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Introduction Arenaria kansuensis Maxim. (AKM) is one of the most valued medicinal and edible herbs widely used in Qinghai-Tibet Plateau and there is also a large number of AKM bioactive constituents for health benefits of human beings. However, few works have referred to phytochemical content, fingerprint analysis and quality control of AKM. Therefore, the establishment of validated analytical methods is urgently needed for fingerprint comparison and quantitative analysis of AKM multicomponent. Objectives To determine quantitatively and compare the phytochemical constituents of AKM located at different areas. Methodology The chemical constituents in AKM samples were separated, identified, and quantified by high-performance liquid chromatography (HPLC) with a diode array detector. The discrimination and separation models for the chemical constituents were developed by chemometric analysis. Results The flavones and β-carboline alkaloids were rich in AKM herbs, and the overall pattern of phytochemical profiles was the same, while the significant differences were detected in the total flavonoids, total β-carboline alkaloids and individual contents, especially the predominant compounds such as tricin and arenarine B. This demonstrated that ecogeographical origin gave an important impact on phytochemical compositions which could be considered as reliable parameters for classifying the AKM resources. Moreover, the contents of AKM constituents were higher in July and/or August than other months of the year, and there were no significant differences in the main phytochemical contents between cultivated and wild AKM herbs. Conclusion This study could provide credible data and method for geographical origin trace, comprehensive evaluation and further utilization of AKM resources.

Background Anxiety is a global public health problem affecting the lives of large numbers of patients (pts) and their families. Some studies suggest that pts with MINOCA (myocardial infarction with non-obstructive coronary arteries) have more anxiety levels than the rest of pts with myocardial infarction (MI). The aim of this study is to compare anxiety levels between pts with MINOCA and the rest of MI pts using a validated scale: The State Trait Anxiety Inventory (STAI). Methods An analytical and observational study was developed in a University Hospital. We analysed the clinical data of all consecutive MI pts admitted to our centre from July 2017 to December 2020. Inclusion criteria were determined by the 4th Universal Definition of Myocardial Infarction and the latest definitions of MINOCA according to the 2020 ESC Guidelines. A group of experts reviewed all MINOCA cases in order to exclude those who did not fulfil the selection standards and takotsubo syndrome and myocarditis pts were excluded. STAI questionnaire was completed by each patient during admission. Data collected included “trait anxiety”, that refers to relatively stable individual differences in anxiety-proneness, whereas “state anxiety” is a temporary emotion due to a particular situation (hospitalization in this case). Total score in each subgroup ranges from 0 to 60 points, where a higher score correlates with major anxiety levels. Follow up analysis included major adverse cardiovascular events (MACE: cardiovascular readmission, myocardial reinfarction, stroke and death from any cause). Survival analysis is based on Cox regression. Median follow-up was 25±23 months. Results From a total of 413 consecutive MI pts, 243 (58.8%) completed the questionnaire. Of them, 32 (13%) were MINOCA pts. There were no significant differences in trait anxiety between both groups (MINOCA mean value 21±14 points vs rest of MI 19±10 points, p=0.9), nor in state anxiety (20±14 vs 19±10 points, p=0.8). There were also no significant differences when data were analysed by percentiles: 37.5% of MINOCA pts were below P25 and 28% of the rest of MI were also in this percentile. At the other end of the scale, 31.3% of MINOCA pts were above P75, as well as 22.7% of the rest of MI pts (table 1). During follow up, punctuation in trait anxiety in MINOCA pts showed no differences in MACE (HR 1.01, CI 95% (0.9–1.07)), nor did punctuation in state anxiety (HR 1.01, CI 95% (0.9–1.06)). This was also observed in the rest of MI pts: trait anxiety HR 1.01, CI 95% (0.9–1.04) and state anxiety HR 0.9, CI 95% (0.9–1.01). Conclusion In this study, there were no significant differences in anxiety levels between MINOCA pts and the rest of MI pts. The lack of takotsubo pts in this study reflect real data from MINOCA according to the latest definitions. Subjective emotions could lead to mistaken findings, making it necessary to determine emotional disorders with validated and objective tools. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): European Society of Cardiology Table 1. Percentile values of STAI.

