Gregg E. Homanics

Mice Lacking the β3 Subunit of the GABAA Receptor Have the Epilepsy Phenotype and Many of the Behavioral Characteristics of Angelman Syndrome

Angelman syndrome (AS) is a severe neurodevelopmental disorder resulting from a deletion/mutation in maternal chromosome 15q11–13. The genes in 15q11–13 contributing to the full array of the clinical phenotype are not fully identified. This study examines whether a loss or reduction in the GABAA receptor β3subunit (GABRB3) gene, contained within the AS deletion region, may contribute to the overall severity of AS. Disrupting the gabrb3 gene in mice produces electroencephalographic abnormalities, seizures, and behavior that parallel those seen in AS. The seizures that are observed in these mice showed a pharmacological response profile to antiepileptic medications similar to that observed in AS. Additionally, these mice exhibited learning and memory deficits, poor motor skills on a repetitive task, hyperactivity, and a disturbed rest–activity cycle, features all common to AS. The loss of the single gene, gabrb3, in these mice is sufficient to cause phenotypic traits that have marked similarities to the clinical features of AS, indicating that impaired expression of the GABRB3 gene in humans probably contributes to the overall phenotype of Angelman syndrome. At least one other gene, the E6-associated protein ubiquitin-protein ligase (UBE3A) gene, has been implicated in AS, so the relative contribution of the GABRB3 gene alone or in combination with other genes remains to be established.

Inhaled anesthetics and immobility: Mechanisms, mysteries, and minimum alveolar anesthetic concentration

Studies using molecular modeling, genetic engineering, neurophysiology/pharmacology, and whole animals have advanced our understanding of where and how inhaled anesthetics act to produce immobility (minimum alveolar anesthetic concentration; MAC) by actions on the spinal cord. Numerous ligand- and voltage-gated channels might plausibly mediate MAC, and specific animo acid sites in certain receptors present likely candidates for mediation. However, in vivo studies to date suggest that several channels or receptors may not be mediators (e.g., &ggr;-aminobutyric acid A, acetylcholine, potassium, 5-hydroxytryptamine-3, opioids, and &agr;2-adrenergic), whereas other receptors/channels (e.g., glycine, N-methyl-d-aspartate, and sodium) remain credible candidates.

GABAA receptor α4 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 α4 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 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 GABAA-Rs containing the α4 subunit and that gaboxadol achieves its effects via the activation of this GABAA-R subtype.

GABAA receptor changes in δ subunit-deficient mice: Altered expression of α4 and γ2 subunits in the forebrain

The δ subunit is a novel subunit of the pentameric γ‐aminobutyric acid (GABA)A receptor that conveys special pharmacological and functional properties to recombinant receptors and may be particularly important in mediating tonic inhibition. Mice that lack the δ subunit have been produced by gene‐targeting technology, and these mice were studied with immunohistochemical and immunoblot methods to determine whether changes in GABAA receptors were limited to deletion of the δ subunit or whether alterations in other GABAA receptor subunits were also present in the δ subunit knockout (δ–/–) mice. Immunohistochemical studies of wild‐type mice confirmed the restricted distribution of the δ subunit in the forebrain. Regions with moderate to high levels of δ subunit expression included thalamic relay nuclei, caudate‐putamen, molecular layer of the dentate gyrus, and outer layers of the cerebral cortex. Virtually no δ subunit labeling was evident in adjacent regions, such as the thalamic reticular nucleus, hypothalamus, and globus pallidus. Comparisons of the expression of other subunits in δ–/– and wild‐type mice demonstrated substantial changes in the α4 and γ2 subunits of the GABAA receptor in the δ–/– mice. γ2 Subunit expression was increased, whereas α4 subunit expression was decreased in δ–/– mice. Importantly, alterations of both the α4 and the γ2 subunits were confined primarily to brain regions that normally expressed the δ subunit. This suggests that the additional subunit changes are directly linked to loss of the δ subunit and could reflect local changes in subunit composition and function of GABAA receptors in δ–/– mice. J. Comp. Neurol. 446:179–197, 2002.

A new naturally occurring GABA A receptor subunit partnership with high sensitivity to ethanol

According to the rules of GABAA receptor (GABAAR) subunit assembly, α4 and α6 subunits are considered to be the natural partners of δ subunits. These GABAARs are a preferred target of low, sobriety-impairing concentrations of ethanol. Here we demonstrate a new naturally occurring GABAAR subunit partnership: δ subunits of hippocampal interneurons are coexpressed and colocalized with α1 subunits, but not with α4, α6 or any other α subunits. Ethanol potentiates the tonic inhibition mediated by such native α1/δ GABAARs in wild-type and in α4 subunit–deficient (Gabra4−/−) mice, but not in δ subunit–deficient (Gabrd−/−) mice. We also ruled out any compensatory upregulation of α6 subunits that might have accounted for the ethanol effect in Gabra4−/− mice. Thus, α1/δ subunit assemblies represent a new neuronal GABAAR subunit partnership present in hippocampal interneurons, mediate tonic inhibitory currents and are highly sensitive to low concentrations of ethanol.

GABAA 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.

