sites could explain the drug-induced inhibitions observed at low receptor occupancy, whereas an additional interaction with a positive allosteric binding site may play a role in the observed potentiations at high receptor occupancy. This is the first study to identify direct modulation of hGABA(A)-Rs as a novel mode of action for several drugs of abuse. Consequently, hGABA(A)-Rs should be considered as target for psychiatric pharmaceuticals and in developing treatment for drug intoxications. PMID:21729720

  1. Estimating the efficiency of benzodiazepines on GABAA receptors comprising γ1 or γ2 subunits

    PubMed Central

    Baburin, I; Khom, S; Timin, E; Hohaus, A; Sieghart, W; Hering, S

    2008-01-01

    Background and purpose: Heterologous expression of α1, β2 and γ2S(γ1) subunits produces a mixed population of GABAA receptors containing α1β2 or α1β2γ2S(γ1) subunits. GABA sensitivity (lower in receptors containing γ1 or γ2S subunits) and the potentiation of GABA-activated chloride currents (IGABA) by benzodiazepines (BZDs) are dependent on γ2S(γ1) incorporation. A variable γ subunit incorporation may affect the estimation of IGABA potentiation by BZDs. We propose an approach for estimation of BZD efficiency that accounts for mixed population of α1β2 and α1β2γ2S(γ1) receptors. Experimental approach: We investigated the relation between GABA sensitivity (EC50) and BZD modulation by analysing triazolam-, clotiazepam- and midazolam-induced potentiation of IGABA in Xenopus oocytes under two-microelectrode voltage clamp. Key results: Plotting EC50 versus BZD-induced shifts of GABA concentration-response curves (ΔEC50(BZD)) of oocytes injected with different amounts of α1, β2 and γ2S(γ1) cRNA (1:1:1–1:1:10) revealed a linear regression between γ2S(γ1)-mediated reduction of GABA sensitivity (EC50) and ΔEC50(BZD). The slope factors of the regression were always higher for oocytes expressing α1β2γ1 subunit receptors (1.8±0.1 (triazolam), 1.6±0.1 (clotiazepam), 2.3±0.2 (midazolam)) than for oocytes expressing α1β2γ2S receptors (1.4±0.1 (triazolam), 1.4±0.1 (clotiazepam), 1.3±0.1 (midazolam)). Mutant GABAA receptors (α1β2-R207Cγ2S) with lower GABA sensitivity showed higher drug efficiencies (slope factors=1.1±0.1 (triazolam), 1.1±0.1 (clotiazepam), 1.2±0.1 (midazolam)). Conclusions and implications: Regression analysis enabled the estimation of BZD efficiency when variable mixtures of α1β2 and α1β2γ2S(γ1) receptors are expressed and provided new insights into the γ2S(γ1) dependency of BZD action. PMID:18604239

  2. 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

  3. Abuse Potential of Soma®: the GABAA Receptor as a Target

    PubMed Central

    Gonzalez, Lorie A.; Gatch, Michael B.; Forster, Michael J.; Dillon, Glenn H.

    2010-01-01

    Soma® (carisoprodol) is an increasingly abused, centrally-acting muscle relaxant. Despite the prevalence of carisoprodol abuse, its mechanism of action remains unclear. Its sedative effects, which contribute to its therapeutic and recreational use, are generally attributed to the actions of its primary metabolite, meprobamate, at GABAA receptors (GABAAR). Meprobamate is a controlled substance at the federal level; ironically, carisoprodol is not currently classified as such. Using behavioral and molecular pharmacological approaches, we recently demonstrated carisoprodol, itself, is capable of modulating GABAAR function in a manner similar to central nervous system depressants. Its functional similarities with this highly addictive class of drugs may contribute to the abuse potential of carisoprodol. The site of action of carisoprodol has not been identified; based on our studies, interaction with benzodiazepine or barbiturate sites is unlikely. These recent findings, when coupled with numerous reports in the literature, support the contention that the non-controlled status of carisoprodol should be reevaluated. PMID:20419052

