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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.

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.

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.

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.

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.

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.

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.

Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties.

Chronic alcohol exposure-induced changes in reinforcement mechanisms and motivational state are thought to contribute to the development of cravings and relapse during protracted withdrawal. The nucleus accumbens (NAcc) is a key structure of the mesolimbic dopaminergic reward system and plays an important role in mediating alcohol-seeking behaviors. Here we describe the long-lasting alterations of γ-aminobutyric acid type A receptors (GABAARs) of medium spiny neurons (MSNs) in the NAcc after chronic intermittent ethanol (CIE) treatment, a rat model of alcohol dependence. CIE treatment and withdrawal (>40 days) produced decreases in the ethanol and Ro15-4513 potentiation of extrasynaptic GABAARs, which mediate the picrotoxin-sensitive tonic current ( Itonic), while potentiation of synaptic receptors, which give rise to miniature inhibitory postsynaptic currents (mIPSCs), was increased. Diazepam sensitivity of both Itonicand mIPSCs was decreased by CIE treatment. The average magnitude of Itonicwas unchanged, but mIPSC amplitude and frequency decreased and mIPSC rise time increased after CIE treatment. Rise-time histograms revealed decreased frequency of fast-rising mIPSCs after CIE treatment, consistent with possible decreases in somatic GABAergic synapses in MSNs from CIE rats. However, unbiased stereological analysis of NeuN-stained NAcc neurons did not detect any decreases in NAcc volume, neuronal numbers, or neuronal cell body volume. Western blot analysis of surface subunit levels revealed selective decreases in α1 and δ and increases in α4, α5, and γ2 GABAAR subunits after CIE treatment and withdrawal. Similar, but reversible, alterations occurred after a single ethanol dose (5 g/kg). These data reveal CIE-induced long-lasting neuroadaptations in the NAcc GABAergic neurotransmission.

The δ subunit of the GABAAR is highly expressed in the dentate gyrus of the hippocampus where it mediates a tonic extrasynaptic inhibitory current that is sensitive to neurosteroids. In female mice, the expression level of the d subunit within the dentate gyrus is elevated in the diestrous relative to estrous phase of the estrous cycle. Previous work in our lab found that female δ-GABAAR KO mice showed enhanced hippocampus-dependent trace but normal hippocampus-independent delay fear conditioning. Wild-type females in this study showed a wide range of freezing levels, whereas δ-GABAAR KO mice expressed only high levels of fear. We hypothesized that the variability in the wild-type mice may have been due to estrous cycle-mediated changes in the expression of the δ-GABAAR, with low levels of freezing in mice that were in the diestrous phase when dentate gyrus tonic inhibition is high. In the present study we tested this hypothesis by utilizing contextual, delay, and trace fear conditioning protocols in mice that were trained and tested in either the diestrous or estrous phases. Consistent with our hypothesis, we found a significant impairment of hippocampus-dependent learning and memory during diestrus relative to estrus in wild-type mice and this impairment was absent in δ-GABAAR mice. These findings argue that the δ-GABAAR plays an important role in estrous cycle-mediated fluctuations in hippocampus-dependent learning and memory. © Springer Science+Business Media 2014.

Pharmacological and genetic evidence reveals that GABAAreceptor (GABAA-R) expression and localization are modulated in response to acute and chronic ethanol (EtOH) exposure. To determine molecular mechanisms of GABAA-R plasticity in response toin vivoacute EtOH, we measured early time changes in GABAA-R subunit localization. Single doses of EtOH (3 g/kg via i.p. injection in rats) produced decreases in surface levels of GABAA-R α4 and δ subunits at 5–15 min post-EtOH in hippocampus CA1 and dentate gyrus, verifying our earlier report (Liang et al., 2007). Here we also examined the β3 subunit and its phosphorylation state during internalization. β3 also was internalized during 5–15 min after EtOH exposure, while phosphorylation of β3 was increased, then decreased at later times, ruling out β3 dephosphorylation-dependent endocytosis. As early as 5 min post-EtOH, there is an initial increase in association between the δ subunits with clathrin adaptor proteins AP2-μ2 revealed by coimmunoprecipitation, followed by a decrease in association 15 min post-EtOH.In vitrostudies using glutathioneS-transferase fused to the δ subunit intracellular domain (ICD) show that two regions, one containing a classical YxxΦ motif and the other an atypical R/K-rich motif, directly and differentially bind to AP2-μ2, with the former YRSV exhibiting higher affinity. Mutating both regions in the δ-ICD abolishes μ2 binding, providing a possible mechanism that can explain the rapid downregulation of extrasynaptic α4βδ-GABAA-R followingin vivoEtOH administration, in which the δ-ICD increases in affinity for clathrin AP2-μ2 leading to endocytosis.