Background : Our previous studies on Asterothamnus centrali-asiaticus Novopokr. (ACN) and Arenaria kansuensis Maxim. (AKM) had led to the isolation of some phytochemical constituents and evaluation of anticonvulsant effect based on their extracts. ACN and AKM have been widely used in traditional Tibetan herbs for neuropsychiatric diseases and cardiopulmonary disorders. Purpose : The purpose is to investigate structure-activity relationships of flavonoids isolated from ACN and AKM, for binding to the benzodiazepine site (BZ-S) of γ-aminobutyric acid type A (GABAA) receptor complex, and to search for anticonvulsant compounds without undesirable effects such as myorelaxation and sedation. Study design and methods : The affinities of these flavonoids for the BZ-S of GABAA receptors were determined by [3H]flunitrazepam binding to mouse cerebellum membranes in vitro. And the anticonvulsant, myorelaxant and sedative effects were determined by pentylenetetrazol (PTZ)-induced seizure and electrogenic seizure protection, rotarod test and locomotor activity test, respectively. Results : Fifteen and thirteen flavonoids were isolated from ACN and AKM, respectively. Structure-activity relationships analysis indicated that 6-and/or 8-OMe flavones exhibited the most potent binding affinity to GABAA receptors. Furthermore, 2′,4′,5,7-tetrahydroxy-5′,6-dimethoxyflavone (DMF, IC50 value of 0.10 μM), a flavone isolated from ACN, presented high anticonvulsant activity against chemical-induced seizures and electrogenic seizures, without myorelaxation and sedation. Conclusion : This study suggested that these flavones, especially DMF, are new BZ receptor ligands and prospective therapeutic candidates for seizures.

Previously, the phytochemical constituents of Biebersteinia heterostemon Maxim (BHM) and Arenaria kansuensis Maxim (AKM) were studied and the evaluation of anxiolytic effect based on their extracts was also investigated. The two traditional Tibetan herbs, BHM and AKM, have been widely used in Qinghai-Tibet Plateau for cardiopulmonary disorders and neuropsychiatric diseases. The anxiolytic activities of a number of agents mediated by α2/3-containing GABAThe flavonoid constituents were separated and purified through the repeatedly silica gel or/and C18 column chromatography. The affinities of the compounds for BZ-S of GABAEleven total flavonoid compounds were obtained from the Tibetan herbs (BHM and AKM). The flavones with 6-and/or 8-OMe possessed the most potent binding affinity to GABAThis present study indicated that the flavones, especially DMS, are novel GABA

Anxiety disorders are the most common mental illness in the U.S. and are estimated to consume one-third of the country's mental health spending. Although anxiolytic therapies are available, many patients exhibit treatment-resistance, relapse, or substantial side effects. An urgent need exists to explore the underlying mechanisms of chronic anxiety and to develop alternative therapies. Presently, we identified dihydromyricetin (DHM), a flavonoid that has anxiolytic properties in a mouse model of isolation-induced anxiety. Socially isolated mice demonstrated increased anxiety levels and reduced exploratory behavior measured by elevated plus-maze and open-field tests. Socially isolated mice showed impaired GABAergic neurotransmission, including reduction in GABAA receptor-mediated extrasynaptic tonic currents, as well as amplitude and frequency of miniature inhibitory postsynaptic currents measured by whole-cell patch-clamp recordings from hippocampal slices. Furthermore, intracellular ATP levels and gephyrin expression decreased in anxious animals. DHM treatment restored ATP and gephyrin expression, GABAergic transmission and synaptic function, as well as decreased anxiety-like behavior. Our findings indicate broader roles for DHM in anxiolysis, GABAergic neurotransmission, and synaptic function. Collectively, our data suggest that reduction in intracellular ATP and gephyrin contribute to the development of anxiety, and represent novel treatment targets. DHM is a potential candidate for pharmacotherapy for anxiety disorders.