  4. Ocean acidification slows retinal function in a damselfish through interference with GABAA receptors.

    PubMed

    Chung, Wen-Sung; Marshall, N Justin; Watson, Sue-Ann; Munday, Philip L; Nilsson, Göran E

    2014-02-01

    Vision is one of the most efficient senses used by animals to catch prey and avoid predators. Therefore, any deficiency in the visual system could have important consequences for individual performance. We examined the effect of CO2 levels projected to occur by the end of this century on retinal responses in a damselfish, by determining the threshold of its flicker electroretinogram (fERG). The maximal flicker frequency of the retina was reduced by continuous exposure to elevated CO2, potentially impairing the capacity of fish to react to fast events. This effect was rapidly counteracted by treatment with a GABA antagonist (gabazine), indicating that GABAA receptor function is disrupted by elevated CO2. In addition to demonstrating the effects of elevated CO2 on fast flicker fusion of marine fishes, our results show that the fish retina could be a model system to study the effects of high CO2 on neural processing. PMID:24477607

  5. Differential Roles of GABAA Receptor Subtypes in Benzodiazepine-Induced Enhancement of Brain-Stimulation Reward

    PubMed Central

    Reynolds, Lauren M; Engin, Elif; Tantillo, Gabriella; Lau, Hew Mun; Muschamp, John W; Carlezon, William A; Rudolph, Uwe

    2012-01-01

    Benzodiazepines such as diazepam are widely prescribed as anxiolytics and sleep aids. Continued use of benzodiazepines, however, can lead to addiction in vulnerable individuals. Here, we investigate the neural mechanisms of the behavioral effects of benzodiazepines using the intracranial self-stimulation (ICSS) test, a procedure with which the reward-enhancing effects of these drugs can be measured. Benzodiazepines bind nonselectively to several different GABAA receptor subtypes. To elucidate the α subunit(s) responsible for the reward-enhancing effects of benzodiazepines, we examined mice carrying a histidine-to-arginine point mutation in the α1, α2, or α3 subunit, which renders the targeted subunit nonresponsive to diazepam, other benzodiazepines and zolpidem. In wild-type and α1-point-mutated mice, diazepam caused a dose-dependent reduction in ICSS thresholds (reflecting a reward-enhancing effect) that is comparable to the reduction observed following cocaine administration. This effect was abolished in α2- and α3-point-mutant mice, suggesting that these subunits are necessary for the reward-enhancing action of diazepam. α2 Subunits appear to be particularly important, since diazepam increased ICSS thresholds (reflecting an aversive-like effect) in α2-point-mutant animals. Zolpidem, an α1-preferring benzodiazepine-site agonist, had no reward-enhancing effects in any genotype. Our findings implicate α2 and α3 subunit containing GABAA receptors as key mediators of the reward-related effects of benzodiazepines. This finding has important implications for the development of new medications that retain the therapeutic effects of benzodiazepines but lack abuse liability. PMID:22763624

  6. 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

  7. Juvenile stress-induced alteration of maturation of the GABAA receptor alpha subunit in the rat.

    PubMed

    Jacobson-Pick, Shlomit; Elkobi, Alina; Vander, Shelly; Rosenblum, Kobi; Richter-Levin, Gal

    2008-11-01

    Profound evidence indicates that GABAA receptors are important in the control of physiological response to stress and anxiety. The alpha subunit type composition contributes significantly to the functional characterization of the GABAA receptors. The alpha2, alpha3, alpha5 subunits are predominately expressed in the brain during embryonic and early postnatal periods of normal rats, whilst alpha1 are most prominent during later developmental stages. In the present study, we examined the long-term effects of juvenile stress on GABA alpha subunit expression in adulthood in the amygdala and hippocampus. We applied the elevated platform stress paradigm at juvenility and used the open-field and startle response tests to assess anxiety level in adulthood. Juvenile stress effects without behavioural tests in adulthood were also examined since previous studies indicated that the mere exposure to these tests might be stressful for rats, enhancing the effects of the juvenile exposure to stress. In adulthood, we quantitatively determined the level of expression of alpha1, alpha2 and alpha3 in the hippocampus and amygdala. Our results indicate that subjecting juvenile stressed rats to additional challenges in adulthood results in an immature-like expression profile of these subunits. To test for potential functional implications of these alterations we examined the effects of the anxiolytic (diazepam) and the sedative (brotizolam) benzodiazepines on juvenile stressed and control rats following additional challenges in adulthood. Juvenile stressed rats were more sensitive to diazepam and less sensitive to brotizolam, suggesting that the alterations in GABA alpha subunit expression in these animals have functional consequences. PMID:18364065