Summary Purpose: The β3 subunit of the γ-aminobutyric acid type A receptors (GABAA–Rs) is an essential component of GABAA–Rs in fetal, perinatal, and adult mammalian brain. Various transcripts of the β3 subunit gene (GABRB3) produce various proteins with different N-termini. Rare variants in this N-terminus (exon 1A and exon 2) of GABRB3 protein segregate in affected family members of two multigeneration-multiplex families with remitting childhood absence epilepsy (rCAE), suggesting GABRB3 is a major Mendelian epilepsy gene for rare families with CAE. Therefore, the N-terminus of GABRB3 could be important for GABRB3 regulation in development, and its alteration could produce rCAE. Herein we determine if single nucleotide polymorphisms (SNPs) within the 1,148-bp region upstream from exon 1A influence the expression of GABRB3. Methods: We studied luciferase reporter expression for promoter activity, 1,148-bp upstream from exon 1A, using human embryonic kidney 293 cells. We generated constructs of the promoter region and compared different SNP haplotypes in 48 patients with rCAE. Next, we compared frequencies of rs20317, located in the core promoter region, and rs4906902, located in the enhancer region between 48 patients with rCAE and >500 healthy controls matched for ethnicity and ancestral origin. Key Findings: Highest luciferase expression occurred 230-bp upstream of exon 1A. The construct that excluded this region lost luciferase activity. Therefore, this region contains the core promoter of exon 1A. Allele C but not allele G (rs20317) significantly increased luciferase expression activity. Allele C creates binding motifs for cMYB and EGR-3. Longer constructs overlapping this region have a binding motif for REST (RE1-silencing transcription factor), a critical epigenetic modulator for neuronal genes. REST represses expression of neuronal genes in nonneuronal tissues, resulting in reduced luciferase expression activity. Even in the suppressed condition, the longer construct enhanced luciferase expression activity of the shorter construct, which excluded the distal end containing rs4906902. However, allele frequencies of rs20317 and rs4906902 were not significantly associated with 48 rCAE patients in comparison to >500 controls matched for ethnicity and ancestral origin. Significance: Common SNPs in the promoter region increase luciferase expression activity. An epigenetic modulator, REST, specifically alters expression of GABRB3 exon 1A transcripts, suggesting epigenetic regulation by REST dominantly controls the expression of GABRB3 variant 2 transcript in early life GABAA signaling. Abnormal epigenetic regulation could be involved in absence seizures.

Alcohol use disorders (AUDs) constitute the most common form of substance abuse. The development of AUDs involves repeated alcohol use leading to tolerance, alcohol withdrawal syndrome, and physical and psychological dependence, with loss of ability to control excessive drinking. Currently there is no effective therapeutic agent for AUDs without major side effects. Dihydromyricetin (DHM; 1 mg/kg, i.p. injection), a flavonoid component of herbal medicines, counteracted acute alcohol (EtOH) intoxication, and also withdrawal signs in rats including tolerance, increased anxiety, and seizure susceptibility; DHM greatly reduced EtOH consumption in an intermittent voluntary EtOH intake paradigm in rats. GABAAreceptors (GABAARs) are major targets of acute and chronic EtOH actions on the brain. At the cellular levels, DHM (1 μm) antagonized both acute EtOH-induced potentiation of GABAARs and EtOH exposure/withdrawal-induced GABAAR plasticity, including alterations in responsiveness of extrasynaptic and postsynaptic GABAARs to acute EtOH and, most importantly, increases in GABAAR α4 subunit expression in hippocampus and cultured neurons. DHM anti-alcohol effects on both behavior and CNS neurons were antagonized by flumazenil (10 mg/kgin vivo; 10 μmin vitro), the benzodiazepine (BZ) antagonist. DHM competitively inhibited BZ-site [3H]flunitrazepam binding (IC50, 4.36 μm), suggesting DHM interaction with EtOH involves the BZ sites on GABAARs. In summary, we determined DHM anti-alcoholic effects on animal models and determined a major molecular target and cellular mechanism of DHM for counteracting alcohol intoxication and dependence. We demonstrated pharmacological properties of DHM consistent with those expected to underlie successful medical treatment of AUDs; therefore DHM is a therapeutic candidate.

Background: A major effect of low-dose ethanol is impairment of hippocampus-dependent cognitive function. α4/δ -containing GABAARs are highly expressed within the dentate gyrus region of the hippocampus where they mediate a tonic inhibitory current that is sensitive to the enhancement by low ethanol concentrations. These receptors are also powerful modulators of learning and memory, suggesting that they could play an important role in ethanol’s cognitive impairing effects. The goal of this study was to develop a high-throughput cognitive ethanol assay, amenable to use in genetically modified mice that could be used to test this hypothesis. Methods: We developed a procedure where preexposure to a conditioning chamber is used to rescue the “immediate shock deficit.” Using this task, ethanol can be specifically targeted at the hippocampus-dependent process of contextual learning without interfering with pain sensitivity or behavioral performance. Results: Validation of this task in C57BL/6 mice indicated that 1.0 g/kg ethanol and 10 mg/kg allopregnanolone disrupt contextual learning. Ro15-4513 reversed the effects of ethanol but not allopregnanolone, whereas it produced an impairment when given alone. The high-throughput nature of this task allowed for its application in a large cohort of α4 GABAAR KO mice. Loss of the α4 GABAAR subunit produced an enhanced sensitivity to the cognitive impairing effects of ethanol. This is consistent with the enhanced ethanol sensitivity of synaptic GABAARs that has been previously observed in the dentate gyrus in these mice, but inconsistent with the reduced ethanol sensitivity of extrasynaptic GABAARs observed in the same cells. Conclusions: Overall, these findings are consistent with our hypothesis that ethanol acts directly at GABAA receptors to impair hippocampus-dependent cognitive function. Furthermore, validation of this high-throughput assay will allow for future studies to use anatomically and temporally restricted genetic manipulations to probe more deeply into the neural mechanisms of ethanol action on learning and memory circuits.