gamma-Aminobutyric acid (GABA)-mediated inhibitory neurotransmission and the gene products involved were discovered during the mid-twentieth century. Historically, myriad existing nervous system drugs act as positive and negative allosteric modulators of these proteins, making GABA a major component of modern neuropharmacology, and suggesting that many potential drugs will be found that share these targets. Although some of these drugs act on proteins involved in synthesis, degradation, and membrane transport of GABA, the GABA receptors Type A (GABAAR) and Type B (GABABR) are the targets of the great majority of GABAergic drugs. This discovery is due in no small part to Professor Norman Bowery. Whereas the topic of GABABR is appropriately emphasized in this special issue, Norman Bowery also made many insights into GABAAR pharmacology, the topic of this article. GABAAR are members of the ligand-gated ion channel receptor superfamily, a chloride channel family of a dozen or more heteropentameric subtypes containing 19 possible different subunits. These subtypes show different brain regional and subcellular localization, age-dependent expression, and potential for plastic changes with experience including drug exposure. Not only are GABAAR the targets of agonist depressants and antagonist convulsants, but most GABAAR drugs act at other (allosteric) binding sites on the GABAAR proteins. Some anxiolytic and sedative drugs, like benzodiazepine and related drugs, act on GABAAR subtype-dependent extracellular domain sites. General anesthetics including alcohols and neurosteroids act at GABAAR subunit-interface trans-membrane sites. Ethanol at high anesthetic doses acts on GABAAR subtype-dependent trans-membrane domain sites. Ethanol at low intoxicating doses acts at GABAAR subtype-dependent extracellular domain sites. Thus GABAAR subtypes possess pharmacologically specific receptor binding sites for a large group of different chemical classes of clinically important neuropharmacological agents. This article is part of the "Special Issue Dedicated to Norman G. Bowery".

While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABAA receptors (GABAARs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABAAR, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABAARs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with immunolabeling at perisynaptic locations in Fmr1 KO mice. While α4 immunogold particles were also reduced at perisynaptic locations in the Fmr1 KO mice, the labeling was increased at synaptic sites. Together these findings suggest that, in the dentate gyrus, altered surface expression of the δ subunit, rather than a decrease in δ subunit expression alone, could be limiting δ subunit-mediated tonic inhibition in this model of FXS. Finding ways to increase surface expression of the δ subunit of the GABAAR could be a novel approach to treatment of hyperexcitability-related alterations in FXS.

Alcohol (EtOH) intoxication causes changes in the rodent brain γ-aminobutyric acid receptor (GABAAR) subunit composition and function, playing a crucial role in EtOH withdrawal symptoms and dependence. Building evidence indicates that withdrawal from acute EtOH and chronic intermittent EtOH (CIE) results in decreased EtOH-enhanced GABAAR δ subunit-containing extrasynaptic and EtOH-insensitive α1βγ2 subtype synaptic GABAARs but increased synaptic α4βγ2 subtype, and increased EtOH sensitivity of GABAAR miniature postsynaptic currents (mIPSCs) correlated with EtOH dependence. Here we demonstrate that after acute EtOH intoxication and CIE, upregulation of hippocampal α4βγ2 subtypes, as well as increased cell-surface levels of GABAAR α2 and γ1 subunits, along with increased α2β1γ1 GABAAR pentamers in hippocampal slices using cell-surface cross-linking, followed by Western blot and coimmunoprecipitation. One-dose and two-dose acute EtOH treatments produced temporal plastic changes in EtOH-induced anxiolysis or withdrawal anxiety, and the presence or absence of EtOH-sensitive synaptic currents correlated with cell surface peptide levels of both α4 and γ1(new α2) subunits. CIE increased the abundance of novel mIPSC patterns differing in activation/deactivation kinetics, charge transfer, and sensitivity to EtOH. The different mIPSC patterns in CIE could be correlated with upregulated highly EtOH-sensitive α2βγ subtypes and EtOH-sensitive α4βγ2 subtypes. Naïve α4 subunit knockout mice express EtOH-sensitive mIPSCs in hippocampal slices, correlating with upregulated GABAAR α2 (and not α4) subunits. Consistent with α2, β1, and γ1 subunits genetically linked to alcoholism in humans, our findings indicate that these new α2-containing synaptic GABAARs could mediate the maintained anxiolytic response to EtOH in dependent individuals, rat or human, contributing to elevated EtOH consumption.