  8. 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

  9. ROBUST PHOTO-REGULATION OF GABAA RECEPTORS BY ALLOSTERIC MODULATION WITH A PROPOFOL ANALOG

    PubMed Central

    Yue, Lan; Pawlowski, Michal; Dellal, Shlomo S.; Xie, An; Feng, Feng; Otis, Thomas S.; Bruzik, Karol S.; Qian, Haohua; Pepperberg, David R.

    2013-01-01

    Photochemical switches represent a powerful method for improving pharmacological therapies and controlling cellular physiology. Here we report the photo-regulation of GABAA receptors (GABAARs) by a derivative of propofol (2,6-diisopropylphenol), a GABAAR allosteric modulator, that we have modified to contain photo-isomerizable azobenzene. Using α1β2γ2 GABAARs expressed in Xenopus laevis oocytes and native GABAARs of isolated retinal ganglion cells, we show that the trans-azobenzene isomer of the new compound (trans-MPC088), generated by visible light (wavelengths ~440 nm), potentiates the GABA-elicited response and at higher concentrations directly activates the receptors. cis-MPC088, generated from trans-MPC088 by UV light (~365 nm), produces little if any receptor potentiation/activation. In cerebellar slices, MPC088 co-applied with GABA affords bidirectional photo-modulation of Purkinje cell membrane current and spike-firing rate. The findings demonstrate photo-control of GABAARs by an allosteric ligand and open new avenues for fundamental and clinically oriented research on GABAARs, a major class of neurotransmitter receptors in the central nervous system. PMID:23033071

  10. 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

  11. Capturing state-dependent dynamic events of GABAA-receptors: a microscopic look into the structural and functional insights.

    PubMed

    Payghan, Pavan V; Bera, Indrani; Bhattacharyya, Dhananjay; Ghoshal, Nanda

    2016-08-01

    The γ-amino butyric acid type A receptors (GABAA-Rs) are the key players in the mammalian brain that meditate fast inhibitory neurotransmission events. The structural integrity of these ligand-gated ion channel controls chloride ion permeability, which in turn monitors important pharmacological functions. Despite ample studies on GABAA-Rs, there was a need for a study on full-length receptor structures, devoted to track structure-function correlations based on their dynamic behavior consideration. We have employed molecular dynamics simulations accompanied by other biophysical methods to shed light on sequential and unaddressed questions like How GABAA-R structure facilitates the entry of GABA molecules at its two orthosteric binding sites? After entry, what structural features and changes monitor site-wise GABA binding differences? In the same context, what are the roles and responsibilities of loops such as C and F? On physiologically relevant time scales, how open to close state transition occurs? How salt bridges such as E155-R207 and E153-R207 maintain state-dependent C-loop structures? In an attempt, our simulation study unravels the complete course of GABA binding-unbinding pathway. This provides us with the relevant understanding of state-dependent dynamic events of GABAA-Rs. PMID:26372345

  12. 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

  13. GABAA receptors in the dorsal raphé nucleus of mice: escalation of aggression after alcohol consumption

    PubMed Central

    Takahashi, Aki; Kwa, Carolyn; DeBold, Joseph F.