The endoribonuclease, Dicer, is indispensable for generating the majority of mature microRNAs (miRNAs), which are posttranscriptional regulators of gene expression involved in a wide range of developmental and pathological processes in the mammalian CNS. Although functions of Dicer-dependent miRNA pathways in neurons and oligodendrocytes have been extensively investigated, little is known about the role of Dicer in astrocytes. Here, we report the effect of Cre-loxP-mediated conditional deletion ofDicerselectively from postnatal astroglia on brain development. Dicer-deficient mice exhibited normal motor development and neurological morphology before postnatal week 5. Thereafter, mutant mice invariably developed a rapidly fulminant neurological decline characterized by ataxia, severe progressive cerebellar degeneration, seizures, uncontrollable movements, and premature death by postnatal week 9–10. Integrated transcription profiling, histological, and functional analyses of cerebella showed that deletion ofDicerin cerebellar astrocytes altered the transcriptome of astrocytes to be more similar to an immature or reactive-like state before the onset of neurological symptoms or morphological changes. As a result, critical and mature astrocytic functions including glutamate uptake and antioxidant pathways were substantially impaired, leading to massive apoptosis of cerebellar granule cells and degeneration of Purkinje cells. Collectively, our study demonstrates the critical involvement of Dicer in normal astrocyte maturation and maintenance. Our findings also reveal non-cell-autonomous roles of astrocytic Dicer-dependent pathways in regulating proper neuronal functions and implicate that loss of or dysregulation of astrocytic Dicer-dependent pathways may be involved in neurodegeneration and other neurological disorders.

Alcohol use causes many physiological changes in brain with behavioral sequelae. We previously observed (J Neurosci 27:12367-12377, 2007) plastic changes in hippocampal slice recordings paralleling behavioral changes in rats treated with a single intoxicating dose of ethanol (EtOH). Here, we were able to reproduce in primary cultured hippocampal neurons many of the effects of in vivo EtOH exposure on GABA(A) receptors (GABA(A)Rs). Cells grown 11 to 15 days in vitro demonstrated GABA(A)R δ subunit expression and sensitivity to enhancement by short-term exposure to EtOH (60 mM) of GABA(A)R-mediated tonic current (I(tonic)) using whole-cell patch-clamp techniques. EtOH gave virtually no enhancement of mIPSCs. Cells pre-exposed to EtOH (60 mM) for 30 min showed, 1 h after EtOH withdrawal, a 50% decrease in basal I(tonic) magnitude and tolerance to short-term EtOH enhancement of I(tonic), followed by reduced basal mIPSC area at 4 h. At 24 h, we saw considerable recovery in mIPSC area and significant potentiation by short-term EtOH; in addition, GABA(A)R currents exhibited reduced enhancement by benzodiazepines. These changes paralleled significant decreases in cell-surface expression of normally extrasynaptic δ and α4 GABA(A)R subunits as early as 20 min after EtOH exposure and reduced α5-containing GABA(A)Rs at 1 h, followed by a larger reduction of normally synaptic α1 subunit at 4 h, and then by increases in α4γ2-containing cell-surface receptors by 24 h. Measuring internalization of biotinylated GABA(A)Rs, we showed for the first time that the EtOH-induced loss of I(tonic) and cell-surface δ/α4 20 min after withdrawal results from increased receptor endocytosis rather than decreased exocytosis.

In 1969, H.H. Jasper, A.A. Ward, and A. Pope and the Public Health Service Advisory Committee on the Epilepsies of the National Institutes of Health (NIH) published the first edition on Basic Mechanisms of the Epilepsies (BME). Since then, basic and clinical researchers in epilepsy have gathered together each decade to assess where epilepsy research has been, what it has accomplished, and where it should go. In 1999, the third edition of BME was named in honor of H.H. Jasper. Projected for publication in 2011, the fourth edition of Jasper's BME will (1) synthesize the role of interactions between neurons, synapses, and glia in the initiation, spread, and arrest of seizures; (2) examine the molecular, cellular, and network plasticity mechanisms that subserve excitability, seizure susceptibility, and ultimately epileptogenesis; (3) provide a framework for expanding the genome of rare mendelian epilepsies and understanding the complex heredity responsible for common epilepsies; (4) explore cellular mechanisms of the two main groups of presently known Mendelian epilepsy genes, namely ion channelopathies and developmental epilepsy genes; and (5) for the first time, describe the current efforts to translate the discoveries in epilepsy disease mechanisms into molecular and cellular therapeutic strategies in order to repair and cure the epilepsies. For an expanded treatment of this topic see Jasper's Basic Mechanisms of the Epilepsies, Fourth Edition (Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado-Escueta AV, eds) published by Oxford University Press (available on the National Library of Medicine Bookshelf [NCBI] at http://www.ncbi.nlm.nih.gov/books).

Small-molecule target identification is a vital and daunting task for the chemical biology community as well as for researchers interested in applying the power of chemical genetics to impact biology and medicine. To overcome this “target ID” bottleneck, new technologies are being developed that analyze protein–drug interactions, such as drug affinity responsive target stability (DARTS), which aims to discover the direct binding targets (and off targets) of small molecules on a proteome scale without requiring chemical modification of the compound. Here, we review the DARTS method, discuss why it works, and provide new perspectives for future development in this area.