The first cryoEM high-resolution structure of a recombinant full-length heterotrimeric γ-aminobutyric acid (GABA) type A receptor (GABAR) subtype α1β3γ2L in complex with important GABAR ligands was published by the Aricescu lab in January 2019 (1,2). GABARs are the major receptors mediating rapid inhibitory neurotransmission in the central nervous system and members of the pentameric ligand-gated ion channel (pLGIC) gene superfamily.

Benzodiazepines are among the most potent and efficacious compounds ever discovered for anticonvulsant action. This includes the 1,4 benzodiazepines such as diazepam and clonazepam and the 1,5 benzodiazepines such as clobazam. This includes the 1,4 benzodiazepines such as diazepam and clonazepam and the 1,5 benzodiazepines such as clobazam. The use of benzodiazepines as antiepileptics is described in later chapters, which should be consulted for drug names, structures, and treatment details. Benzodiazepines have shown some success in adjunct therapy for refractory partial epilepsy and refractory generalized seizures. The activity of benzodiazepines as anticonvulsants against a variety of seizure types, e.g., maximal electroshock, pentylenetetrazole, picrotoxin, bicuculline, and kindled seizures decreases over a period of weeks, i.e., tolerance develops to the anticonvulsant action. The central nervous system depressant activity of benzodiazepines had been considered for many years to involve enhancement of inhibitory synaptic transmission rather than blockade of action potentials or of excitatory synaptic transmission.

Background Duplications of 15q11.2-q13.1 (Dup15q syndrome), including the paternally imprinted gene UBE3A and three nonimprinted gamma-aminobutyric acid type-A (GABAA) receptor genes, are highly penetrant for neurodevelopmental disorders such as autism spectrum disorder (ASD). To guide targeted treatments of Dup15q syndrome and other forms of ASD, biomarkers are needed that reflect molecular mechanisms of pathology. We recently described a beta EEG phenotype of Dup15q syndrome, but it remains unknown which specific genes drive this phenotype. Methods To test the hypothesis that UBE3A overexpression is not necessary for the beta EEG phenotype, we compared EEG from a reference cohort of children with Dup15q syndrome (n = 27) to (1) the pharmacological effects of the GABAA modulator midazolam (n = 12) on EEG from healthy adults, (2) EEG from typically developing (TD) children (n = 14), and (3) EEG from two children with duplications of paternal 15q (i.e., the UBE3A-silenced allele). Results Peak beta power was significantly increased in the reference cohort relative to TD controls. Midazolam administration recapitulated the beta EEG phenotype in healthy adults with a similar peak frequency in central channels (f = 23.0 Hz) as Dup15q syndrome (f = 23.1 Hz). Both paternal Dup15q syndrome cases displayed beta power comparable to the reference cohort. Conclusions Our results suggest a critical role for GABAergic transmission in the Dup15q syndrome beta EEG phenotype, which cannot be explained by UBE3A dysfunction alone. If this mechanism is confirmed, the phenotype may be used as a marker of GABAergic pathology in clinical trials for Dup15q syndrome. Electronic supplementary material The online version of this article (10.1186/s13229-019-0280-6) contains supplementary material, which is available to authorized users.

Following publication of the original article [1], we have been notified that the Ethics statement of the articles should be changed. The Ethics statement now reads

