Mutation of novel ethanol-responsive lncRNA Gm41261 impacts ethanol-related behavioral responses in mice.

Chronic alcohol exposure results in widespread dysregulation of gene expression that contributes to the pathogenesis of Alcohol Use Disorder (AUD). Long noncoding RNAs are key regulators of the transcriptome that we hypothesize coordinate alcohol-induced transcriptome dysregulation and contribute to AUD. Based on RNA-Sequencing data of human prefrontal cortex, basolateral amygdala and nucleus accumbens of AUD versus non-AUD brain, the human LINC01265 and its predicted murine homolog Gm41261 (i.e., TX2) were selected for functional interrogation. We tested the hypothesis that TX2 contributes to ethanol drinking and behavioral responses to ethanol. CRISPR/Cas9 mutagenesis was used to create a TX2 mutant mouse line in which 306 base-pairs were deleted from the locus. RNA analysis revealed that an abnormal TX2 transcript was produced at an unchanged level in mutant animals. Behaviorally, mutant mice had reduced ethanol, gaboxadol and zolpidem-induced loss of the righting response and reduced tolerance to ethanol in both sexes. In addition, a male-specific reduction in two-bottle choice every-other-day ethanol drinking was observed. Male TX2 mutants exhibited evidence of enhanced GABA release and altered GABAA receptor subunit composition in neurons of the nucleus accumbens shell. In C57BL6/J mice, TX2 within the cortex was cytoplasmic and largely present in Rbfox3+ neurons and IBA1+ microglia, but not in Olig2+ oligodendrocytes or in the majority of GFAP+ astrocytes. These data support the hypothesis that TX2 mutagenesis and dysregulation impacts ethanol drinking behavior and ethanol-induced behavioral responses in mice, likely through alterations in the GABAergic system.

Hippocampal ceRNA networks from chronic intermittent ethanol vapor-exposed male mice and functional analysis of top-ranked lncRNA genes for ethanol drinking phenotypes.

The molecular mechanisms regulating the development and progression of alcohol use disorder (AUD) are largely unknown. While noncoding RNAs have previously been implicated as playing key roles in AUD, long-noncoding RNA (lncRNA) remains understudied in relation to AUD. In this study, we first identified ethanol-responsive lncRNAs in the mouse hippocampus that are transcriptional network hub genes. Microarray analysis of lncRNA, miRNA, circular RNA, and protein coding gene expression in the hippocampus from chronic intermittent ethanol vapor- or air- (control) exposed mice was used to identify ethanol-responsive competing endogenous RNA (ceRNA) networks. Highly interconnected lncRNAs (genes that had the strongest overall correlation to all other dysregulated genes identified) were ranked. The top four lncRNAs were novel, previously uncharacterized genes named Gm42575, 4930413E15Rik, Gm15767, and Gm33447, hereafter referred to as Pitt1, Pitt2, Pitt3, and Pitt4, respectively. We subsequently tested the hypothesis that CRISPR/Cas9 mutagenesis of the putative promoter and first exon of these lncRNAs in C57BL/6J mice would alter ethanol drinking behavior. The Drinking in the Dark (DID) assay was used to examine binge-like drinking behavior, and the Every-Other-Day Two-Bottle Choice (EOD-2BC) assay was used to examine intermittent ethanol consumption and preference. No significant differences between control and mutant mice were observed in the DID assay. Female-specific reductions in ethanol consumption were observed in the EOD-2BC assay for Pitt1, Pitt3, and Pitt4 mutant mice compared to controls. Male-specific alterations in ethanol preference were observed for Pitt1 and Pitt2. Female-specific increases in ethanol preference were observed for Pitt3 and Pitt4. Total fluid consumption was reduced in Pitt1 and Pitt2 mutants at 15% v/v ethanol and in Pitt3 and Pitt4 at 20% v/v ethanol in females only. We conclude that all lncRNAs targeted altered ethanol drinking behavior, and that lncRNAs Pitt1, Pitt3, and Pitt4 influenced ethanol consumption in a sex-specific manner. Further research is necessary to elucidate the biological mechanisms for these effects. These findings add to the literature implicating noncoding RNAs in AUD and suggest lncRNAs also play an important regulatory role in the disease.