    2010-01-01

    Rationale The dorsal raphé nucleus (DRN), the origin for serotonin (5-HT) in forebrain areas, has been implicated in the neural control of escalated aggression. Gamma aminobutyric acid type-A (GABAA) and type-B (GABAB) receptors are expressed in the DRN and modulate 5-HT neuronal activity, and both play a role in the behavioral effect of alcohol. Objective The purpose of this study is to examine the interaction between drugs acting on GABA receptors in the DRN and alcohol in their effects on aggressive behaviors. Method Male CFW mice, housed with a female, were trained to self-administer ethanol (1.0 g/kg) or water via an operant conditioning panel in their home cage. Immediately after they drank either ethanol or water, the animals were microinfused with a GABAergic drug into the DRN, and their aggressive behaviors were assessed 10 min later. Muscimol (0.006 nmol), a GABAA receptor agonist, escalated alcohol-heightened aggression but had no effect in the absence of ethanol. This effect of muscimol was prominent in the animals that showed alcohol-heightened aggression, but not the animals that reduced or did not change aggressive behavior after ethanol infusion compared to water. On the other hand, the GABAB agonist baclofen (0.06 nmol) increased aggressive behavior similarly in both water and ethanol conditions. Antagonists of the GABAA and GABAB receptors, bicuculline (0.006 nmol) and phaclofen (0.3 nmol) respectively, did not suppress heightened-aggressive behavior induced by ethanol self-administration. Conclusion GABAA receptors in the DRN are one of the neurobiological targets of alcohol-heightened aggression. Activation of the GABAB receptors in the DRN also produced escalated aggression, but that is independent of the effect of alcohol. PMID:20589493

  14. 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

  15. 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

  16. Diazepam-bound GABAA receptor models identify new benzodiazepine binding-site ligands

    PubMed Central

    Richter, Lars; de Graaf, Chris; Sieghart, Werner; Varagic, Zdravko; Mörzinger, Martina; de Esch, Iwan J P; Ecker, Gerhard F; Ernst, Margot

    2012-01-01

    Benzodiazepines exert their anxiolytic, anticonvulsant, muscle-relaxant and sedative-hypnotic properties by allosterically enhancing the action of GABA at GABAA receptors via their benzodiazepine-binding site. Although these drugs have been used clinically since 1960, the molecular basis of this interaction is still not known. By using multiple homology models and an un biased docking protocol, we identified a binding hypothesis for the diazepam-bound structure of the benzodiazepine site, which was confirmed by experimental evidence. Moreover, two independent virtual screening approaches based on this structure identified known benzodiazepine-site ligands from different structural classes and predicted potential new ligands for this site. Receptor-binding assays and electrophysiological studies on recombinant receptors confirmed these predictions and thus identified new chemotypes for the benzodiazepine-binding site. Our results support the validity of the diazepam-bound structure of the benzodiazepine-binding pocket, demonstrate its suitability for drug discovery and pave the way for structure-based drug design. PMID:22446838

  17. Opposite effects of GABAA and NMDA receptor antagonists on ethanol-induced behavioral sleep in rats.

    PubMed

    Beleslin, D B; Djokanović, N; Jovanović Mićić, D; Samardzić, R

    1997-01-01

    The effects of the GABAA receptor antagonists, pentylenetetrazol, bicuculline, and picrotoxin, the glycine antagonist, strychnine, and the NMDA receptor antagonist, memantine, on ethanol-induced behavioral sleep and body temperature were investigated. Pentylenetetrazol, bicuculline, and picrotoxin given prior and following ethanol reduced the behavioral sleep and potentiated the hypothermia caused by ethanol. However, convulsions appeared when bicuculline, but not pentylenetetrazol and picrotoxin, were given following ethanol. After the reversal of unconsciousness in rats without convulsions the animals remained awake throughout the experiments without motor incoordination, hyperexcitability, and sedation, but they were in hypothermia within 12 h. The glycine antagonist, strychnine, given prior or after ethanol had virtually no effect on ethanol-induced behavioral sleep and hypothermia. Ethanol given prior or following strychnine failed to antagonize strychnine-induced convulsions. The NMDA receptor antagonist, memantine, given following ethanol potentiated the behavioral sleep and had virtually no effect on hypothermia induced by ethanol. It is suggested that the ethanol-induced behavioral sleep may be attributed to its ability to enhance the GABAergic mechanisms and to inhibit NMDA-mediated excitatory responses. However, the ethanol-induced hypothermia may be ascribed solely to the facilitation of GABAergic transmission. Further, it is postulated that a bidirectional inhibitory system subserves the regulation of behavioral sleep and convulsions. However, one-way inhibitory system underlies the ethanol-induced hypothermia. PMID:9085718