There is a need for treatments that can safely promote regulatory lymphocyte responses. T cells express GABA receptors (GABAA-Rs) and GABA administration can inhibit Th1-mediated processes such as type 1 diabetes and rheumatoid arthritis in mouse models. Whether GABAA-R agonists can also inhibit Th17-driven processes such as experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), is an open question. GABA does not pass through the blood-brain barrier (BBB) making it ill-suited to inhibit the spreading of autoreactivity within the CNS. Homotaurine is a BBB-permeable amino acid that antagonizes amyloid fibril formation and was found to be safe but ineffective in long-term Alzheimer’s disease clinical trials. Homotaurine also acts as GABAA-R agonist with better pharmacokinetics than that of GABA. Working with both monophasic and relapsing-remitting mouse models of EAE, we show that oral administration of homotaurine can (1) enhance CD8+CD122+PD-1+ and CD4+Foxp3+ Treg, but not Breg, responses, (2) inhibit autoreactive Th17 and Th1 responses, and (3) effectively ameliorate ongoing disease. These observations demonstrate the potential of BBB-permeable GABAA-R agonists as a new class of treatment to enhance CD8+ and CD4+ Treg responses and limit Th17 and Th1-medaited inflammation in the CNS.

Ethnopharmacological relevance Previously, the phytochemical constituents of Biebersteinia heterostemon Maxim (BHM) and Arenaria kansuensis Maxim (AKM) were studied and the evaluation of anxiolytic effect based on their extracts was also investigated. The two traditional Tibetan herbs, BHM and AKM, have been widely used in Qinghai-Tibet Plateau for cardiopulmonary disorders and neuropsychiatric diseases. The anxiolytic activities of a number of agents mediated by α2/3-containing GABAA receptors (GABAARs) have been demonstrated through the genetic and pharmacological studies. Flavonoids, such as flavones and flavanols, are a class of ligands that act at GABAARs and exhibit anxiolytic effects in vivo. Here, the flavonoids are the predominant constituents isolated from BHM and AKM. And our purpose is to investigate structure-activity relationships of the flavonoid compounds with binding to BZ-S of GABAAR complexes, and to search for anxiolytic constituents that lack undesirable-effects such as sedation and myorelaxation. Materials and methods The flavonoid constituents were separated and purified through the repeatedly silica gel or/and C18 column chromatography. The affinities of the compounds for BZ-S of GABAARs were detected by the radioreceptor binding assay with bovine cerebellum membranes, in which the different recombinant subunits-containing GABAARs were expressed in HEK 293T cells. The behavior tests, including elevated plus maze, locomotor activity, holeboard, rotarod and horizontal wire, were used to determine and evaluate the anxiolytic, sedative, and myorelaxant effects of these flavonoids. Results Eleven total flavonoid compounds were obtained from the Tibetan herbs (BHM and AKM). The flavones with 6-and/or 8-OMe possessed the most potent binding affinity to GABAARs, which were based on the result of structure-activity relationships analysis. Demethoxysudachitin (DMS, Ki = 0.59 μM), a flavone that binds to recombinant α1-3/5 subunit-containing GABAARs, was isolated from BHM, and exhibited high anxiolytic activity, without inducing sedation and myorelaxation. Moreover, the anxiolytic effect of DMS was antagonized by flumazenil, suggesting that a mode of action was mediated via the BZ-S of GABAARs. Conclusions This present study indicated that the flavones, especially DMS, are novel GABAAR ligands and therapeutic potential candidates for anxiety.

Our previous studies on Asterothamnus centrali-asiaticus Novopokr. (ACN) and Arenaria kansuensis Maxim. (AKM) had led to the isolation of some phytochemical constituents and evaluation of anticonvulsant effect based on their extracts. ACN and AKM have been widely used in traditional Tibetan herbs for neuropsychiatric diseases and cardiopulmonary disorders.The purpose is to investigate structure-activity relationships of flavonoids isolated from ACN and AKM, for binding to the benzodiazepine site (BZ-S) of γ-aminobutyric acid type A (GABAThe affinities of these flavonoids for the BZ-S of GABAFifteen and thirteen flavonoids were isolated from ACN and AKM, respectively. Structure-activity relationships analysis indicated that 6-and/or 8-OMe flavones exhibited the most potent binding affinity to GABAThis study suggested that these flavones, especially DMF, are new BZ receptor ligands and prospective therapeutic candidates for seizures.

