␥-Aminobutyric acid (GABA) type A receptors mediate fast inhibitory synaptic transmission and have been implicated in responses to sedative͞hypnotic agents (including neuroactive steroids), anxiety, and learning and memory. Using gene targeting technology, we generated a strain of mice deficient in the ␦ subunit of the GABA type A receptors. In vivo testing of various behavioral responses revealed a strikingly selective attenuation of responses to neuroactive steroids, but not to other modulatory drugs. Electrophysiological recordings from hippocampal slices revealed a significantly faster miniature inhibitory postsynaptic current decay time in null mice, with no change in miniature inhibitory postsynaptic current amplitude or frequency. Learning and memory assessed with fear conditioning were normal. These results begin to illuminate the novel contributions of the ␦ subunit to GABA pharmacology and sedative͞hypnotic responses and behavior and provide insights into the physiology of neurosteroids.

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 ␣4 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 ␣4 subunit by targeted disruption of the Gabra4 gene. ␣4 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 ␣4 subunit and that gaboxadol achieves its effects via the activation of this GABA A-R subtype.ABA is the major inhibitory neurotransmitter in the mammalian CNS. Its primary target, GABA A receptors (GABA A -Rs), are pentameric complexes that function as ligandgated chloride ion channels. Two types of inhibitory neurotransmission are mediated via GABA A -Rs (1, 2). Phasic inhibition results from the activation of receptors at the synapse by intermittent release of high concentrations of GABA from presynaptic terminals. Tonic inhibition, in contrast, is mediated by the continuous activation of receptors located outside the synaptic cleft by low concentrations of ambient GABA. These ''extrasynaptic'' GABA A -Rs have a higher affinity for GABA and have faster channel deactivation rates (3, 4) and, more importantly, slower rates of desensitization (1-5), relative to the classical ''synaptic'' GABA A -Rs.There are a variety of subunit families that make up GABA ARs; a total of 19 distinct subunits have been cloned, ␣1-6, ␤1-3, ␥1-3, ␦, , , , and 1-3 (6). This diversity in subunit composition results in substantial anatomical, functional, and pharmacological heterogeneity. GABA A -Rs containing the ␣4 subunit are highly expressed in the thalamus and dentate gyrus, with lower levels in cortex, striatum, and other brain areas (7-9). GABA A -Rs containing ␣4 subunits often are found with the ␥2 or ␦ subunits, in combination with ␤ subunits; the ␣4␤␦ subtypes are proposed to be localized to extrasynaptic sites and contribute to tonic inhibition (5, 10-13). Other extrasynaptic receptor subtypes include ␣5␤3␥2 in hippocampal CA1 pyramidal cells (14) and ␣6␤␦ in cerebellar granule cells (15). Notably, the ␣4 subunit containing GABA A -Rs, especially ␣4␤␥2, are not exclusively extrasynaptic; some are found within dentate gyrus synapses and others are located perisynaptically, wher...

ABSTRACT␥-Aminobutyric acid type A receptors (GABA A -Rs) mediate the bulk of rapid inhibitory synaptic transmission in the central nervous system. The ␤3 subunit is an essential component of the GABA A -R in many brain regions, especially during development, and is implicated in several pathophysiologic processes. We examined mice harboring a ␤3 gene inactivated by gene targeting. GABA A -R density is approximately halved in brain of ␤3-deficient mice, and GABA A -R function is severely impaired. Most ␤3-deficient mice die as neonates; some neonatal mortality, but not all, is accompanied by cleft palate. ␤3-deficient mice that survive are runted until weaning but achieve normal body size by adulthood, although with reduced life span. These mice are fertile but mothers fail to nurture offspring. Brain morphology is grossly normal, but a number of behaviors are abnormal, consistent with the widespread location of the ␤3 subunit. The mice are very hyperactive and hyperresponsive to human contact and other sensory stimuli, and often run continuously in tight circles. When held by the tail, they hold all paws in like a ball, which is frequently a sign of neurological impairment. They have difficulty swimming, walking on grids, and fall off platforms and rotarods, although they do not have a jerky gait. ␤3-deficient mice display frequent myoclonus and occasional epileptic seizures, documented by electroencephalographic recording. Hyperactivity, lack of coordination, and seizures are consistent with reduced presynaptic inhibition in spinal cord and impaired inhibition in higher cortical centers and͞or pleiotropic developmental defects.

Neuronal rhythmic activities within thalamocortical circuits range from partially synchronous oscillations during normal sleep to hypersynchrony associated with absence epilepsy. It has been proposed that recurrent inhibition within the thalamic reticular nucleus serves to reduce synchrony and thus prevents seizures. Inhibition and synchrony in slices from mice devoid of the gamma-aminobutyric acid type-A (GABAA) receptor beta3 subunit were examined, because in rodent thalamus, beta3 is largely restricted to reticular nucleus. In beta3 knockout mice, GABAA-mediated inhibition was nearly abolished in reticular nucleus, but was unaffected in relay cells. In addition, oscillatory synchrony was dramatically intensified. Thus, recurrent inhibitory connections within reticular nucleus act as "desynchronizers."