Alcohol tolerance resulting from chronic administration is well known to be accompanied by cross-tolerance to sedative/anesthetic drugs, especially those acting on the γ-aminobutyric acid type A receptors (GABAARs). Rats treated with chronic intermittent ethanol (CIE) show decreased function and altered pharmacology of GABAARs in hippocampal neurons, consistent with cell- and location-specific changes in GABAAR subunit composition. We previously observed variably altered sensitivity to GABAergic drugs in vivo and in hippocampal neurons using whole cell patch-clamp recording in brain slices. Here, we examined additional clinical GABAergic drugs to correlate CIE-induced tolerance to potentiation of neuronal GABAAR-mediated currents with tolerance of these agents to sedative/anesthetic effects in vivo. Typical of several drug classes and two cell types, in CA1 pyramidal neurons, the benzodiazepine diazepam doubled the total charge transfer (TCT) of miniature postsynaptic inhibitory currents (mIPSCs), whereas it quadrupled the TCT of tonic currents. CIE treatment altered these responses to variable extent, as it did to loss of righting reflex (LORR) induced by these same drugs: 90–95% tolerance to flurazepam, the neuroactive steroid alphaxalone, and ethanol; 30–40% to pentobarbital, etomidate, and the GABA agonist gaboxadol; and no tolerance to propofol. There was a strong correlation between tolerance in the LORR assay and tolerance to enhancement of tonic currents, but not mIPSCs. The striking correlation suggests that the sedative/anesthetic actions of GABAergic drugs may be mediated primarily via the potentiation of extrasynaptic GABAARs. This requires the reasonable assumption that the same types of GABAARs in other brain regions involved directly in hypnotic drug actions show similar tolerance.

General anesthetics, once thought to exert their effects through non-specific membrane effects, have highly specific ion channel targets that can silence neuronal populations in the nervous system, thereby causing unconsciousness and immobility, characteristic of general anesthesia. Inhibitory GABA(A) receptors (GABA(A)Rs), particularly highly GABA-sensitive extrasynaptic receptor subtypes that give rise to sustained inhibitory currents, are uniquely sensitive to GABA(A)R-active anesthetics. A prominent population of extrasynaptic GABA(A)Rs is made up of alpha4, beta2 or beta3, and delta subunits. Considering the demonstrated importance of GABA receptor beta3 subunits for in vivo anesthetic effects of etomidate and propofol, we decided to investigate the effects of GABA anesthetics on "extrasynaptic" alpha4beta3delta and also binary alpha4beta3 receptors expressed in human embryonic kidney (HEK) cells. Consistent with previous work on similar receptor subtypes we show that maximal GABA currents through "extrasynaptic" alpha4beta3delta receptors, receptors defined by sensitivity to EtOH (30mM) and the beta-carboline beta-CCE (1microM), are enhanced by the GABA(A)R-active anesthetics etomidate, propofol, and the neurosteroid anesthetic THDOC. Furthermore, we show that receptors formed by alpha4beta3 subunits alone also show high GABA sensitivity and that saturating GABA responses of alpha4beta3 receptors are increased to the same extent by etomidate, propofol, and THDOC as are alpha4beta3delta receptors. Therefore, both alpha4beta3 and alpha4beta3delta receptors show low GABA efficacy, and GABA is also a partial agonist on certain binary alphabeta receptor subtypes. Increasing GABA efficacy on alpha4/6beta3delta and alpha4beta3 receptors is likely to make an important contribution to the anesthetic effects of etomidate, propofol and the neurosteroid THDOC.

Functional and behavioral tolerance to chronic benzodiazepine (BZ) exposure has been associated with an uncoupling of the BZ and GABA binding sites. As in rats exposed to BZ for periods of a week or longer, recombinant GABA(A) receptors (GABARs) expressed in Sf9 cells lose the normally observed allosteric enhancement of [3H]flunitrazepam binding by GABA agonists, which is measured in homogenized membranes after a few hours exposure to pharmacological doses of agonist BZ. Treatment of Sf9 cells expressing recombinant GABAR with various drugs that inhibit protein kinase A (PKA), but not protein kinase C (PKC), resulted in an uncoupling of the BZ and GABA binding sites; whereas promotion of phosphorylation by PKA, but not PKC, favored coupling and recoupling. However, mutation of the only PKA phosphorylation site expressed from among the subunits proved that direct phosphorylation of the GABAR was not involved in either coupling after chronic BZ exposure or reversal of uncoupling after exposure to the competitive BZ antagonist, flumazenil. Osmotic-shock of cell membrane homogenates to lyse intracellular compartments reversed uncoupling, and uncoupling can be replicated in untreated cells by performing membrane binding assays in an acidic environment, suggesting that GABARs become internalized into an acidic intracellular environment where normal BZ binding occurs, but that potentiation by GABA is hindered. The internalization of receptors was shown by immunofluorescence after chronic exposure to either BZ or the PKA inhibitor H-89.