GABAA receptors (GABAARs) are the main inhibitory neurotransmitter receptors and mediate rapid synaptic as well as slow extrasynaptic inhibitory neurotransmission. Structurally, GABAARs are ligand-gated ion channels formed by a total of 19 homologous subunits, each with four transmembrane domains assembled as pentamers, forming a GABA-gated Cl– channels. The major classical synaptic GABAAR subtypes are formed by 2α2β and a γ subunit, with six different possible α subunits, three different β subunits, and three γ subunits, with the most abundant subtype, α1β2γ2 receptors. More recently, highly GABA-sensitive extrasynaptic δ subunit-containing receptors that are persistently (tonically) activated by low ambient levels of GABA have entered the limelight. GABAARs are targets for sedative/hypnotic and anxiolytic drugs (e.g., benzodiazepines [BZs] and other BZ site ligands), as well as general anesthetics (e.g., etomidate, propofol, barbiturate, and neurosteroid anesthetics, and possibly volatile agents and long-chain alcohols), and also are important targets for alcohol actions.

Alcohol (ethanol, EtOH) is one of the most widely abused drugs with profound effects on brain function and behavior. GABAA receptors (GABAARs) are one of the major targets for EtOH in the brain. Temporary plastic changes in GABAARs after withdrawal from a single EtOH exposure occur both in vivo and in vitro, which may be the basis for chronic EtOH addiction, tolerance and withdrawal symptoms. Extrasynaptic δ-GABAAR endocytosis is implicated in EtOH-induced GABAAR plasticity, but the mechanisms by which the relative abundance and localization of specific GABAARs are altered by EtOH exposure and withdrawal remain unclear. In this study, we investigated the mechanisms underlying rapid regulation of extrasynaptic δ-GABAAR by a single EtOH withdrawal in cultured rat hippocampal neurons. Thirty-minutes EtOH (60 mM) exposure increased extrasynaptic tonic current (Itonic) amplitude without affecting synaptic GABAAR function in neurons. In contrast, at 30 min after withdrawal, Itonic amplitude and responsiveness to acute EtOH were both reduced. Similar results occurred in neurons with okadaic acid (OA) or phorbol 12,13-dibutyrate (PDBu) exposure. Protein kinase C (PKC) inhibition prevented the reduction of Itonic amplitude and the tolerance to acute EtOH, as well as the reduction of GABAAR-δ subunit abundance induced by a single EtOH withdrawal. Moreover, EtOH withdrawal selectively increased PKCδ level, whereas PKCδ inhibition specifically rescued the EtOH-induced alterations in GABAAR-δ subunit level and δ-GABAAR function. Together, we provided strong evidence for the important roles of PKCδ in the rapid regulation of extrasynaptic δ-GABAAR induced by a single EtOH withdrawal.

gamma-Aminobutyric acid (GABA)-mediated inhibitory neurotransmission and the gene products involved were discovered during the mid-twentieth century. Historically, myriad existing nervous system drugs act as positive and negative allosteric modulators of these proteins, making GABA a major component of modern neuropharmacology, and suggesting that many potential drugs will be found that share these targets. Although some of these drugs act on proteins involved in synthesis, degradation, and membrane trasnsport of GABA, the GABA receptors Type A (GABAAR) and Type B (GABABR) are the targets of the great majority of GABAergic drugs. This discovery is due in no small part to Professor Norman Bowery. Whereas the topic of GABABR is appropriately emphasized in this special issue, Norman Bowery also made many insights into GABAAR pharmacology, the topic of this article. GABAAR are members of the ligand-gated ion channel receptor superfamily, a chloride channel family of a dozen or more heteropentameric subtypes containing 19 possible different subunits. These subtypes show different brain regional and subcellular localization, age-dependent expression, and potential for plastic changes with experience including drug exposure. Not only are GABAAR the targets of agonist depressants and antagonist convulsants, but most GABAAR drugs act at other (allosteric) binding sites on the GABAAR proteins. Some anxiolytic and sedative drugs, like benzodiazepine and related drugs, act on GABAAR subtype-dependent extracellular domain sites. General anesthetics including alcohols and neurosteroids act at GABAAR subunit-interface trans-membrane sites. Ethanol at high anesthetic doses acts on GABAAR subtype-dependent trans-membrane domain sites. Ethanol at low intoxicating doses acts at GABAAR subtype-dependent extracellular domain sites. Thus GABAAR subtypes possess pharmacologically specific receptor binding sites for a large group of different chemical classes of clinically important neuropharmacological agents.