MAP2 is differentially phosphorylated in schizophrenia, altering its function.

Schizophrenia (Sz) is a highly polygenic disorder, with common, rare, and structural variants each contributing only a small fraction of overall disease risk. Thus, there is a need to identify downstream points of convergence that can be targeted with therapeutics. Reduction of microtubule-associated protein 2 (MAP2) immunoreactivity (MAP2-IR) is present in individuals with Sz, despite no change in MAP2 protein levels. MAP2 is phosphorylated downstream of multiple receptors and kinases identified as Sz risk genes, altering its immunoreactivity and function. Using an unbiased phosphoproteomics approach, we quantified 18 MAP2 phosphopeptides, 9 of which were significantly altered in Sz subjects. Network analysis grouped MAP2 phosphopeptides into three modules, each with a distinct relationship to dendritic spine loss, synaptic protein levels, and clinical function in Sz subjects. We then investigated the most hyperphosphorylated site in Sz, phosphoserine1782 (pS1782). Computational modeling predicted phosphorylation of S1782 reduces binding of MAP2 to microtubules, which was confirmed experimentally. We generated a transgenic mouse containing a phosphomimetic mutation at S1782 (S1782E) and found reductions in basilar dendritic length and complexity along with reduced spine density. Because only a limited number of MAP2 interacting proteins have been previously identified, we combined co-immunoprecipitation with mass spectrometry to characterize the MAP2 interactome in mouse brain. The MAP2 interactome was enriched for proteins involved in protein translation. These associations were shown to be functional as overexpression of wild type and phosphomimetic MAP2 reduced protein synthesis in vitro. Finally, we found that Sz subjects with low MAP2-IR had reductions in the levels of synaptic proteins relative to nonpsychiatric control (NPC) subjects and to Sz subjects with normal and MAP2-IR, and this same pattern was recapitulated in S1782E mice. These findings suggest a new conceptual framework for Sz-that a large proportion of individuals have a "MAP2opathy"-in which MAP function is altered by phosphorylation, leading to impairments of neuronal structure, synaptic protein synthesis, and function.

Reduced sedation and increased ethanol consumption in knock-in mice expressing an ethanol insensitive alpha 2 subunit of the glycine receptor.

Previous studies have shown the presence of several subunits of the inhibitory glycine receptor (GlyR) in the reward system, specifically in medium spiny neurons (MSNs) of the nucleus Accumbens (nAc). It was suggested that GlyR α1 subunits regulate nAc excitability and ethanol consumption. However, little is known about the role of the α2 subunit in the adult brain since it is a subunit highly expressed during early brain development. In this study, we used genetically modified mice with a mutation (KR389-390AA) in the intracellular loop of the GlyR α2 subunit which results in a heteromeric α2ß receptor that is insensitive to ethanol. Using this mouse model denoted knock-in α2 (KI α2), our electrophysiological studies showed that neurons in the adult nAc expressed functional KI GlyRs that were rather insensitive to ethanol when compared with WT GlyRs. In behavioral tests, the KI α2 mice did not show any difference in basal motor coordination, locomotor activity, or conditioned place preference compared with WT littermate controls. In terms of ethanol response, KI α2 male mice recovered faster from the administration of ataxic and sedative doses of ethanol. Furthermore, KI α2 mice consumed higher amounts of ethanol in the first days of the drinking in the dark protocol, as compared with WT mice. These results show that the α2 subunit is important for the potentiation of GlyRs in the adult brain and this might result in reduced sedation and increased ethanol consumption.

Role of TLR4 in the Modulation of Central Amygdala GABA Transmission by CRF Following Restraint Stress.