According to the rules of GABA(A) receptor (GABA(A)R) subunit assembly, alpha4 and alpha6 subunits are considered to be the natural partners of delta subunits. These GABA(A)Rs are a preferred target of low, sobriety-impairing concentrations of ethanol. Here we demonstrate a new naturally occurring GABA(A)R subunit partnership: delta subunits of hippocampal interneurons are coexpressed and colocalized with alpha1 subunits, but not with alpha4, alpha6 or any other alpha subunits. Ethanol potentiates the tonic inhibition mediated by such native alpha1/delta GABA(A)Rs in wild-type and in alpha4 subunit-deficient (Gabra4(-/-)) mice, but not in delta subunit-deficient (Gabrd(-/-)) mice. We also ruled out any compensatory upregulation of alpha6 subunits that might have accounted for the ethanol effect in Gabra4(-/-) mice. Thus, alpha1/delta subunit assemblies represent a new neuronal GABA(A)R subunit partnership present in hippocampal interneurons, mediate tonic inhibitory currents and are highly sensitive to low concentrations of ethanol.

The delta subunit is a novel subunit of the pentameric gamma-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 delta subunit have been produced by gene-targeting technology, and these mice were studied with immunohistochemical and immunoblot methods to determine whether changes in GABA(A) receptors were limited to deletion of the delta subunit or whether alterations in other GABA(A) receptor subunits were also present in the delta subunit knockout (delta-/-) mice. Immunohistochemical studies of wild-type mice confirmed the restricted distribution of the delta subunit in the forebrain. Regions with moderate to high levels of delta subunit expression included thalamic relay nuclei, caudate-putamen, molecular layer of the dentate gyrus, and outer layers of the cerebral cortex. Virtually no delta 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 delta-/- and wild-type mice demonstrated substantial changes in the alpha4 and gamma2 subunits of the GABA(A) receptor in the delta-/- mice. gamma2 Subunit expression was increased, whereas alpha4 subunit expression was decreased in delta-/- mice. Importantly, alterations of both the alpha4 and the gamma2 subunits were confined primarily to brain regions that normally expressed the delta subunit. This suggests that the additional subunit changes are directly linked to loss of the delta subunit and could reflect local changes in subunit composition and function of GABA(A) receptors in delta-/- mice.

GABA A receptors mediate fast inhibitory neurotransmission in the central nervous system (CNS), and approximately half of these receptors contain ␣1 subunits. GABA A receptor ␣1 subunits are important for receptor assembly and specific pharmacological responses to benzodiazepines. Plasticity in GABA A receptor ␣1 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 ␣1 subunitcontaining 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 ␣1 subunit. The mice are viable but exhibit an intention tremor. Western blot analysis confirms the complete loss of ␣1 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 ␤2/3 and ␥2 subunits and increased expression of ␣2 and ␣3 subunits. There was no change in ␣4, ␣5, or ␦ subunit peptide levels in cerebral cortex. Knockout mice exhibit loss of over half of GABA A receptors measured by [ 3

Targeted deletion of the alpha1 subunit gene results in a profound loss of gamma-aminobutyric acid type A (GABA(A)) receptors in adult mouse brain but has only moderate behavioral consequences. Mutant mice exhibit several adaptations in GABA(A) receptor subunit expression, as measured by Western blotting. By using immunohistochemistry, we investigated here whether these adaptations serve to replace the missing alpha1 subunit or represent compensatory changes in neurons that normally express these subunits. We focused on cerebellum and thalamus and distinguished postsynaptic GABA(A) receptor clusters by their colocalization with gephyrin. In the molecular layer of the cerebellum, alpha1 subunit clusters colocalized with gephyrin disappeared from Purkinje cell dendrites of mutant mice, whereas alpha3 subunit/gephyrin clusters, presumably located on dendrites of Golgi interneurons, increased sevenfold, suggesting profound network reorganization in the absence of the alpha1 subunit. In thalamus, a prominent increase in alpha3 and alpha4 subunit immunoreactivity was evident, but without change in regional distribution. In the ventrobasal complex, which contains primarily postsynaptic alpha1- and extrasynaptic alpha4-GABA(A) receptors, the loss of alpha1 subunit was accompanied by disruption of gamma2 subunit and gephyrin clustering, in spite of the increased alpha4 subunit expression. However, in the reticular nucleus, which lacks alpha1-GABA(A) receptors in wild-type mice, postsynaptic alpha3/gamma2/gephyrin clusters were unaffected. These results demonstrate that adaptive responses in the brain of alpha1(0/0) mice involve reorganization of GABAergic circuits and not merely replacement of the missing alpha1 subunit by another receptor subtype. In addition, clustering of gephyrin at synaptic sites in cerebellum and thalamus appears to be dependent on expression of a GABA(A) receptor subtype localized postsynaptically.