Dev Chandra

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.

Taurine Is a Potent Activator of Extrasynaptic GABAA Receptors in the Thalamus

Taurine is one of the most abundant free amino acids in the brain. In a number of studies, taurine has been reported to activate glycine receptors (Gly-Rs) at moderate concentrations (≥100 μm), and to be a weak agonist at GABAA receptors (GABAA-Rs), which are usually activated at high concentrations (≥1 mm). In this study, we show that taurine reduced the excitability of thalamocortical relay neurons and activated both extrasynaptic GABAA-Rs and Gly-Rs in neurons in the mouse ventrobasal (VB) thalamus. Low concentrations of taurine (10–100 μm) decreased neuronal input resistance and firing frequency, and elicited a steady outward current under voltage clamp, but had no effects on fast inhibitory synaptic currents. Currents elicited by 50 μm taurine were abolished by gabazine, insensitive to midazolam, and partially blocked by 20 μm Zn2+, consistent with the pharmacological properties of extrasynaptic GABAA-Rs (α4β2δ subtype) involved in tonic inhibition in the thalamus. Tonic inhibition was enhanced by an inhibitor of taurine transport, suggesting that taurine can act as an endogenous activator of these receptors. Taurine-evoked currents were absent in relay neurons from GABAA-R α4 subunit knock-out mice. The amplitude of the taurine current was larger in neurons from adult mice than juvenile mice. Taurine was a more potent agonist at recombinant α4β2δ GABAA-Rs than at α1β2γ2 GABAA-Rs. We conclude that physiological concentrations of taurine can inhibit VB neurons via activation of extrasynaptic GABAA-Rs and that taurine may function as an endogenous regulator of excitability and network activity in the thalamus.

Spatiotemporal specificity of GABAA receptor-mediated regulation of adult hippocampal neurogenesis

GABAergic transmission regulates adult neurogenesis by exerting negative feedback on cell proliferation and enabling dendrite formation and outgrowth. Further, GABAergic synapses target differentiating dentate gyrus granule cells prior to formation of glutamatergic connections. GABAA receptors (GABAARs) mediating tonic (extrasynaptic) and phasic (synaptic) transmission are molecularly and functionally distinct, but their specific role in regulating adult neurogenesis is unknown. Using global and single‐cell targeted gene deletion of subunits contributing to the assembly of GABAARs mediating tonic (α4, δ) or phasic (α2) GABAergic transmission, we demonstrate here in the dentate gyrus of adult mice that GABAARs containing α4, but not δ, subunits mediate GABAergic effects on cell proliferation, initial migration and early dendritic development. In contrast, α2‐GABAARs cell‐autonomously signal to control positioning of newborn neurons and regulate late maturation of their dendritic tree. In particular, we observed pruning of distal dendrites in immature granule cells lacking the α2 subunit. This alteration could be prevented by pharmacological inhibition of thrombospondin signaling with chronic gabapentin treatment, shown previously to reduce glutamatergic synaptogenesis. These observations point to homeostatic regulation of inhibitory and excitatory inputs onto newborn granule cells under the control of α2‐GABAARs. Taken together, the availability of distinct GABAAR subtypes provides a molecular mechanism endowing spatiotemporal specificity to GABAergic control of neuronal maturation in adult brain.

Ethanol modulates synaptic and extrasynaptic GABAA receptors in the thalamus.

Drinking alcohol is associated with the disturbance of normal sleep rhythms, and insomnia is a major factor in alcoholic relapse. The thalamus is a brain structure that plays a pivotal role in sleep regulation and rhythmicity. A number of studies have implicated GABAA receptors (GABAA-Rs) in the anxiolytic, amnestic, sedative, and anesthetic effects of ethanol. In the present study, we examined the effects of ethanol on both synaptic and extrasynaptic GABAA-Rs of relay neurons in the thalamus. We found that ethanol (≥50 mM) elicits a sustained current in thalamocortical relay neurons from the mouse ventrobasal thalamus, and this current is associated with a decrease in neuronal excitability and firing rate in response to depolarization. The steady current induced by ethanol was totally abolished by gabazine and was absent in relay neurons from GABAA-R α4 subunit knockout mice, indicating that the effect of ethanol is to enhance tonic GABA-mediated inhibition. Ethanol (50 mM) enhanced the amplitude of tonic inhibition by nearly 50%. On the other hand, ethanol had no effect on spontaneous or evoked inhibitory postsynaptic currents (IPSCs) at 50 mM but did prolong IPSCs at 100 mM. Ethanol had no effect on the paired-pulse depression ratio, suggesting that the release of GABA from presynaptic terminals is insensitive to ethanol. We conclude that ethanol, at moderate (50 mM) but not low (10 mM) concentrations, can inhibit thalamocortical relay neurons and that this occurs mainly via the actions of ethanol at extrasynaptic GABAA-Rs containing GABAA-R α4 subunits.