AIMS: Stress induces neuroimmune responses via Toll-like receptor 4 (TLR4) activation. Here, we investigated the role of TLR4 in the effects of the stress peptide corticotropin-releasing factor (CRF) on GABAergic transmission in the central nucleus of the amygdala (CeA) following restraint stress. METHODS: Tlr4 knock out (KO) and wild-type rats were exposed to no stress (naïve), a single restraint stress (1 h) or repeated restraint stress (1 h per day for 3 consecutive days). After 1 h recovery from the final stress session, whole-cell patch-clamp electrophysiology was used to investigate the effects of CRF (200 nM) on CeA GABAA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs). RESULTS: TLR4 does not regulate baseline GABAergic transmission in the CeA of naive and stress-treated animals. However, CRF significantly increased the mean sIPSC frequencies (indicating enhanced GABA release) across all genotypes and stress treatments, except for the Tlr4 KO rats that experienced repeated restraint stress. CONCLUSIONS: Overall, our results suggest a limited role for TLR4 in CRF's modulation of CeA GABAergic synapses in naïve and single stress rats, though TLR4-deficient rats that experienced repeated psychological stress exhibit a blunted CRF cellular response. SHORT SUMMARY: TLR4 has a limited role in CRF's activation of the CeA under basal conditions, but interacts with the CRF system to regulate GABAergic synapse function in animals that experience repeated psychological stress.

Manipulations of extracellular Loop 2 in α1 GlyR ultra-sensitive ethanol receptors (USERs) enhance receptor sensitivity to isoflurane, ethanol, and lidocaine, but not propofol.

We recently developed ultra-sensitive ethanol receptors (USERs) as a novel tool for investigation of single receptor subunit populations sensitized to extremely low ethanol concentrations that do not affect other receptors in the nervous system. To this end, we found that mutations within the extracellular Loop 2 region of glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABAARs) can significantly increase receptor sensitivity to micro-molar concentrations of ethanol resulting in up to a 100-fold increase in ethanol sensitivity relative to wild-type (WT) receptors. The current study investigated: (1) Whether structural manipulations of Loop 2 in α1 GlyRs could similarly increase receptor sensitivity to other anesthetics; and (2) If mutations exclusive to the C-terminal end of Loop 2 are sufficient to impart these changes. We expressed α1 GlyR USERs in Xenopus oocytes and tested the effects of three classes of anesthetics, isoflurane (volatile), propofol (intravenous), and lidocaine (local), known to enhance glycine-induced chloride currents using two-electrode voltage clamp electrophysiology. Loop 2 mutations produced a significant 10-fold increase in isoflurane and lidocaine sensitivity, but no increase in propofol sensitivity compared to WT α1 GlyRs. Interestingly, we also found that structural manipulations in the C-terminal end of Loop 2 were sufficient and selective for α1 GlyR modulation by ethanol, isoflurane, and lidocaine. These studies are the first to report the extracellular region of α1 GlyRs as a site of lidocaine action. Overall, the findings suggest that Loop 2 of α1 GlyRs is a key region that mediates isoflurane and lidocaine modulation. Moreover, the results identify important amino acids in Loop 2 that regulate isoflurane, lidocaine, and ethanol action. Collectively, these data indicate the commonality of the sites for isoflurane, lidocaine, and ethanol action, and the structural requirements for allosteric modulation on α1 GlyRs within the extracellular Loop 2 region.

Linking GABA(A) receptor subunits to alcohol-induced conditioned taste aversion and recovery from acute alcohol intoxication.

GABA type A receptors (GABA(A)-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABA(A)-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011). All together, they indicate that aversive property of ethanol is dependent on ethanol action on α2-containing GABA(A)-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABA(A)-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 (-/-) and α3 (-/Y) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism.

Inhaled anesthetic responses of recombinant receptors and knockin mice harboring α2(S270H/L277A) GABA(A) receptor subunits that are resistant to isoflurane.