  18. A neurosteroid potentiation site can be moved among GABAA receptor subunits.

    PubMed

    Bracamontes, John R; Li, Ping; Akk, Gustav; Steinbach, Joe Henry

    2012-11-15

    Endogenous neurosteroids are among the most potent and efficacious potentiators of activation of GABA(A) receptors. It has been proposed that a conserved glutamine residue in the first membrane-spanning region (TM1 region) of the α subunits is required for binding of potentiating neurosteroids. Mutations of this residue can reduce or remove the ability of steroids to potentiate function. However, it is not known whether potentiation requires that a steroid interact with the α subunit, or not. To examine this question we mutated the homologous residue in the β2 and γ2L subunits to glutamine, and found that these mutations could not confer potentiation by allopregnanolone (3α5αP) when expressed in receptors containing ineffective α1 subunits. However, potentiation is restored when the entire TM1 region from the α1 subunit is transferred to the β2 or γ2L subunit. Mutations in the TM1 region that affect potentiation when made in the α1 subunit have similar effects when made in transferred TM1 region. Further, the effects of 3α5αP on single-channel kinetics are similar for wild-type receptors and receptors with moved TM1 regions. These results support the idea that steroids bind in the transmembrane regions of the receptor. The observations are consistent with previous work indicating that neurosteroid potentiation is mediated by an action that affects the receptor as a whole, rather than an individual subunit or pair of subunits, and in addition demonstrate that the mechanism is independent of the nature of the subunit that interacts with steroid. PMID:22988137

  19. Possible involvement of the CA1 GABAA receptors upon acquisition and expression of the ACPA-induced place preference in mice.

    PubMed

    Nasehi, Mohammad; Kamali-Dolatabadi, Leila; Torabi-Nami, Mohammad; Zarrindast, Mohammad-Reza

    2016-07-01

    A plethora of investigations has substantiated a close relationship between cannabinoidergic and GABAergic systems in hippocampal CA1. The crucial role of these two systems in regulation of the addictive behaviors is well described. The aim of the current study was to investigate whether the CA1 GABAA receptors are involved in ACPA (a selective CB1 cannabinoid receptor agonist)-induced rewarding effects using the condition place preference (CPP) protocol. Moreover, the hole-board paradigm was used to measure the exploratory behaviors which may potentially influence this phenomenon. Results showed that ACPA (0.02mg/kg, i.p.) induced CPP. Applying a 3-day conditioning schedule, we found that the sole administration of the GABAA receptor agonist, muscimol (0.125, 0.25 and 0.5μg/mouse; intra-CA1), or the GABAA receptor antagonist, bicuculline (0.0635, 0.125 and 0.25μg/mouse; intra-CA1), fail to induce CPP or CPA (condition place aversion). Similarly, injection of the subthreshold dose of muscimol (0.125μg/mouse, intra-CA1) decreased the CPP acquisition induced by ACPA. Similar intervention with the subthreshold dose of bicuculline (0.125μg/mouse, intra-CA1) did not alter the CPP acquisition induced by ACPA. Furthermore, the sole intra-CA1 administration of muscimol (0.125, 0.25 and 0.5μg/mouse) and bicuculline (0.0635, 0.125 and 0.25μg/mouse; intra-CA1) prior to testing, did not induce CPP or CPA. Similar interventions revealed that muscimol and bicuculline increase and decrease CPP expression induced by ACPA, respectively. The ACPA- and muscimol-induced CPP could be blocked by bicuculline. Taken together, the CA1 GABAA receptors seem to be possibly involved in the modulation of acquisition or expression process upon ACPA-induced CPP. PMID:27090228