Inhibition of thalamic excitability by 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol: a selective role for delta-GABA(A) receptors.

The sedative and hypnotic agent 4,5,6,7‐tetrahydroisoxazolo[4,5‐c]pyridine‐3‐ol (THIP) is a GABAA receptor (GABAAR) agonist that preferentially activates δ‐subunit‐containing GABAARs (δ‐GABAARs). To clarify the role of δ‐GABAARs in mediating the sedative actions of THIP, we utilized mice lacking the α1‐ or δ‐subunit in a combined electrophysiological and behavioural analysis. Whole‐cell patch‐clamp recordings were obtained from ventrobasal thalamic nucleus (VB) neurones at a holding potential of −60 mV. Application of bicuculline to wild‐type (WT) VB neurones revealed a GABAAR‐mediated tonic current of 92 ± 19 pA, which was greatly reduced (13 ± 5 pA) for VB neurones of δ0/0 mice. Deletion of the δ‐ but not the α1‐subunit dramatically reduced the THIP (1 μm)‐induced inward current in these neurones (WT, −309 ± 23 pA; δ0/0, −18 ± 3 pA; α10/0, −377 ± 45 pA). Furthermore, THIP selectively decreased the excitability of WT and α10/0 but not δ0/0 VB neurones. THIP did not affect the properties of miniature inhibitory post‐synaptic currents in any of the genotypes. No differences in rotarod performance and locomotor activity were observed across the three genotypes. In WT mice, performance of these behaviours was impaired by THIP in a dose‐dependent manner. The effect of THIP on rotarod performance was blunted for δ0/0 but not α10/0 mice. We previously reported that deletion of the α1‐subunit abolished synaptic GABAA responses of VB neurones. Therefore, collectively, these findings suggest that extrasynaptic δ‐GABAARs vs. synaptic α1‐subunit‐containing GABAARs of thalamocortical neurones represent an important molecular target underpinning the sedative actions of THIP.

Isoflurane Is a Potent Modulator of Extrasynaptic GABAA Receptors in the Thalamus

Volatile anesthetics are used clinically to produce analgesia, amnesia, unconsciousness, blunted autonomic responsiveness, and immobility. Previous work has shown that the volatile anesthetic isoflurane, at concentrations that produce unconsciousness (250–500 μM), enhances fast synaptic inhibition in the brain mediated by GABAA receptors (GABAA-Rs). In addition, isoflurane causes sedation at concentrations lower than those required to produce unconsciousness or analgesia. In this study, we found that isoflurane, at low concentrations (25–85 μM) associated with its sedative actions, elicits a sustained current associated with a conductance increase in thalamocortical neurons in the mouse ventrobasal (VB) nucleus. These isoflurane-evoked currents reversed polarity close to the Cl– equilibrium potential and were totally blocked by the GABAA-R antagonist gabazine. Isoflurane (25–250 μM) produced no sustained current in VB neurons from GABAA-R α4-subunit knockout (Gabra4–/–) mice, although 250 μM isoflurane enhanced synaptic inhibition in VB neurons from both wild-type and Gabra4–/– mice. These data indicate an obligatory requirement for α4-subunit expression in the generation of the isoflurane-activated current. In addition, isoflurane directly activated α4β2δ GABAA-Rs expressed in human embryonic kidney 293 cells, and it was more potent at α4β2δ than at α1β2γ2 receptors (the presumptive extrasynaptic and synaptic GABAA-R subtypes in VB neurons). We conclude that the extrasynaptic GABAA-Rs of thalamocortical neurons are sensitive to low concentrations of isoflurane. In view of the crucial role of the thalamus in sensory processing, sleep, and cognition, the modulation of these extrasynaptic GABAA-Rs by isoflurane may contribute to the sedation and hypnosis associated with low doses of this anesthetic agent.

GABA A receptor γ2 subunit knockdown mice have enhanced anxiety-like behavior but unaltered hypnotic response to benzodiazepines