The mechanism by which the inhaled anesthetic isoflurane produces amnesia and immobility is not understood. Isoflurane modulates GABA(A) receptors (GABA(A)-Rs) in a manner that makes them plausible targets. We asked whether GABA(A)-R α2 subunits contribute to a site of anesthetic action in vivo. Previous studies demonstrated that Ser270 in the second transmembrane domain is involved in the modulation of GABA(A)-Rs by volatile anesthetics and alcohol, either as a binding site or a critical allosteric residue. We engineered GABA(A)-Rs with two mutations in the α2 subunit, changing Ser270 to His and Leu277 to Ala. Recombinant receptors with these mutations demonstrated normal affinity for GABA, but substantially reduced responses to isoflurane. We then produced mutant (knockin) mice in which this mutated subunit replaced the wild-type α2 subunit. The adult mutant mice were overtly normal, although there was evidence of enhanced neonatal mortality and fear conditioning. Electrophysiological recordings from dentate granule neurons in brain slices confirmed the decreased actions of isoflurane on mutant receptors contributing to inhibitory synaptic currents. The loss of righting reflex EC(50) for isoflurane did not differ between genotypes, but time to regain the righting reflex was increased in N(2) generation knockins. This effect was not observed at the N(4) generation. Isoflurane produced immobility (as measured by tail clamp) and amnesia (as measured by fear conditioning) in both wild-type and mutant mice, and potencies (EC(50)) did not differ between the strains for these actions of isoflurane. Thus, immobility or amnesia does not require isoflurane potentiation of the α2 subunit.

Loss of ethanol conditioned taste aversion and motor stimulation in knockin mice with ethanol-insensitive α2-containing GABA(A) receptors.

GABA type A receptors (GABA(A)-Rs) are potential targets of ethanol. However, there are multiple subtypes of this receptor, and, thus far, individual subunits have not been definitively linked with specific ethanol behavioral actions. Interestingly, though, a chromosomal cluster of four GABA(A)-R subunit genes, including α2 (Gabra2), was associated with human alcoholism (Am J Hum Genet 74:705-714, 2004; Pharmacol Biochem Behav 90:95-104, 2008; J Psychiatr Res 42:184-191, 2008). The goal of our study was to determine the role of receptors containing this subunit in alcohol action. We designed an α2 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity in a recombinant expression system. Knockin mice containing this mutant subunit were tested in a range of ethanol behavioral tests. These mutant mice did not develop the typical conditioned taste aversion in response to ethanol and showed complete loss of the motor stimulant effects of ethanol. Conversely, they also demonstrated changes in ethanol intake and preference in multiple tests. The knockin mice showed increased ethanol-induced hypnosis but no difference in anxiolytic effects or recovery from acute ethanol-induced motor incoordination. Overall, these studies demonstrate that the effects of ethanol at GABAergic synapses containing the α2 subunit are important for specific behavioral effects of ethanol that may be relevant to the genetic linkage of this subunit with human alcoholism.

Testing the silence of mutations: Transcriptomic and behavioral studies of GABA(A) receptor α1 and α2 subunit knock-in mice.

Knock-in mice were constructed with mutations in the α1 (H(270), A(277)) and α2 (H(270), A(277)) subunits of the GABAA receptor, which resulted in receptors that lacked modulation by ethanol but retained normal responses to GABA in vitro. A key question is whether these mutant receptors also function normally in vivo. Perturbation of brain function was evaluated by gene expression profiling in the cerebral cortex and by behavioral pharmacology experiments with GABAergic drugs. Analysis of individual transcripts found only six transcripts that were changed in α1 knock-in mice and three in the α2 mutants (p<0.05, corrected for multiple comparisons). Two transcripts that are sensitive to neuronal activity, Arc and Fos, increased about 250% in the α2 mutants, and about 50% in the α1 mutants. Behavioral effects (loss of righting reflex, rotarod) of flurazepam and pentobarbital were not different between α2 mutants and wild-type, but they were enhanced for α1 knock-in mice. These results indicate that introduction of these mutations in the α2 subunit of the GABAA receptor does not produce marked perturbation of brain function, as measured by gene expression and GABAergic behavioral responses, but the same mutations in the α1 subunit produce more pronounced changes, especially in GABAergic function.

Trace and contextual fear conditioning is enhanced in mice lacking the alpha4 subunit of the GABA(A) receptor.