Alcohol (ethanol, EtOH) has pleiotropic actions and induces a number of acute and long-term effects due to direct actions on alcohol targets, and effects of alcohol metabolites and metabolism. Many detrimental health consequences are due to EtOH metabolism and metabolites, in particular acetaldehyde, whose high reactivity leads to nonspecific chemical modifications of proteins and nucleic acids. Like acetaldehyde, alcohol has been widely considered a nonspecific drug, despite rather persuasive evidence implicating inhibitory GABAA receptors (GABAARs) in acute alcohol actions, for example, a GABAAR ligand, the imidazobenzodiazepine Ro15-4513 antagonizes many low-to-moderate dose alcohol actions in mammals. It was therefore rather surprising that abundant types of synaptic GABAARs are generally not responsive to relevant low concentrations of EtOH. In contrast, δ-subunit-containing GABAARs and extrasynaptic tonic GABA currents mediated by these receptors are sensitive to alcohol concentrations that are reached in blood and tissues during low-to-moderate alcohol consumption. We recently showed that low-dose alcohol enhancement on highly alcohol-sensitive GABAAR subtypes is antagonized by Ro15-4513 in an apparently competitive manner, providing a molecular explanation for behavioural Ro15-4513 alcohol antagonism. The identification of a Ro15-4513/EtOH binding site on unique GABAAR subtypes opens the possibility to characterize this alcohol site(s) and screen for compounds that modulate the function of EtOH/Ro15-4513-sensitive GABAARs. The utility of such drugs might range from novel alcohol antagonists that might be useful in the emergency room, to drugs for the treatment of alcoholism, as well as alcohol-mimetic drugs to harness acute positive effects of alcohol.

Background gamma-Aminobutyric acid type A receptors (GABA(A)-Rs) have been implicated in mediating some of the behavioral effects of ethanol (EtOH), but the contribution of specific GABA(A)-R subunits is not yet fully understood. The GABA(A)-R alpha 4 subunit often partners with beta2/3 and delta subunits to form extrasynaptic GABA(A)-Rs that mediate tonic inhibition. Several in vitro studies have suggested that these extrasynaptic GABA(A)-Rs may be particularly relevant to the intoxicating effects of low doses of EtOH. In alpha 4 subunit knockout mice, tonic inhibition was greatly reduced, as were the potentiating effects of EtOH. We therefore hypothesized that those behavioral responses to EtOH that are mediated by alpha 4-containing GABA(A)-Rs would be diminished in alpha 4 knockout mice. Methods We investigated behavioral responses to acute administration of moderate/high dose EtOH or pentylenetetrazol in alpha 4 subunit knockout mice. We compared behavioral responses to EtOH in alpha 4 knockout and wild-type littermates in the elevated plus maze (0.0, 1.0 g/kg EtOH), screen test (1.5, 2.0 g/kg), hypothermia (1.5, 2.0 g/kg), fixed speed rotarod (1.5, 2.0, 2.5 g/kg), open field (0.0, 1.0, 2.0 g/kg), radiant tail flick (2.0 g/kg), loss of righting reflex (3.5 g/kg), and EtOH metabolism and clearance assays. Sensitivity to pentylenetetrazol-induced seizures was also analyzed. Results No differences were observed between alpha 4 knockout mice and wild-type controls in terms of the baseline behavior in the absence of EtOH treatment or in the behavioral effects of EtOH in the assays tested. In contrast, alpha 4 knockout mice were significantly more sensitive to pentylenetetrazol-induced seizures. Conclusions We conclude that GABA(A)-Rs containing the alpha 4 subunit are not absolutely required for the acute behavioral responses to moderate/high dose EtOH that were assessed with the elevated plus maze, screen test, hypothermia, fixed speed rotarod, open field, radiant tail flick, and loss of right reflex assays. We further suggest that these findings are complicated by the demonstrated compensatory alterations in synaptic GABA(A)-R EtOH sensitivity and function in alpha 4 knockout mice.

We investigated the ubiquitin-like modification of GABA(A) receptor-associated protein (GABARAP) and its function. A fusion protein of GABARAP with v5 in the N terminus and myc in the C terminus was expressed in rat cultured hippocampal neurons and PC12 cells. Western blotting with antibodies to v5 and myc revealed that the C terminus of GABARAP was cleaved off. Cleavage was blocked by mutating the C-terminal Gly116 to Ala, suggesting that G116 is required for the processing. Unlike ubiquitin, GABARAP was not incorporated covalently into higher-molecular-weight protein complexes. Nor was GABARAP degraded by ubiquitinylation through the proteasome, although GABARAP formed noncovalent dimers. Immunofluorescent confocal microscopy demonstrated that recombinantly expressed GABARAP was diffusely localized in PC12 cells. However, prevention of C-terminal processing in the mutant GABARAP(G116A) resulted in redistribution to the Golgi. In neurons, punctate cytoplasmic staining of GABARAP was seen in soma and processes, whereas GABARAP(G116A) was limited to soma. Compared with wild-type GABARAP, the colocalization and interaction of GABARAP(G116A) with GABA(A) receptors were significantly reduced, resulting in a reduction in expression of receptors in the plasma membrane. When alpha1beta2gamma2S-containing GABA(A) receptors were expressed in oocytes, the increased surface expression of GABA(A) receptors, as shown by increased GABA currents and surface-accessible GABA(A) receptor subunit polypeptides resulting from GABARAP coexpression, was prevented by mutation G116A. In addition, the distribution of NSF (N-ethylmaleimide-sensitive factor) was affected in GABARAP(G116A)-expressing neurons. These results suggest that glycine 116 is required for C-terminal processing of GABARAP and that processing is essential for the localization of GABARAP and its functions as a trafficking protein.

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 results into a coherent and highly plausible paradigm on how alcohol exerts an important part of its action in the brain, we are confident about our findings and conclusions. However, many important open questions remain to be answered.