GABAergic inhibitory transmission is involved in the acute and chronic effects of ethanol on the brain and behavior. One-dose ethanol exposure induces transient plastic changes in GABAA receptor subunit levels, composition, and regional and subcellular localization. Rapid down-regulation of early responder δ subunit-containing GABAA receptor subtypes mediating ethanol-sensitive tonic inhibitory currents in critical neuronal circuits corresponds to rapid tolerance to ethanol’s behavioral responses. Slightly slower, α1 subunit-containing GABAA receptor subtypes mediating ethanol-insensitive synaptic inhibition are down-regulated, corresponding to tolerance to additional ethanol behaviors plus cross-tolerance to other GABAergic drugs including benzodiazepines, anesthetics, and neurosteroids, especially sedative-hypnotic effects. Compensatory up-regulation of synaptically localized α4 and α2 subunit-containing GABAA receptor subtypes, mediating ethanol-sensitive synaptic inhibitory currents follow, but exhibit altered physio-pharmacology, seizure susceptibility, hyperexcitability, anxiety, and tolerance to GABAergic positive allosteric modulators, corresponding to heightened alcohol withdrawal syndrome. All these changes (behavioral, physiological, and biochemical) induced by ethanol administration are transient and return to normal in a few days. After chronic intermittent ethanol (CIE) treatment the same changes are observed but they become persistent after 30 or more doses, lasting for at least 120 days in the rat, and probably for life. We conclude that the ethanol-induced changes in GABAA receptors represent aberrant plasticity contributing critically to ethanol dependence and increased voluntary consumption. We suggest that the craving, drug-seeking, and increased consumption in the rat model are tied to ethanol-induced plastic changes in GABAA receptors, importantly the development of ethanol-sensitive synaptic GABAA receptor-mediating inhibitory currents that participate in maintained positive reward actions of ethanol on critical neuronal circuits. These probably disinhibit nerve endings of inhibitory GABAergic neurons on dopamine reward circuit cells, and limbic system circuits mediating anxiolysis in hippocampus and amygdala. We further suggest that the GABAA receptors contributing to alcohol dependence in the rat and presumably in human alcohol use disorders (AUD) are the ethanol-induced up-regulated subtypes containing α4 and most importantly α2 subunits. These mediate critical aspects of the positive reinforcement of ethanol in the dependent chronic user while alleviating heightened withdrawal symptoms experienced whenever ethanol is absent. The speculative conclusions based on firm observations are readily testable.

GABAergic inhibitory transmission is involved in the acute and chronic effects of ethanol on the brain and behavior. One-dose ethanol exposure induces transient plastic changes in GABAA receptor subunit levels, composition, and regional and subcellular localization. Rapid down-regulation of early responder δ subunit-containing GABAA receptor subtypes mediating ethanol-sensitive tonic inhibitory currents in critical neuronal circuits corresponds to rapid tolerance to ethanol’s behavioral responses. Slightly slower, α1 subunit-containing GABAA receptor subtypes mediating ethanol-insensitive synaptic inhibition are down-regulated, corresponding to tolerance to additional ethanol behaviors plus cross-tolerance to other GABAergic drugs including benzodiazepines, anesthetics, and neurosteroids, especially sedative-hypnotic effects. Compensatory up-regulation of synaptically localized α4 and α2 subunit-containing GABAA receptor subtypes, mediating ethanol-sensitive synaptic inhibitory currents follow, but exhibit altered physio-pharmacology, seizure susceptibility, hyperexcitability, anxiety, and tolerance to GABAergic positive allosteric modulators, corresponding to heightened alcohol withdrawal syndrome. All these changes (behavioral, physiological, and biochemical) induced by ethanol administration are transient and return to normal in a few days. After chronic intermittent ethanol (CIE) treatment the same changes are observed but they become persistent after 30 or more doses, lasting for at least 120 days in the rat, and probably for life. We conclude that the ethanol-induced changes in GABAA receptors represent aberrant plasticity contributing critically to ethanol dependence and increased voluntary consumption. We suggest that the craving, drug-seeking, and increased consumption in the rat model are tied to ethanol-induced plastic changes in GABAA receptors, importantly the development of ethanol-sensitive synaptic GABAA receptor-mediating inhibitory currents that participate in maintained positive reward actions of ethanol on critical neuronal circuits. These probably disinhibit nerve endings of inhibitory GABAergic neurons on dopamine reward circuit cells, and limbic system circuits mediating anxiolysis in hippocampus and amygdala. We further suggest that the GABAA receptors contributing to alcohol dependence in the rat and presumably in human alcohol use disorders (AUD) are the ethanol-induced up-regulated subtypes containing α4 and most importantly α2 subunits. These mediate critical aspects of the positive reinforcement of ethanol in the dependent chronic user while alleviating heightened withdrawal symptoms experienced whenever ethanol is absent. The speculative conclusions based on firm observations are readily testable.

