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Two types of GABAA receptor-mediated inhibition (phasic and tonic) have been described in cerebellar granule cells, although these cells receive GABAergic input only from a single cell type, the Golgi cell. In adult rats, granule cells express six GABAA receptor subunits abundantly (alpha1, alpha6, beta2, beta3, gamma2, and delta), which are coassembled into at least four to six distinct GABAA receptor subtypes. We tested whether a differential distribution of GABAA receptors on the surface of granule cells could play a role in the different forms of inhibition, assuming that phasic inhibition originates from the activation of synaptic receptors, whereas tonic inhibition is provided mainly by extrasynaptic receptors. The alpha1, alpha6, beta2/3, and gamma2 subunits have been found by immunogold localizations to be concentrated in GABAergic Golgi synapses and also are present in the extrasynaptic membrane at a lower concentration. In contrast, immunoparticles for the delta subunit could not be detected in synaptic junctions, although they were abundantly present in the extrasynaptic dendritic and somatic membranes. Gold particles for the alpha6, gamma2, and beta2/3, but not the alpha1 and delta, subunits also were concentrated in some glutamatergic mossy fiber synapses, where their colocalization with AMPA-type glutamate receptors was demonstrated. The exclusive extrasynaptic presence of the delta subunit-containing receptors, together with their kinetic properties, suggests that tonic inhibition could be mediated mainly by extrasynaptic alpha6beta2/3delta receptors, whereas phasic inhibition is attributable to the activation of synaptic alpha1beta2/3gamma2, alpha6beta2/3gamma2, and alpha1alpha6beta2/3gamma2 receptors.

GABA(A) receptors are the major inhibitory neurotransmitter receptors in the brain and are the site of action of many clinically important drugs. These receptors are composed of five subunits that can belong to eight different subunit classes. Depending on their subunit composition, these receptors exhibit distinct pharmacological and electrophysiological properties. Recent studies on recombinant and native GABA(A) receptors suggest the existence of far more receptor subtypes than previously assumed. Thus, receptors composed of one, two, three, four, or five different subunits might exist in the brain. Studies on the regional, cellular and subcellular distribution of GABA(A) receptor subunits, and on the co-localization of these subunits at the light and electron microscopic level for the first time provide information on the distribution of GABA(A) receptor subtypes in the brain. These studies will have to be complemented by electrophysiological and pharmacological studies on the respective recombinant and native receptors to finally identify the receptor subtypes present in the brain. The distinct cellular and subcellular location of individual receptor subtypes suggests that they exhibit specific functions in the brain that can be selectively modulated by subtype specific drugs. This conclusion is supported by the recent demonstration that different GABA(A) receptor subtypes mediate different effects of benzodiazepines. Together, these results should cause a revival of GABA(A) receptor research and strongly stimulate the development of drugs with a higher selectivity for alpha2-, alpha3-, or alpha5-subunit-containing receptor subtypes. Such drugs might exhibit quite selective clinical effects.

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␥-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.

GABA A receptors are ligand-gated chloride ion channels that are presumed to be pentamers composed of ␣, ␤, and ␥ subunits. The subunit stoichiometry, however, is controversial, and the subunit arrangement presently is not known. In this study the ratio of subunits in recombinant ␣1␤3␥2 receptors was determined in Western blots from the relative signal intensities of antibodies directed against the N terminus or the cytoplasmic loop of different subunits after the relative reactivity of these antibodies had been determined with GABA A receptor subunit chimeras composed of the N-terminal domain of one and the remaining part of the other subunit. Via this method a subunit stoichiometry of two ␣ subunits, two ␤ subunits, and one ␥ subunit was derived. Similar experiments investigating the composition of ␣1␤3 receptors expressed on the surface of human embryonic kidney (HEK) 293 cells cotransfected with ␣1 and ␤3 subunits resulted in a stoichiometry of two ␣ and three ␤ subunits. Density gradient centrifugation studies indicated that combinations of ␣1␤3␥2 or ␣1␤3 subunits expressed in HEK 293 cells are able to form pentamers, whereas combinations of ␣1␥2 or ␤3␥2 subunits predominantly form heterodimers. These results provide valuable information on the mechanism of GABA A receptor assembly and support the conclusion that GABA A receptors are pentamers in which a total of four alternating ␣ and ␤ subunits are connected by a ␥ subunit. Key words: GABA A receptor; stoichiometry; assembly; subunit arrangement; human embryonic kidney 293 cells; chimeric subunits; density gradient centrifugation; Western blotGABA, the major inhibitory transmitter in the CNS, mediates fast synaptic inhibition by opening the chloride ion channel intrinsic to the GABA A receptor. This receptor is a hetero-oligomeric protein and the site of action of a variety of pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, steroids, anesthetics, and convulsants (Sieghart, 1995). So far, six ␣, three ␤, three ␥, one ␦, and two subunits of these receptors, as well as several alternatively spliced isoforms of some of these subunits, have been identified in mammalian brain (Macdonald and Olsen, 1994;Sieghart, 1995). Expression studies have indicated that an ␣, a ␤, and a ␥ subunit have to combine to produce GABA A receptors with a pharmacology resembling that of receptors found in the brain and that, depending on the subunits used for transfection of cells, receptors with distinct pharmacological and electrophysiological properties do arise (Sieghart, 1995). Overall it is assumed, however, that a total of five subunits have to combine to form functional GABA A receptors (Nayeem et al., 1994).A variety of subunit-specific antibodies has been raised to investigate the subunit composition of GABA A receptors. Immunocytochemical studies demonstrating the colocalization of subunits in GABA A receptor clusters on neuronal membranes (Fritschy et al., 1992;Caruncho and Costa, 1994;Fritschy and Möhler, 1995;Somogyi et al., 199...