General anesthetics, including etomidate, act by binding to and enhancing the function of GABA type A receptors (GABAARs), which mediate inhibitory neurotransmission in the brain. Here, we used a radiolabeled, photoreactive etomidate analog ([3H]azietomidate), which retains anesthetic potencyin vivoand enhances GABAAR functionin vitro, to identify directly, for the first time, amino acids that contribute to a GABAAR anesthetic binding site. For GABAARs purified by affinity chromatography from detergent extracts of bovine cortex, [3H]azietomidate photoincorporation was increased by GABA and inhibited by etomidate in a concentration-dependent manner (IC50= 30 μm). Protein microsequencing of fragments isolated from proteolytic digests established photolabeling of two residues: one within the αM1 transmembrane helix at α1Met-236 (and/or the homologous methionines in α2,3,5), not previously implicated in etomidate function, and one within the βM3 transmembrane helix at β3Met-286 (and/or the homologous methionines in β1,2), an etomidate sensitivity determinant. The pharmacological specificity of labeling indicates that these methionines contribute to a single binding pocket for etomidate located in the transmembrane domain at the interface between β and α subunits, in what is predicted by structural models based on homology with the nicotinic acetylcholine receptor to be a water-filled pocket ∼50 Å below the GABA binding site. The localization of the etomidate binding site to an intersubunit, not an intrasubunit, binding pocket is a novel conclusion that suggests more generally that the localization of drug binding sites to subunit interfaces may be a feature not only for GABA and benzodiazepines but also for etomidate and other intravenous and volatile anesthetics.

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

Although it is now more than two decades since it was first reported that the imidazobenzodiazepine Ro15-4513 reverses behavioral alcohol effects, the molecular target(s) of Ro15-4513 and the mechanism of alcohol antagonism remain elusive. Here, we show that Ro15-4513 blocks the alcohol enhancement on recombinant “extrasynaptic” α4/6β3δ GABA A receptors at doses that do not reduce the GABA-induced Cl − current. At low ethanol concentrations (≤30 mM), the Ro15-4513 antagonism is complete. However, at higher ethanol concentrations (≥100 mM), there is a Ro15-4513-insensitive ethanol enhancement that is abolished in receptors containing a point mutation in the second transmembrane region of the β3 subunit (β3N265M). Therefore, α4/6β3δ GABA receptors have two distinct alcohol modulation sites: ( i ) a low-dose ethanol site present in α4/6β3δ receptors that is antagonized by the behavioral alcohol antagonist Ro15-4513 and ( ii ) a site activated at high (anesthetic) alcohol doses, defined by mutations in membrane-spanning regions. Receptors composed of α4β3N265Mδ subunits that lack the high-dose alcohol site show a saturable ethanol dose-response curve with a half-maximal enhancement at 16 mM, close to the legal blood alcohol driving limit in most U.S. states (17.4 mM). Like in behavioral experiments, the alcohol antagonist effect of Ro15-4513 on recombinant α4β3δ receptors is blocked by flumazenil and β-carboline-ethyl ester (β-CCE). Our findings suggest that ethanol/Ro15-4513-sensitive GABA A receptors are important mediators of behavioral alcohol effects.

GABAAreceptors (GABARs) are heteromultimeric proteins composed of five subunits. The specific subunit composition determines critical properties of a GABAR such as pharmacological sensitivities and whether the receptor contributes to synaptic or extrasynaptic forms of inhibition. Classically, synaptic but not extrasynaptic GABARs are thought to respond to benzodiazepines, whereas the reverse has been suggested for ethanol. To examine the effects of subunit composition on GABAR functionin situ, we took advantage of two naturally occurring alleles of the rat gene for GABAR subunit α6 (Gabra6100RandGabra6100Q). Depending on their subunit partners, these two variants of α6 can lead to differential sensitivities to benzodiazepines and ethanol. An examination of synaptic and extrasynaptic GABA-mediated currents in cerebellar granule cells fromGabra6100R/100RandGabra6100Q/100Qrats uncovered marked allele-dependent differences in benzodiazepine sensitivity. Unexpectedly, we found that the benzodiazepines flunitrazepam and diazepam enhanced extrasynaptic inhibition mediated by δ subunit-containing GABARs inGabra6100Q/100Qrats. Complementary experiments on recombinant GABARs confirmed that, at subsaturating [GABA], flunitrazepam potentiates α6/δ subunit-containing GABARs. Based on data and a simple theoretical analysis, we estimate that the average extrasynaptic [GABA] is ∼160 nmin perfused slices. These results (1) demonstrate contributions of α6 subunits to both synaptic and extrasynaptic GABA responses, (2) establish that δ subunit-containing GABARs are benzodiazepine sensitive at subsaturating [GABA] and, (3) provide an empirical estimate of extrasynaptic [GABA] in slices.