While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABAA receptors (GABAARs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABAAR, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABAARs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with immunolabeling at perisynaptic locations in Fmr1 KO mice. While α4 immunogold particles were also reduced at perisynaptic locations in the Fmr1 KO mice, the labeling was increased at synaptic sites. Together these findings suggest that, in the dentate gyrus, altered surface expression of the δ subunit, rather than a decrease in δ subunit expression alone, could be limiting δ subunit-mediated tonic inhibition in this model of FXS. Finding ways to increase surface expression of the δ subunit of the GABAAR could be a novel approach to treatment of hyperexcitability-related alterations in FXS.

GABAA receptors (GABA(A)Rs) mediate rapid inhibitory transmission in the brain. GABA(A)Rs are ligand-gated chloride ion channel proteins and exist in about a dozen or more heteropentameric subtypes exhibiting variable age and brain regional localization and thus participation in differing brain functions and diseases. GABA(A)Rs are also subject to modulation by several chemotypes of allosteric ligands that help define structure and function, including subtype definition. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABA(A)Rs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Two classes of pharmacologically important allosteric modulatory ligand binding sites reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site and the high-affinity, relevant to intoxication, ethanol site. The benzodiazepine site is specific for certain GABA(A)R subtypes, mainly synaptic, while the ethanol site is found at a modified benzodiazepine site on different, extrasynaptic, subtypes. In the transmembrane domain are allosteric modulatory ligand sites for diverse chemotypes of general anesthetics: the volatile and intravenous agents, barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are endogenous positive allosteric modulators. X-ray crystal structures of prokaryotic and invertebrate pentameric ligand-gated ion channels, and the mammalian GABA(A)R protein, allow homology modeling of GABA(A)R subtypes with the various ligand sites located to suggest the structure and function of these proteins and their pharmacological modulation.

GABAA receptors (GABA(A)Rs) mediate rapid inhibitory transmission in the brain. GABA(A)Rs are ligand-gated chloride ion channel proteins and exist in about a dozen or more heteropentameric subtypes exhibiting variable age and brain regional localization and thus participation in differing brain functions and diseases. GABA(A)Rs are also subject to modulation by several chemotypes of allosteric ligands that help define structure and function, including subtype definition. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABA(A)Rs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Two classes of pharmacologically important allosteric modulatory ligand binding sites reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site and the high-affinity, relevant to intoxication, ethanol site. The benzodiazepine site is specific for certain GABA(A)R subtypes, mainly synaptic, while the ethanol site is found at a modified benzodiazepine site on different, extrasynaptic, subtypes. In the transmembrane domain are allosteric modulatory ligand sites for diverse chemotypes of general anesthetics: the volatile and intravenous agents, barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are endogenous positive allosteric modulators. X-ray crystal structures of prokaryotic and invertebrate pentameric ligand-gated ion channels, and the mammalian GABA(A)R protein, allow homology modeling of GABA(A)R subtypes with the various ligand sites located to suggest the structure and function of these proteins and their pharmacological modulation.