The GABA(A)R alpha4 subunit is highly expressed in the dentate gyrus region of the hippocampus at predominantly extra synaptic locations where, along with the GABA(A)R delta subunit, it forms GABA(A) receptors that mediate a tonic inhibitory current. The present study was designed to test hippocampus-dependent and hippocampus-independent learning and memory in GABA(A)R alpha4 subunit-deficient mice using trace and delay fear conditioning, respectively. Mice were of a mixed C57Bl/6J X 129S1/X1 genetic background from alpha4 heterozygous breeding pairs. The alpha4-knockout mice showed enhanced trace and contextual fear conditioning consistent with an enhancement of hippocampus-dependent learning and memory. These enhancements were sex-dependent, similar to previous studies in GABA(A)R delta knockout mice, but differences were present in both males and females. The convergent findings between alpha4 and delta knockout mice suggests that tonic inhibition mediated by alpha4betadelta GABA(A) receptors negatively modulates learning and memory processes and provides further evidence that tonic inhibition makes important functional contributions to learning and behavior.

GABAA receptor alpha 4 subunits mediate extrasynaptic inhibition in thalamus and dentate gyrus and the action of gaboxadol.

The neurotransmitter GABA mediates the majority of rapid inhibition in the CNS. Inhibition can occur via the conventional mechanism, the transient activation of subsynaptic GABAA receptors (GABAA-Rs), or via continuous activation of high-affinity receptors by low concentrations of ambient GABA, leading to "tonic" inhibition that can control levels of excitability and network activity. The GABAA-R alpha4 subunit is expressed at high levels in the dentate gyrus and thalamus and is suspected to contribute to extrasynaptic GABAA-R-mediated tonic inhibition. Mice were engineered to lack the alpha4 subunit by targeted disruption of the Gabra4 gene. alpha4 Subunit knockout mice are viable, breed normally, and are superficially indistinguishable from WT mice. In electrophysiological recordings, these mice show a lack of tonic inhibition in dentate granule cells and thalamic relay neurons. Behaviorally, knockout mice are insensitive to the ataxic, sedative, and analgesic effects of the novel hypnotic drug, gaboxadol. These data demonstrate that tonic inhibition in dentate granule cells and thalamic relay neurons is mediated by extrasynaptic GABAA-Rs containing the alpha4 subunit and that gaboxadol achieves its effects via the activation of this GABAA-R subtype.

An isoflurane- and alcohol-insensitive mutant GABA(A) receptor alpha(1) subunit with near-normal apparent affinity for GABA: characterization in heterologous systems and production of knockin mice.

Volatile anesthetics and alcohols enhance transmission mediated by gamma-aminobutyric acid type A receptors (GABA(A)Rs) in the central nervous system, an effect that may underlie some of the behavioral actions of these agents. Substituting a critical serine residue within the GABA(A)R alpha(1) subunit at position 270 with the larger residue histidine eliminated receptor modulation by isoflurane, but it also affected receptor gating (increased GABA sensitivity). To correct the shift in GABA sensitivity of this mutant, we mutated a second residue, leucine at position 277 to alanine. The double mutant alpha(1)(S270H,L277A)beta(2)gamma(2S) GABA(A)R was expressed in Xenopus laevis oocytes and human embryonic kidney (HEK)293 cells, and it had near-normal GABA sensitivity. However, rapid application of a brief GABA pulse to receptors expressed in HEK293 cells revealed that the deactivation was faster in double mutant than in wild-type receptors. In all heterologous systems, the enhancing effect of isoflurane and ethanol was greatly decreased in the double mutant receptor. Homozygous knockin mice harboring the double mutation were viable and presented no overt abnormality, except hyperactivity. This knockin mouse line should be useful in determining which behavioral actions of volatile anesthetics and ethanol are mediated by the GABA(A)Rs containing the alpha(1) subunit.

A gain-of-function mutation in the GABA receptor produces synaptic and behavioral abnormalities in the mouse.