Summary: Purpose:γ-Aminobutyric acid receptor (GABAAr) subunit β3–deficient mice model Angelman syndrome by displaying impaired learning, abnormal EEG with interictal spikes and slowing, myoclonus, and convulsions. The β3-subunit deficiency causes a failure of intrathalamic reticular nucleus inhibition, leading to abnormally synchronized thalamocortical oscillations. We postulated that this pathophysiology underlies the abnormal cortical EEG and triggers interictal spikes and seizures, but extrathalamic regions also contribute to interictal spikes and seizures, so that the EEG slowing should reveal an absence-like response profile, whereas spikes and seizures have dual responsiveness to absence and partial-seizure drugs. Methods: Recording electrodes were implanted over the parietal cortices of wild-type, heterozygotes, and homozygous null mice. In each experiment, EEG was recorded for 45 min, either drug or vehicle administered, and EEG recorded for another 3 h. Each EEG was scored for slow-wave activity, interictal spikes, and seizures by a reader blinded to treatments. Results: Interictal spiking and percentage of time in EEG slowing in heterozygotes were increased by the proabsence drug baclofen (GABAB-receptor agonist), whereas CGP 35348 (GABAB-receptor antagonist) had the opposite effect. The antiabsence drug ethosuximide markedly suppressed EEG slowing and interictal spiking in heterozygote and null mice. Broad-spectrum clonazepam and valproate were more effective on interictal spiking than on EEG slowing, and fosphenytoin suppressed only interictal spiking. Conclusions: The results suggest that this model of Angelman syndrome, although not expressing typical absence seizures, is characterized by hypersynchronous thalamocortical oscillations that possess absence-like pharmacologic responsiveness and promote EEG slowing, interictal spikes, and convulsive seizures.

The trafficking of GABAAreceptors is an important component of the pathway that regulates plasticity of inhibitory synapses. The 17 kDa GABAAreceptor-associated protein (GABARAP) has been implicated in the trafficking of GABAAreceptors because of its ability to interact not only with the γ2 subunit of the receptor but also with microtubules and theN-ethylmaleimide-sensitive factor (NSF). To elucidate the role of GABARAP in the trafficking of GABAAreceptors, we have constructed a yellow fluorescent protein (YFP) fusion protein of GABARAP and expressed it in neurons using adenovirus, so that its function may be examined. YFP-GABARAP colocalized with γ2 subunit-containing GABAAreceptors and NSF to the perinuclear cytoplasm in cultured hippocampal neurons and to the proximal regions of dendrites that are making synaptic contact. Expression of YFP-GABARAP in Cos7 cells and cultured hippocampal neurons was able to increase the level of GABAAreceptors detected at the plasma membrane, even at low levels of YFP-GABARAP expression. This effect is specific to the function of GABARAP on GABAAreceptor trafficking, because point mutations in the γ2-binding domain of YFP-GABARAP interfered with the ability of YFP-GABARAP to increase GABAAreceptor surface levels. These mutations also disrupted the colocalization of YFP-GABARAP with the γ2 subunit and with NSF in hippocampal neurons. The results of this study show for the first time that GABARAP has a functional effect on the trafficking of GABAAreceptors and provide decisive evidence for the role of GABARAP in transporting GABAAreceptors to the plasma membrane in neurons.

γ-Aminobutyric acid type A receptors (GABARs) have long been implicated in mediating ethanol (EtOH) actions, but so far most of the reported recombinant GABAR combinations have shown EtOH responses only at fairly high concentrations (≥60 mM). We show that GABARs containing the δ-subunit, which are highly sensitive to γ-aminobutyric acid, slowly inactivating, and thought to be located outside of synapses, are enhanced by EtOH at concentrations that are reached with moderate, social EtOH consumption. Reproducible ethanol enhancements occur at 3 mM, a concentration six times lower than the legal blood-alcohol intoxication (driving) limit in most states (0.08% wt/vol or 17.4 mM). GABARs responsive to these low EtOH concentrations require the GABAR δ-subunit, which is thought to be associated exclusively with α 4 - and α 6 -subunits in vivo , and the β 3 -subunit, which has recently been shown to be essential for the in vivo anesthetic actions of etomidate and propofol. GABARs containing β 2 -instead of β 3 -subunits in α 4 βδ- and α 6 βδ-receptor combinations are almost 10 times less sensitive to EtOH, with threshold enhancement at 30 mM. GABARs containing γ 2 -instead of δ-subunits with α 4 β and α 6 β are three times less sensitive to EtOH, with threshold responses at 100 mM, a concentration not usually reached with social EtOH consumption. These combined findings suggest that “extrasynaptic” δ-subunit-containing GABARs, but not their “synaptic” γ-subunit-containing counterparts, are primary targets for EtOH.

The δ subunit of the γ-aminobutyric acid (A) receptor (GABAAR) is expressed postnatally mostly in the cerebellum, thalamus, and dentate gyrus. Previous studies in mice with a targeted disruption of the δ subunit revealed a considerable attenuation of behavioral responses to neuroactive steroids but not to other neuromodulatory drugs. Here we show that δ subunit loss leads to a concomitant reduction in hippocampal α4 subunit levels. These changes were accompanied by faster decay of evoked inhibitory postsynaptic potentials (IPSPs) in dentate granule neurons of –/– mutants (decay τ = 25 ms) compared with +/+ controls (τ = 50 ms). Furthermore, the GABAAR-mediated miniature inhibitory postsynaptic currents (mIPSCs) also decayed faster in δ-mutants (τ = 6.3 ms) than controls (τ = 7.2 ms) and had decreased frequency (controls, 10.5 Hz; mutants, 6.6 Hz). Prolongation of mIPSCs by the neuroactive steroid anesthetic, alphaxalone (1–10 μM), was smaller in δ-mutants (at 10 μM, 65% increase) compared with +/+ littermates (308% increase). In competition binding experiments, alphaxalone (0.03–1 μM) modulation of [35S]t-butylbicyclophosphorothionate binding was reduced in δ-mutant brain homogenates, indicating that the decreased alphaxalone effects on mIPSCs were due to changes in the GABAAR protein. Faster decay of evoked IPSPs and mIPSCs in δ-mutants suggests presence of the δ subunit at both synaptic and extrasynaptic GABAARs. Decreased synaptic and extrasynaptic inhibition likely contributes to the pro-epileptic phenotype of δ-mutants. Reduced neurosteroid sensitivity might also contribute to seizure susceptibility. While the simplest explanation is that δ subunit-containing GABAARs represent the actual target of neurosteroids, it is possible that the behavioral and physiological sensitivity to neuroactive steroids is indirectly altered in the δ –/– mice.

