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.

Chronic ethanol consumption enhances internalization of alpha1 subunit-containing GABAA receptors in cerebral cortex.

The molecular mechanisms that underlie ethanol dependence involve alterations in the functional properties and subunit expression of GABAA receptors. Chronic ethanol exposure decreases GABAA receptor alpha1 subunits and increases alpha4 subunit levels in cerebral cortical membranes. This study explored the effect of chronic ethanol exposure on internalization of GABAA/benzodiazepine receptors. Chronic ethanol exposure increased alpha1 subunit levels by 46 +/- 12% and [3H]flunitrazepam binding by 35 +/- 9% in the clathrin-coated vesicle (CCV) fraction. There was a corresponding 34 +/- 8% decrease in alpha1 peptide expression and 37 +/- 6% decrease in [3H]flunitrazepam binding in the synaptic fraction. Chronic ethanol consumption also increased the alpha1 subunit immunoprecipitate in the cytosolic fraction (77 +/- 22%), measured by western blot analysis. Moreover, co-immunoprecipitation of both clathrin and adaptin-alpha with alpha1 subunits was increased in the cytosolic fraction, suggesting that alpha1 subunit endocytosis is enhanced by chronic ethanol consumption. In contrast, alpha4 subunit peptide levels were not altered in the CCV fraction despite a 39 +/- 13% increase in peptide levels in the synaptic fraction of cortex. Moreover, acute ethanol exposure did not alter alpha1 subunit peptide expression or [3H]flunitrazepam binding in the synaptic or CCV fractions. These results suggest that chronic ethanol consumption selectively increases internalization of alpha1 subunit-containing GABAA receptors in cerebral cortex.

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.

GABA(A)-receptor delta subunit knockout mice have multiple defects in behavioral responses to ethanol.

BACKGROUND: The gamma-aminobutyric acid type A receptors (GABARs) are involved in mediating some of the behavioral effects of beverage alcohol (ethanol). However, the unique pharmacological and behavioral responses conferred by each of the various receptor subunits are not well understood. METHODS: To address the role of the GABAR delta subunit in mediating ethanol responses, gene knockout mice that lack this subunit were tested for a variety of ethanol-induced behavioral responses. RESULTS: Our results indicate that, compared with controls, delta-deficient mice (delta-/-) have (1) reduced ethanol consumption, (2) attenuated withdrawal from chronic ethanol exposure, and (3) reduced anticonvulsant (seizure-protective) effects of ethanol. These mice demonstrate a normal anxiolytic response to ethanol and a normal hypothermic response to ethanol, and they develop both chronic and acute tolerance. CONCLUSIONS: These results further establish the link between GABARs and specific behavioral responses to ethanol and begin to reveal the role of the delta subunit in these responses.

Chronic blockade of N-methyl-D-aspartate receptors alters gamma-aminobutyric acid type A receptor peptide expression and function in the rat.

Chronic in vivo or in vitro application of GABA(A) receptor agonists alters GABA(A) receptor peptide expression and function. Furthermore, chronic in vitro application of N-methyl-D-aspartate (NMDA) agonists and antagonists alters GABA(A) receptor function and mRNA expression. However, it is unknown if chronic in vivo blockade of NMDA receptors alters GABA(A) receptor function and peptide expression in brain. Male Sprague-Dawley rats were chronically administered the noncompetitive NMDA receptor antagonist MK-801 (0.40 mg/kg, twice daily) for 14 days. Chronic blockade of NMDA receptors significantly increased hippocampal GABA(A) receptor alpha4 and gamma2 subunit expression while significantly decreasing hippocampal GABA(A) receptor alpha2 and beta2/3 subunit expression. Hippocampal GABA(A) receptor alpha1 subunit peptide expression was not altered. In contrast, no significant alterations in GABA(A) receptor subunit expression were found in cerebral cortex. Chronic MK-801 administration also significantly decreased GABA(A) receptor-mediated hippocampal Cl- uptake, whereas no change was found in GABA(A) receptor-mediated cerebral cortical Cl- uptake. Finally, chronic MK-801 administration did not alter NMDA receptor NR1, NR2A, or NR2B subunit peptide expression in either the cerebral cortex or the hippocampus. These data demonstrate heterogeneous regulation of GABA(A) receptors by glutamatergic activity in rat hippocampus but not cerebral cortex, suggesting a new mechanism of GABA(A) receptor regulation in brain.