  20. 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

  1. Identification of both GABAA receptors and voltage-activated Na+ channels as molecular targets of anticonvulsant α-asarone

    PubMed Central

    Wang, Ze-Jun; Levinson, Simon R.; Sun, Liqin; Heinbockel, Thomas

    2014-01-01

    Alpha (α)-asarone, a major effective component isolated from the Chinese medicinal herb Acorus tatarinowii, is clinically used as medication for treating epilepsy, cough, bronchitis, and asthma. In the present study, we demonstrated that α-asarone targets central nervous system GABAA receptor as well as voltage-gated Na+ channels. Using whole-cell patch-clamp recording, α-asarone inhibited spontaneous firing of output neurons, mitral cells (MCs), in mouse olfactory bulb brain slice preparation and hyperpolarized the membrane potential of MCs. The inhibitory effect of α-asarone persisted in the presence of ionotropic glutamate receptor blockers but was eliminated after adding a GABAA receptor blocker, suggesting that GABAA receptors mediated the inhibition of MCs by α-asarone. This hypothesis was supported by the finding that α-asarone evoked an outward current, but did not influence inhibitory postsynaptic currents (IPSCs). In addition to inhibiting spontaneous firing, α-asarone also inhibited the Nav1.2 channel, a dominant rat brain Na+ channel subtype. The effects of α-asarone on a defined Nav1.2 were characterized using transfected cells that stably expressed the Nav1.2 channel isoform. α-Asarone displayed strong tonic inhibition of Nav1.2 currents in a concentration- and membrane potential-dependent fashion. α-Asarone reduced channel availability in steady-state inactivation protocols by enhancing or stabilizing Na+ channel inactivation. Both Na+ channel blockade and activation of GABAA receptors provide a possible mechanism for the known anti-epileptic effects of α-asarone. It also suggests that α-asarone could benefit patients with cough possibly through inhibiting a Na+ channel subtype to inhibit peripheral and/or central sensitization of cough reflexes. PMID:24653701

  2. Human locus coeruleus neurons express the GABA(A) receptor gamma2 subunit gene and produce benzodiazepine binding.

    PubMed

    Hellsten, Kati S; Sinkkonen, Saku T; Hyde, Thomas M; Kleinman, Joel E; Särkioja, Terttu; Maksimow, Anu; Uusi-Oukari, Mikko; Korpi, Esa R

    2010-06-21

    Noradrenergic neurons of the locus coeruleus project throughout the cerebral cortex and multiple subcortical structures. Alterations in the locus coeruleus firing are associated with vigilance states and with fear and anxiety disorders. Brain ionotropic type A receptors for gamma-aminobutyric acid (GABA) serve as targets for anxiolytic and sedative drugs, and play an essential regulatory role in the locus coeruleus. GABA(A) receptors are composed of a variable array of subunits forming heteropentameric chloride channels with different pharmacological properties. The gamma2 subunit is essential for the formation of the binding site for benzodiazepines, allosteric modulators of GABA(A) receptors that are clinically often used as sedatives/hypnotics and anxiolytics. There are contradictory reports in regard to the gamma2 subunit's expression and participation in the functional GABA(A) receptors in the mammalian locus coeruleus. We report here that the gamma2 subunit is transcribed and participates in the assembly of functional GABA(A) receptors in the tyrosine hydroxylase-positive neuromelanin-containing neurons within postmortem human locus coeruleus as demonstrated by in situ hybridization with specific gamma2 subunit oligonucleotides and autoradiographic assay for flumazenil-sensitive [(3)H]Ro 15-4513 binding to benzodiazepine sites. These sites were also sensitive to the alpha1 subunit-preferring agonist zolpidem. Our data suggest a species difference in the expression profiles of the alpha1 and gamma2 subunits in the locus coeruleus, with the sedation-related benzodiazepine sites being more important in man than rodents. This may explain the repeated failures in the transition of novel drugs with a promising neuropharmacological profile in rodents to human clinical usage, due to intolerable sedative effects. PMID:20417252

  3. Activation-induced regulation of GABAA receptors: Is there a link with the molecular basis of benzodiazepine tolerance?

