GABAA-receptors display an extensive structural heterogeneity based on the differential assembly of a family of at least 15 subunits (alpha 1-6, beta 1-3, gamma 1-3, delta, rho 1-2) into distinct heteromeric receptor complexes. The subunit composition of receptor subtypes is expected to determine their physiological properties and pharmacological profiles, thereby contributing to flexibility in signal transduction and allosteric modulation. In heterologous expression systems, functional receptors require a combination of alpha-, beta-, and gamma-subunit variants, the gamma 2-subunit being essential to convey a classical benzodiazepine site to the receptor. The subunit composition and stoichiometry of native GABAA-receptor subtypes remain unknown. The aim of this study was to identify immunohistochemically the main subunit combinations expressed in the adult rat brain and to allocate them to identified neurons. The regional and cellular distribution of seven major subunits (alpha 1, alpha 2, alpha 3, alpha 5, beta 2,3, gamma 2, delta) was visualized by immunoperoxidase staining with subunit-specific antibodies (the beta 2- and beta 3-subunits were covisualized with the monoclonal antibody bd-17). Putative receptor subtypes were identified on the basis of colocalization of subunits within individual neurons, as analyzed by confocal laser microscopy in double- and triple-immunofluorescence staining experiments. The results reveal an extraordinary heterogeneity in the distribution of GABAA-receptor subunits, as evidenced by abrupt changes in immunoreactivity along well-defined cytoarchitectonic boundaries and by pronounced differences in the cellular distribution of subunits among various types of neurons. Thus, functionally and morphologically diverse neurons were characterized by a distinct GABAA-receptor subunit repertoire. The multiple staining experiments identified 12 subunit combinations in defined neurons. The most prevalent combination was the triplet alpha 1/beta 2,3/gamma 2, detected in numerous cell types throughout the brain. An additional subunit (alpha 2, alpha 3, or delta) sometimes was associated with this triplet, pointing to the existence of receptors containing four subunits. The triplets alpha 2/beta 2,3/gamma 2, alpha 3/beta 2,3/gamma 2, and alpha 5/beta 2,3/gamma 2 were also identified in discrete cell populations. The prevalence of these seven combinations suggest that they represent major GABAA-receptor subtypes. Five combinations also apparently lacked the beta 2,3-subunits, including one devoid of gamma 2-subunit (alpha 1/alpha 2/gamma 2, alpha 2/gamma 2, alpha 3/gamma 2, alpha 2/alpha 3/gamma 2, alpha 2/alpha 5/delta).(ABSTRACT TRUNCATED AT 400 WORDS)

GABA(A) (gamma-aminobutyric acid(A)) receptors are molecular substrates for the regulation of vigilance, anxiety, muscle tension, epileptogenic activity and memory functions, which is evident from the spectrum of actions elicited by clinically effective drugs acting at their modulatory benzodiazepine-binding site. Here we show, by introducing a histidine-to-arginine point mutation at position 101 of the murine alpha1-subunit gene, that alpha1-type GABA(A) receptors, which are mainly expressed in cortical areas and thalamus, are rendered insensitive to allosteric modulation by benzodiazepine-site ligands, whilst regulation by the physiological neurotransmitter gamma-aminobutyric acid is preserved. alpha1(H101R) mice failed to show the sedative, amnesic and partly the anticonvulsant action of diazepam. In contrast, the anxiolytic-like, myorelaxant, motor-impairing and ethanol-potentiating effects were fully retained, and are attributed to the nonmutated GABA(A) receptors found in the limbic system (alpha2, alpha5), in monoaminergic neurons (alpha3) and in motoneurons (alpha2, alpha5). Thus, benzodiazepine-induced behavioural responses are mediated by specific GABA(A) receptor subtypes in distinct neuronal circuits, which is of interest for drug design.

Most fast inhibitory neurotransmission in the brain is mediated by GABAA receptors, which are mainly postsynaptic and consist of diverse alpha and beta subunits together with the gamma 2 subunit. Although the gamma 2 subunit is not necessary for receptor assembly and translocation to the cell surface, we show here that it is required for clustering of major postsynaptic GABAA receptor subtypes. Loss of GABAA receptor clusters in mice deficient in the gamma 2 subunit, and in cultured cortical neurons from these mice, is paralleled by loss of the synaptic clustering molecule gephyrin and synaptic GABAergic function. Conversely, inhibiting gephyrin expression causes loss of GABAA receptor clusters. The gamma 2 subunit and gephyrin are thus interdependent components of the same synaptic complex that is critical for postsynaptic clustering of abundant subtypes of GABAA receptors in vivo.