In mammalian species, inhibition in the brain is mediated predominantly by the activation of GABAA receptors. We report here changes in inhibitory synaptic function and behavior in a mouse line harboring a gain-of-function mutation at Serine 270 (S270) in the GABAA receptor alpha1 subunit. In recombinant alpha1beta2gamma2 receptors, replacement of S270 by Histidine (H) results in an increase in sensitivity to gamma-aminobutyric acid (GABA), and slowing of deactivation following transient activation by saturating concentrations of GABA. Heterozygous mice expressing the S270H mutation are hyper-responsive to human contact, exhibit intention tremor, smaller body size and reduced viability. These mice also displayed reduced motor coordination, were hypoactive in the home cage, but paradoxically were hyperactive in a novel open field environment. Heterozygous knockin mice of both sexes were fertile but females failed to care for offspring. This deficit in maternal behavior prevented production of homozygous animals. Recordings from brain slices prepared from these animals revealed a substantial prolongation of miniature inhibitory postsynaptic currents (IPSCs) and a loss of sensitivity to the anesthetic isoflurane, in neurons that express a substantial amount of the alpha1 subunit. The results suggest that the biophysical properties of GABAA receptors are important in determining the time-course of inhibition in vivo, and suggest that the duration of synaptic inhibition is a critical determinant that influences a variety of behaviors in the mouse.

Mouse models of Angelman syndrome, a neurodevelopmental disorder, display different brain regional GABA(A) receptor alterations.

Angelman syndrome is a severe neurodevelopmental disorder with cognitive impairment and neurological deficits. It results from a maternal deletion of human chromosome 15q11-13 containing two candidate genes E6-P ubiquitin-protein ligase (UBE3A) and GABA(A) receptor beta3 subunit (GABRB3), the latter of which has been also linked to autism. To clarify the potential role of GABA(A) beta3 subunit-containing inhibitory receptors in these disorders, we applied ligand autoradiography on brain sections from mice with inactivated GABRB3 or maternal UBE3A genes. Binding of GABA(A) receptor channel ([(35)S]t-butylbicyclophosphorothionate) and benzodiazepine ([(3)H]Ro 15-4513) site ligands was reduced in selected brain regions of the beta3-deficient mice as compared to controls, while the UBE3A-deficient mice failed to show reduced GABA(A) receptors. The results, suggesting two different pathophysiological mechanisms, are in agreement with positron emission tomography results from Angelman syndrome patients of the corresponding genetic backgrounds.

Deletion of GABAA receptor alpha 1 subunit-containing receptors alters responses to ethanol and other anesthetics.

GABA(A) receptors have been implicated in mediating several acute effects of ethanol including anxiolysis, ataxia, sedation/hypnosis, and anticonvulsant activity. Ethanol sensitivity of neurons has been associated with expression of alpha1 subunit-containing receptors. The objective of this study was to determine the contribution of alpha1 subunit containing receptors to ethanol responses in comparison to neurosteroids and other anesthetics using GABA(A) receptor alpha1 subunit knockout mice. Deletion of alpha1 subunit-containing receptors did not alter the anxiolytic, ataxic, anticonvulsant, or hypnotic effects of ethanol or acute functional tolerance to ethanol but did increase sensitivity to the locomotor-stimulating effects of ethanol. The ability of ethanol to potentiate muscimol-stimulated chloride uptake and ethanol clearance was also not altered following alpha1 subunit deletion. The anticonvulsant and hypnotic effects of neurosteroids as well as their potentiating effect on GABA-mediated Cl(-) uptake were unaltered in alpha1(-/-) mice. The hypnotic effect of pentobarbital, etomidate, and midazolam were reduced, whereas the effect of ketamine was enhanced in alpha1(-/-) mice. Thus, GABA(A) receptor alpha1 subunit-containing receptors appear to influence the motor-stimulating effect of ethanol and the sedative/hypnotic effects of some anesthetics, but not ethanol. These receptors do not appear to be necessary for most ethanol responses, suggesting involvement of other GABA(A) receptor subtypes or other targets altogether.