    PubMed

    Gravielle, María Clara

    2016-07-01

    Benzodiazepines have been used clinically for more than 50 years to treat disorders such as insomnia, anxiety, and epilepsy, as well as to aid muscle relaxation and anesthesia. The therapeutic index for benzodiazepines if very high and the toxicity is low. However, their usefulness is limited by the development of either or both tolerance to most of their pharmacological actions and dependence. Tolerance develops at different rates depending on the pharmacological action, suggesting the existence of distinct mechanisms for each behavioral parameter. Alternatively, multiple mechanisms could coexist depending on the subtype of GABAA receptor expressed and the brain region involved. Because most of the pharmacological actions of benzodiazepines are mediated through GABAA receptor binding, adaptive alterations in the number, structure, and/or functions of these receptors may play an important role in the development of tolerance. This review is focused on the regulation of GABAA receptors induced by long-term benzodiazepine exposure and its relationship with the development of tolerance. Understanding the mechanisms behind benzodiazepine tolerance is critical for designing drugs that could maintain their efficacy during long-term treatments. PMID:26733466

  4. 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

  5. A novel modulatory binding site for zinc on the GABAA receptor complex in cultured rat neurones.

    PubMed Central

    Smart, T G

    1992-01-01

    1. The properties of gamma-aminobutyric acidA (GABAA) receptor-ion channel complexes and the interaction with the transition metal zinc, were studied on rat sympathetic and cerebellar neurones in dissociated culture using patch clamp recording techniques. 2. The antagonism of GABA-induced membrane currents by zinc on sympathetic neurones was subject to developmental influence. Using embryonic sympathetic neurones acutely cultured for 24-72 h, GABA responses were more depressed by zinc when compared to responses evoked on adult neurones cultured for the same period. For neurones developing in vivo, the percentage inhibition of GABA responses produced by zinc in embryonic neurones was estimated to decline by 50% after 48.2 days following birth. 3. Embryonic sympathetic neurones maintained in culture for prolonged periods (40-50 days in vitro, DIV) became less sensitive to zinc when compared to neurones cultured for shorter periods (10-20 DIV). The decrease in the zinc inhibition for neurones maintained in vitro proceeded at an apparent rate of 0.55% per day. 4. Activation of the GABA receptor by muscimol (0.2-2 microM) was also antagonized by zinc (50-100 microM). 5. Lowering the pH of the perfusing Krebs solution did not affect the inhibition of GABA responses by zinc on sympathetic neurones. 6. Modulation of the GABAA receptor by some benzodiazepines, a barbiturate, a steroid based on pregnanolone, or antagonists bicuculline and picrotoxinin, did not interfere with the antagonism exerted by zinc on sympathetic neurones. A novel binding site for zinc on the GABAA receptor is proposed. 7. Analysis of the GABA-activated current noise on sympathetic neurones revealed two kinetic components to the power spectra requiring a double Lorentzian fit. The time constant describing the fast component (tau 2, 2.1 ms) was unaffected by zinc, whereas the slow component time constant (tau 1, 21.7 ms) was slightly reduced to 17.1 ms. 8. The apparent single-channel conductance for

  6. Oxytocin prevents ethanol actions at δ subunit-containing GABAA receptors and attenuates ethanol-induced motor impairment in rats

    PubMed Central

    Bowen, Michael T.; Peters, Sebastian T.; Absalom, Nathan; Chebib, Mary; Neumann, Inga D.; McGregor, Iain S.