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Benzodiazepine tranquilizers are used in the treatment of anxiety disorders. To identify the molecular and neuronal target mediating the anxiolytic action of benzodiazepines, we generated and analyzed two mouse lines in which the alpha2 or alpha3 GABAA (gamma-aminobutyric acid type A) receptors, respectively, were rendered insensitive to diazepam by a knock-in point mutation. The anxiolytic action of diazepam was absent in mice with the alpha2(H101R) point mutation but present in mice with the alpha3(H126R) point mutation. These findings indicate that the anxiolytic effect of benzodiazepine drugs is mediated by alpha2 GABAA receptors, which are largely expressed in the limbic system, but not by alpha3 GABAA receptors, which predominate in the reticular activating system.

The heterogeneity of ␥-aminobutyric acid type A (GABAA) receptors contributes to the diversity of neuronal inhibition in the regulation of information processing. Although most GABA A receptors are located synaptically, the small population of ␣5GABAA receptors is largely expressed extrasynaptically. To clarify the role of the ␣5GABAA receptors in the control of behavior, a histidineto-arginine point mutation was introduced in position 105 of the murine ␣5 subunit gene, which rendered the ␣5GABAA receptors diazepam-insensitive. Apart from an incomplete muscle relaxing effect, neither the sedative, anticonvulsant, nor anxiolytic-like activity of diazepam was impaired in ␣5(H105R) mice. However, in hippocampal pyramidal cells, the point mutation resulted in a selective reduction of ␣5GABAA receptors, which altered the drugindependent behavior. In line with the role of the hippocampus in certain forms of associative learning, trace fear conditioning, but not delay conditioning or contextual conditioning, was facilitated in the mutant mice. Trace fear conditioning differs from delay conditioning in that the conditioned and unconditioned stimulus are separated by a time interval. Thus, the largely extrasynaptic ␣5GABAA receptors in hippocampal pyramidal cells are implicated as control elements of the temporal association of threat cues in trace fear conditioning.

Classical benzodiazepine drugs are in wide clinical use as anxiolytics, hypnotics, anticonvulsants, and muscle relaxants. They act by enhancing the ␥-aminobutyric acid A (GABA A ) receptor function in the central nervous system. The pharmacological relevance of the multitude of structurally diverse GABA A receptor subtypes has only recently been identified. Based on an in vivo point mutation strategy, ␣ 1 -GABA A receptors were found to mediate sedation, anterograde amnesia, and part of the seizure protection, whereas ␣ 2 -GABA A receptors, but not ␣ 3 -receptors, mediate anxiolysis. Rational drug targeting to specific receptor subtypes has now become possible. Only restricted neuronal networks will be modulated by the new subtype-selective drugs. Promising new anxiolytics have already been developed. A new pharmacology of the benzodiazepine site is on the horizon.GABAergic inhibition is one of the most rapidly developing topics in neuropharmacology. New therapeutic opportunities arise due to increasing insights into the molecular architecture and diversity of the components involved in signal transduction such as GABA A receptors, GABA B receptors, and GABA transporters (Fig. 1). GABA A receptors are important drug targets representing the sites of action of benzodiazepines, barbiturates, and neurosteroids. The present article focuses on the pharmacological distinction of GABA A receptor subtypes as a basis for the development of new drugs that target restricted neuronal networks. In particular, new ligands of the benzodiazepine site acting selectively on GABA A receptor subtypes are expected to dissect the pharmacological spectrum of classical benzodiazepines and display a minimum of side effects. For further information on GABA A receptor subtypes other recent reviews may be consulted (Barnard et al

Although glycine is a major inhibitory transmitter in the mammalian CNS, the role of glycinergic neurons in defined neuronal circuits remains ill defined. This is due in part to difficulties in identifying these cells in living slice preparations for electrophysiological recordings and visualizing their axonal projections. To facilitate the morphological and functional analysis of glycinergic neurons, we generated bacterial artificial chromosome (BAC) transgenic mice, which specifically express enhanced green fluorescent protein (EGFP) under the control of the promotor of the glycine transporter (GlyT) 2 gene, which is a reliable marker for glycinergic neurons. Neurons expressing GlyT2-EGFP were intensely fluorescent, and their dendrites and axons could be visualized in great detail. Numerous positive neurons were detected in the spinal cord, brainstem, and cerebellum. The hypothalamus, intralaminar nuclei of the thalamus, and basal forebrain also received a dense GlyT2-EGFP innervation, whereas in the olfactory bulb, striatum, neocortex, hippocampus, and amygdala positive fibers were much less abundant. No GlyT2-EGFP-positive cell bodies were seen in the forebrain. On the subcellular level, GlyT2-EGFP fluorescence was colocalized extensively with glycine immunoreactivity in somata and dendrites and with both glycine and GlyT2 immunoreactivity in axon terminals, as shown by triple staining at all levels of the neuraxis, confirming the selective expression of the transgene in glycinergic neurons. In slice preparations of the spinal cord, no difference between the functional properties of EGFP-positive and negative neurons could be detected, confirming the utility of visually identifying glycinergic neurons to investigate their functional role in electrophysiological studies.