The majority of fast inhibitory neurotransmission in the CNS is mediated by the GABA type-A (GABAA) receptor, a ligand-gated chloride channel. Of the approximately 20 different subunits composing the hetero-pentameric GABAA receptor, the γ2 subunit in particular seems to be important in several aspects of GABAA receptor function, including clustering of the receptor at synapses. In this study, we report that the intracellular loop of the γ2 subunit interacts with itself as well as with γ1, γ3 and β1–3 subunits, but not with the α subunits. We further show that γ2 subunits interact with photolabeled pentameric GABAA receptors composed of α1, β2/3 and γ2 subunits, and calculate the dissociation constant to be in the micromolar range. By using deletion constructs of the γ2 subunit in a yeast two-hybrid assay, we identified a 23-amino acid motif that mediates self-association, residues 389–411. We confirmed this interaction motif by inhibiting the interaction in a glutathione-S-transferase pull-down assay by adding a corresponding γ2-derived peptide. Using similar approaches, we identified the interaction motif in the γ2 subunit mediating interaction with the β2 subunit as a 47-amino acid motif that includes the γ2 self-interacting motif. The identified γ2 self-association motif is identical to the interaction motif reported between GABAA receptor and GABAA receptor-associated protein (GABARAP). We propose a model for GABAA receptor clustering based on GABARAP and GABAA receptor subunit–subunit interaction.

Chronic administration of ethanol to rats on an intermittent regimen, for 60 repeated intoxicating doses and repeated withdrawal episodes, results in a long-lasting kindling phenomenon. This involves an increasing severity of withdrawal, including a reduced threshold to seizures produced by the GABA(A) antagonist, pentylenetetrazol. We have shown previously that muscimol-evoked 36Cl- efflux and paired-pulse inhibition (involving GABA(A)-mediated recurrent inhibition) were decreased persistently in the CA1 region of hippocampal slices from chronic intermittent ethanol (CIE)-treated rats. We now report elevated levels of mRNA in forebrain for the alpha4 subunit of the GABA(A) receptor (GABAR), considered to be a constituent of pharmacologically and physiologically novel subtypes of GABARs. Using in situ hybridization with digoxigenin-labeled RNA probes, we show that at 2 days withdrawal, 60-dose CIE leads to a significant 30% increase in alpha4 subunit mRNA levels in the dentate gyrus, 46% increase in the CA3, and 26% increase in the CA1 regions. In contrast, there was no significant change in the mRNAs for the alpha5 subunit or glutamic acid decarboxylase 67 in the same regions. This study suggests that GABAR subunit-selective alterations occur after CIE treatment, possibly resulting in the alteration of the subunit composition of GABARs, with presumably altered physiological functions. This plasticity of GABARs may contribute to the increased withdrawal severity, reduced hippocampal inhibition, and increased seizure susceptibility of this animal model of human alcohol dependence.

Summary: Purpose: Mice with a targeted disruption of the γ-aminobutyric acid (A) receptor (GABAAR) δ subunit exhibited spontaneous seizures and a strikingly selective attenuation of responses to neuroactive steroids, but not to other neuromodulators. This study further characterized the behavior and physiology of the δ mutants. Methods: Seizure susceptibility to pentylenetetrazol (PTZ) injection, intracellular recordings, and whole-cell patch recordings in dentate granule neurons was determined. Results: Susceptibility to PTZ-induced convulsive seizures was significantly higher in −/− versus +/+ mice. In intracellular recordings, the evoked inhibitory postsynaptic potentials (IPSPs) had much faster decay τ in −/− mutants (25 ± 3.5 ms) compared with +/+ controls (51 ± 5.2 ms). In whole-cell patch recordings, the decay τ of pharmacologically isolated miniature spontaneous GABAAR-mediated synaptic currents (mIPSCs) from −/− mice was only slightly, but significantly faster (5.7 ± 0.13 ms; n = 18) than that of +/+ controls (6.8 ± 0.32 ms; n = 20). Furthermore, mIPSC prolongation by bath application of alphaxalone (10 μM), was much smaller in −/− mice (52 ± 12%; n = 3) compared with +/+ littermates (192 ± 14%; n = 3). Conclusions: Faster decay of evoked IPSPs and mIPSCs is consistent with altered GABAAR subunit composition in the δ mutants, but suggests predominant extrasynaptic δ subunit location in the dentate gyrus of normal mice. Faster-decaying IPSPs likely contribute to the proepileptic phenotype of the δ mutants. Reduced neurosteroid sensitivity might also contribute to seizure susceptibility. It remains to be determined whether δ subunit–containing GABAARs represent the actual target of neurosteroids or whether the behavioral and physiologic sensitivity to neurosteroids is indirectly altered in the δ−/− mice.