Gabrb3 gene deficient mice exhibit impaired social and exploratory behaviors, deficits in non-selective attention and hypoplasia of cerebellar vermal lobules : A potential model of autism spectrum disorder

OBJECTIVE GABA(A) receptors play an important regulatory role in the developmental events leading to the formation of complex neuronal networks and to the behaviors they govern. The primary aim of this study was to assess whether gabrb3 gene deficient (gabrb3(-/-)) mice exhibit abnormal social behavior, a core deficit associated with autism spectrum disorder. METHODS Social and exploratory behaviors along with non-selective attention were assessed in gabrb3(-/-), littermates (gabrb3(+/+)) and progenitor strains, C57BL/6J and 129/SvJ. In addition, semi-quantitative assessments of the size of cerebellar vermal lobules were performed on gabrb3(+/+) and gabrb3(-/-) mice. RESULTS Relative to controls, gabrb3(-/-) mice exhibited significant deficits in activities related to social behavior including sociability, social novelty and nesting. In addition, gabrb3(-/-) mice also exhibited differences in exploratory behavior compared to controls, as well as reductions in the frequency and duration of rearing episodes, suggested as being an index of non-selective attention. Gabrb3(-/-) mice also displayed significant hypoplasia of the cerebellar vermis compared to gabrb3(+/+) mice. CONCLUSIONS The observed behavioral deficits, especially regarding social behaviors, strengthens the face validity of the gabrb3 gene deficient mouse as being a model of autism spectrum disorder.

Anesthesia Sensitivity in Mice that Lack the β3 Subunit of the γ-Aminobutyric Acid Type A Receptor

BackgroundThe mammalian gamma-aminobutyric acid type A (GABAA) receptor, a likely target of anesthetic action, exhibits remarkable subunit heterogeneity. In vitro expression studies suggest that there is subunit specificity to anesthetic responses at the GABAA receptor. The authors tested whether ge

Pharmacologic and behavioral responses of inbred C57BL/6J and strain 129/SvJ mouse lines.

Gene-targeting technology is creating an explosion in the number of animals available with single gene mutations that affect the function of the central nervous system. Most gene-targeted mice are produced on a mixed genetic background of C57BL/6J and substrains of Strain 129. Understanding the behavioral characteristics and responses to various drugs of these parental strains is vital to interpreting data from gene-targeted mice. We directly compared C57BL/6J and Strain 129/SvJ mouse lines on several behavioral paradigms and in response to several hypnotic and anesthetic drugs. Compared to Strain 129/SvJ mice, C57BL/6J animals are more sensitive to the hypnotic effects of midazolam, zolpidem, and propofol, less sensitive to etomidate and ethanol, and do not differ in sensitivity to Ro15-4513 or pentobarbital. These strains do not differ in their sensitivity to the motor ataxic effects of the volatile anesthetics enflurane or halothane. However, Strain 129/SvJs are more sensitive to the immobilizing effects of halothane but not enflurane. Motor coordination differs initially, but with repeated testing strain differences are no longer apparent. Strain 129/SvJ mice are more anxious on the elevated plus maze and open-field activity assays. Thus, these mouse strains harbor polymorphisms that influence some, but not all, traits of interest to behavioral neuroscientists.

Adenosine A1 receptor knockout mice develop lethal status epilepticus after experimental traumatic brain injury.

Adenosine, acting at A1 receptors, exhibits anticonvulsant effects in experimental epilepsy—and inhibits progression to status epilepticus (SE). Seizures after traumatic brain injury (TBI) may contribute to pathophysiology. Thus, we hypothesized that endogenous adenosine, acting via A1 receptors, mediates antiepileptic benefit after experimental TBI. We subjected A1-receptor knockout (ko) mice, heterozygotes, and wild-type (wt) littermates (n = 115) to controlled cortical impact (CCI). We used four outcome protocols in male mice: (1) observation for seizures, SE, and mortality in the initial 2 h, (2) assessment of seizure score (electroencephalogram (EEG)) in the initial 2 h, (3) assessment of mortality at 24 h across injury levels, and (4) serial assessment of arterial blood pressure, heart rate, blood gases, and hematocrit. Lastly, to assess the influence of gender on this observation, we observed female mice for seizures, SE, and mortality in the initial 2 h. Seizure activity was noted in 83% of male ko mice in the initial 2 h, but was seen in no heterozygotes and only 33% of wt (P < 0.05). Seizures in wt were brief (1 to 2 secs). In contrast, SE involving lethal sustained (>1 h) tonic clonic activity was uniquely seen in ko mice after CCI (50% incidence in males), (P < 0.05). Seizure score was twofold higher in ko mice after CCI versus either heterozygote or wt (P < 0.05). An injury-intensity dose–response for 24 h mortality was seen in ko mice (P < 0.05). Physiologic parameters were similar between genotypes. Seizures were seen in 100% of female ko mice after CCI versus 14% of heterozygotes and 25% wt (P < 0.05) and SE was restricted to the ko mice (83% incidence). Our data suggest a critical endogenous anticonvulsant action of adenosine at A1 receptors early after experimental TBI.