BackgroundGamma-aminobutyric acid type A receptors (GABAA-Rs) are the major inhibitory receptors in the mammalian brain and are modulated by a number of sedative/hypnotic drugs including benzodiazepines and anesthetics. The significance of specific GABAA-Rs subunits with respect to behavior and in vivo drug responses is incompletely understood. The γ2 subunit is highly expressed throughout the brain. Global γ2 knockout mice are insensitive to the hypnotic effects of diazepam and die perinatally. Heterozygous γ2 global knockout mice are viable and have increased anxiety-like behaviors. To further investigate the role of the γ2 subunit in behavior and whole animal drug action, we used gene targeting to create a novel mouse line with attenuated γ2 expression, i.e., γ2 knockdown mice.ResultsKnockdown mice were created by inserting a neomycin resistance cassette into intron 8 of the γ2 gene. Knockdown mice, on average, showed a 65% reduction of γ2 subunit mRNA compared to controls; however γ2 gene expression was highly variable in these mice, ranging from 10–95% of normal. Immunohistochemical studies demonstrated that γ2 protein levels were also variably reduced. Pharmacological studies using autoradiography on frozen brain sections demonstrated that binding of the benzodiazepine site ligand Ro15-4513 was decreased in mutant mice compared to controls. Behaviorally, knockdown mice displayed enhanced anxiety-like behaviors on the elevated plus maze and forced novelty exploration tests. Surprisingly, mutant mice had an unaltered response to hypnotic doses of the benzodiazepine site ligands diazepam, midazolam and zolpidem as well as ethanol and pentobarbital. Lastly, we demonstrated that the γ2 knockdown mouse line can be used to create γ2 global knockout mice by crossing to a general deleter cre-expressing mouse line.ConclusionWe conclude that: 1) insertion of a neomycin resistance gene into intron 8 of the γ2 gene variably reduced the amount of γ2, and that 2) attenuated expression of γ2 increased anxiety-like behaviors but did not lead to differences in the hypnotic response to benzodiazepine site ligands. This suggests that reduced synaptic inhibition can lead to a phenotype of increased anxiety-like behavior. In contrast, normal drug effects can be maintained despite a dramatic reduction in GABAA-R targets.

Prototypic GABA(A) receptor agonist muscimol acts preferentially through forebrain high-affinity binding sites.

Muscimol has been regarded as a universal agonist for all γ-aminobutyric acid type A receptor (GABAA-R) subtypes. However, brain regional distribution of muscimols high-affinity binding sites greatly differs from those of other binding sites of the GABAA-R. To test whether behavioral effects of muscimol correlated with the density of high-affinity [3H]muscimol binding, we examined several GABAA-R subunit gene-modified mouse lines: α1, α4, or δ-knockouts (KO), α4+δ-double KO, and Thy1.2 promoter-driven α6 transgenic mice (Thy1α6). We determined the high-affinity [3H]muscimol binding in brain sections by quantitative autoradiography and sedative/ataxic effects induced in vivo by muscimol using a constant speed rotarod. α4-KO mice had reduced [3H]muscimol binding in the caudate-putamen, thalamus, and hippocampus, and were less sensitive to the behavioral impairment by muscimol. Similarly, δ-KO mice also had reduced binding to forebrain regions and a lower behavioral sensitivity to muscimol than their wild-type controls. In contrast, α1-KO mice had unaltered behavioral sensitivity to muscimol and unaltered [3H]muscimol binding, even though previous studies have demonstrated dramatically reduced binding to various other GABAA-R sites in these mice. Finally, Thy1α6 mice exhibited increased behavioral sensitivity to muscimol, and to another direct GABA-site agonist gaboxadol, and increased [3H]muscimol binding in the cerebral cortex and hippocampus. Thus, the differences in sedative and motor-impairing actions of muscimol in various mouse models correlated with the level of forebrain high-affinity [3H]muscimol binding. These data suggest that a small special population of GABAA-Rs, most likely extrasynaptic non-α1-containing receptors, strongly contributes to the in vivo pharmacological effects of muscimol.

Trace and contextual fear conditioning is enhanced in mice lacking the α4 subunit of the GABAA 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.

Gamma-aminobutyric acid type A receptor alpha 4 subunit knockout mice are resistant to the amnestic effect of isoflurane.

BACKGROUND: General anesthesia produces multiple end points including immobility, hypnosis, sedation, and amnesia. Tonic inhibition via &ggr;-aminobutyric acid type A receptors (GABAA-Rs) may play a role in mediating behavioral end points that are suppressed by low concentrations of anesthetics (e.g., hypnosis and amnesia). GABAA-Rs containing the &agr;4 subunit are highly concentrated in the hippocampus and thalamus, and when combined with &dgr; subunits they mediate tonic inhibition, which is sensitive to low concentrations of isoflurane. METHODS: In this study, we used a GABAA &agr;4 receptor knockout mouse line to evaluate the contribution of &agr;4-containing GABAA-Rs to the effects of immobility, hypnosis, and amnesia produced by isoflurane. Knockout mice and their wild-type counterparts were assessed on 3 behavioral tests: conditional fear (to assess amnesia), loss of righting reflex (to assess hypnosis), and the minimum alveolar concentration of inhaled anesthetic necessary to produce immobility in response to noxious stimulation in 50% of subjects (to assess immobility). RESULTS: Genetic inactivation of the &agr;4 subunit reduced the amnestic effect of isoflurane, minimally affected loss of righting reflex, and had no effect on immobility. CONCLUSIONS: These results lend support to the hypothesis that different sites of action mediate different anesthetic end points and suggest that &agr;4-containing GABAA-Rs are important mediators of the amnestic effect of isoflurane on hippocampal-dependent declarative memory.