GABA(A) receptor alpha-1 subunit deletion alters receptor subtype assembly, pharmacological and behavioral responses to benzodiazepines and zolpidem.

Potentiation of GABA(A) receptor activation through allosteric benzodiazepine (BZ) sites produces the anxiolytic, anticonvulsant and sedative/hypnotic effects of BZs. Using a mouse line lacking alpha1 subunit expression, we investigated the contribution of the alpha1 subunit to GABA(A) receptor pharmacology, function and related behaviors in response to BZ site agonists. Competitive [(3)H]flunitrazepam binding experiments using the Type I BZ site agonist, zolpidem, and the Type I and II BZ site non-specific agonist, diazepam, demonstrated the complete loss of Type I BZ binding sites in alpha1(-/-) mice and a compensatory increase in Type II BZ binding sites (41+/-6%, P<0.002). Chloride uptake analysis in alpha1(-/-) mice revealed an increase (108+/-10%, P<0.001) in the efficacy (E(max)) of flunitrazepam while the EC(50) of zolpidem was increased 495+/-26% (alpha1(+/+): 184+/-56 nM; alpha1(-/-): 1096+/-279 nM, P<0.01). An anxiolytic effect of diazepam was detected in both alpha1(+/+) and alpha1(-/-) mice as measured on the elevated plus maze; however, alpha1(-/-) mice exhibited a greater percentage of open arm entries and percentage of open arm time following 0.6 mg/kg diazepam. Furthermore, alpha1(-/-) mice were more sensitive to the motor impairing/sedative effects of diazepam (1-10 mg/kg) as measured by locomotor activity in the open field. Knockout mice were insensitive to the anticonvulsant effect of diazepam (1-15 mg/kg, P<0.001). The hypnotic effect of zolpidem (60 mg/kg) was reduced by 66% (P<0.001) in alpha1(-/-) mice as measured by loss of righting reflex while the effect of diazepam (33 mg/kg) was increased 57% in alpha1(-/-) mice (P<0.05). These studies demonstrate that compensatory adaptations in GABA(A) receptor subunit expression result in subunit substitution and assembly of functional receptors. Such adaptations reveal important relationships between subunit expression, receptor function and behavioral responses.

Molecular and pharmacological characterization of GABA(A) receptor alpha1 subunit knockout mice.

GABA(A) receptors mediate fast inhibitory neurotransmission in the central nervous system (CNS), and approximately half of these receptors contain alpha1 subunits. GABA(A) receptor alpha1 subunits are important for receptor assembly and specific pharmacological responses to benzodiazepines. Plasticity in GABA(A) receptor alpha1 subunit expression is associated with changes in CNS excitability observed during normal brain development, in animal models of epilepsy, and upon withdrawal from alcohol and benzodiazepines. To examine the role of alpha1 subunit-containing GABA(A) receptors in vivo, we characterized receptor subunit expression and pharmacological properties in cerebral cortex of knockout mice with a targeted deletion of the alpha1 subunit. The mice are viable but exhibit an intention tremor. Western blot analysis confirms the complete loss of alpha1 subunit peptide expression. Stable adaptations in the expression of several GABA(A) receptor subunits are observed in the fifth to seventh generations, including decreased expression of beta2/3 and gamma2 subunits and increased expression of alpha2 and alpha3 subunits. There was no change in alpha4, alpha5, or delta subunit peptide levels in cerebral cortex. Knockout mice exhibit loss of over half of GABA(A) receptors measured by [(3)H]muscimol, [(3)H]2-(3-carboxyl)-3-amino-6-(4-methoxyphenyl)-pyridazinium bromide ([(3)H]SR-95531), and t-butylbicyclophosphoro[(35)S]thionate ([(35)S]TBPS) binding. [(3)H]Ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate ([(3)H]Ro15-4513) binding is reduced by variable amounts in different regions across brain. GABA(A) receptor alpha1(-/-) mice lose all high-affinity [(3)H]zolpidem binding and about half of [(3)H]flunitrazepam binding in the cerebral cortex. The potency and maximal efficacy of muscimol-stimulated (36)Cl(-) uptake in cerebral cortical synaptoneurosomes are reduced in alpha1(-/-) mice. Furthermore, knockout mice exhibit increased bicuculline-induced seizure susceptibility compared with wild-type mice. These data emphasize the significance of alpha1 subunit expression and its involvement in the regulation of CNS excitability.