    2015-01-01

    Even moderate doses of alcohol cause considerable impairment of motor coordination, an effect that substantially involves potentiation of GABAergic activity at δ subunit-containing GABAA receptors (δ-GABAARs). Here, we demonstrate that oxytocin selectively attenuates ethanol-induced motor impairment and ethanol-induced increases in GABAergic activity at δ-GABAARs 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 GABAARs. 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 GABAAR agonist 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, which binds at a different site on δ-GABAARs than ethanol. Vasopressin, which is a nonapeptide with substantial structural similarity to oxytocin, did not alter ethanol effects at δ-GABAARs. This pattern of results confirms the specificity of the interaction between oxytocin and ethanol at δ-GABAARs. Finally, our in vitro constructs did not express any oxytocin receptors, meaning that the observed interactions occur directly at δ-GABAARs. 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. PMID:25713389

  7. 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. PMID:25713389

  8. GABAA and GABAB receptor-mediated effects in guinea-pig ileum.

    PubMed

    Giotti, A; Luzzi, S; Spagnesi, S; Zilletti, L

    1983-03-01

    -pig ileum: a bicuculline-sensitive GABA(A) receptor, which elicits contraction through an excitatory action on cholinergic post-ganglionic neurones; and a bicuculline-insensitive GABA(B) receptor which causes relaxation through an inhibitory presynaptic action on cholinergic post-ganglionic neurones. We confirm that GABA, homotaurine and muscimol are GABA(A) agonists, while GABA and (-)-baclofen are GABA(B) agonists. PMID:6301600

  9. Felbamate is a subunit selective modulator of recombinant gamma-aminobutyric acid type A receptors expressed in Xenopus oocytes.

    PubMed

    Simeone, Timothy A; Otto, James F; Wilcox, Karen S; White, H Steve

    2006-12-15

    Felbamate (2-phenyl-1,3-propanediol dicarbamate) is clinically available for the treatment of refractory epileptic seizures, and is known to modulate several ion channels including gamma-aminobutyric acid type A (GABA(A)) receptors. To determine felbamate subunit selectivity for GABA(A) receptors we expressed 15 different GABA(A) receptor combinations in Xenopus laevis oocytes. Felbamate positively modulated GABA-currents of alpha(1)beta(2)gamma(2S), alpha(1)beta(3)gamma(2S), alpha(2)beta(2)gamma(2S) and alpha(2)beta(3)gamma(2S), whereas felbamate was either ineffective or negatively modulated the other 11 receptor combinations. Regional distributions of GABA(A) receptor subunits suggest that felbamate may differentially modulate distinct inhibitory circuits, a possibility that may have relevance to felbamate efficacy in refractory epilepsies. PMID:17056029

  10. Effects of propofol on the dopamine, metabolites and GABAA receptors in media prefrontal cortex in freely moving rats

    PubMed Central

    Wang, Yuan; Yu, Tian; Yuan, Chengdong; Yuan, Jie; Luo, Zhuxin; Pan, Yunchao; Zhang, Yi; Zhang, Yu; Yu, Buwei

    2016-01-01

    Recent researches indicate that the mechanism of anesthetic induce loss of consciousness (LOC) is related to dopamine dysfunction in the media prefrontal cortex (mPFC). Given GABAA receptors are the main target for commonly intravenous anesthetic propofol, in this study, we test whether that propofol induced LOC mediate by GABAA receptors in mPFC through altering the dopamine and its metabolites. In the present study, we use Loss of righting reflex (LORR) and Recovery of righting reflex (RORR) as measure to respectively reflect the status of unconsciousness and consciousness recovery in rats. We imitate the clinical anesthesia process, found the minimum of induction and maintenance concentration of propofol respectively was 11 mg/kg and 40 mg/kg per hour. Then, microdialysis technique was used to observe the change of dopamine (DA), metabolites 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) before and after intravenous infusion of propofol from caudal vein of freely moving rats. The results showed that propofol can increase the level of DOPAC except HVA, and reduced the level of DA in mPFC during unconsciousness of rats. DOPAC and DA return to the baseline level when the rats began to regain consciousness. Local reverse dialysis infusion of GABAA receptor antagonist GABAzine (50 uM) in mPFC can promote the time of LORR, reduce the time of RORR, and increase the basal level of DOPAC. With this condition, propofol increased HVA instead of DOPAC, whereas the DA was still reduced. These results suggest that propofol may induce unconsciousness by directly inhibiting dopamine release in the mPFC, and this effect does not be mediated by GABAA receptor in mPFC.