Altered receptor subtypes in the forebrain of GABA(A) receptor delta subunit-deficient mice: recruitment of gamma 2 subunits.

A GABA(A) receptor delta subunit-deficient mouse line was created by homologous recombination in embryonic stem cells to investigate the role of the subunit in the brain GABA(A) receptors. High-affinity [(3)H]muscimol binding to GABA sites as studied by ligand autoradiography was reduced in various brain regions of delta(-/-) animals. [(3)H]Ro 15-4513 binding to benzodiazepine sites was increased in delta(-/-) animals, partly due to an increment of diazepam-insensitive receptors, indicating an augmented forebrain assembly of gamma 2 subunits with alpha 4 subunits. In the western blots of forebrain membranes of delta(-/-) animals, the level of gamma 2 subunit was increased and that of alpha 4 decreased, while the level of alpha1 subunits remained unchanged. In the delta(-/-) forebrains, the remaining alpha 4 subunits were associated more often with gamma 2 subunits, since there was an increase in the alpha 4 subunit level immunoprecipitated by the gamma 2 subunit antibody. The pharmacological properties of t-butylbicyclophosphoro[(35)S]thionate binding to the integral ion-channel sites were slightly altered in the forebrain and cerebellum, consistent with elevated levels of alpha 4 gamma 2 and alpha 6 gamma 2 subunit-containing receptors, respectively.The altered pharmacology of forebrain GABA(A) receptors and the decrease of the alpha 4 subunit level in delta subunit-deficient mice suggest that the delta subunit preferentially assembles with the alpha 4 subunit. The delta subunit seems to interfere with the co-assembly of alpha 4 and gamma 2 subunits and, therefore, in its absence, the gamma 2 subunit is recruited into a larger population of alpha 4 subunit-containing functional receptors. These results support the idea of subunit competition during the assembly of native GABA(A) receptors.

Disruption of GABA(A) receptors on GABAergic interneurons leads to increased oscillatory power in the olfactory bulb network.

Synchronized neural activity is believed to be essential for many CNS functions, including neuronal development, sensory perception, and memory formation. In several brain areas GABA(A) receptor-mediated synaptic inhibition is thought to be important for the generation of synchronous network activity. We have used GABA(A) receptor beta3 subunit deficient mice (beta3-/-) to study the role of GABAergic inhibition in the generation of network oscillations in the olfactory bulb (OB) and to reveal the role of such oscillations in olfaction. The expression of functional GABA(A) receptors was drastically reduced (>93%) in beta3-/- granule cells, the local inhibitory interneurons of the OB. This was revealed by a large reduction of muscimol-evoked whole-cell current and the total current mediated by spontaneous, miniature inhibitory postsynaptic currents (mIPSCs). In beta3-/- mitral/tufted cells (principal cells), there was a two-fold increase in mIPSC amplitudes without any significant change in their kinetics or frequency. In parallel with the altered inhibition, there was a significant increase in the amplitude of theta (80% increase) and gamma (178% increase) frequency oscillations in beta3-/- OBs recorded in vivo from freely moving mice. In odor discrimination tests, we found beta3-/- mice to be initially the same as, but better with experience than beta3+/+ mice in distinguishing closely related monomolecular alcohols. However, beta3-/- mice were initially better and then worse with practice than control mice in distinguishing closely related mixtures of alcohols. Our results indicate that the disruption of GABA(A) receptor-mediated synaptic inhibition of GABAergic interneurons and the augmentation of IPSCs in principal cells result in increased network oscillations in the OB with complex effects on olfactory discrimination, which can be explained by an increase in the size or effective power of oscillating neural cell assemblies among the mitral cells of beta3-/- mice.