Five subtypes of type A gamma-aminobutyric acid receptors identified in neurons by double and triple immunofluorescence staining with subunit-specific antibodies.

Abstract

The extraordinary structural diversity of subunits forming type A y-aminobutyric acid (GABAA) (al-6, 1-4, y1-3, 8, p) (1-5) provides the basis for an extraordinary structural diversity of GABAA receptors. The heterogeneity of GABAA receptors is expected to provide a hitherto unexplored diversity in the functions of receptor subtypes affecting their sensitivity to GABA, modulation by allosteric effectors, adaptation to stimulus conditions, distribution within a neuron, ontogenetic development, or alterations in disease states (1).As a prerequisite for a functional analysis of receptor subtypes, elucidation of their cellular location and subunit composition is essential. So far, the distribution of subunits has largely been mapped at the mRNA level (6-12). However, the coassembly of subunits and the presence of multiple receptors in a single neuron can be demonstrated only by detailed immunohistochemical analysis. In this study, the possible colocalization ofthe al, a3, P2,3, and Y2 subunits was assessed in rat brain neurons by double and triple immunofluorescence staining. The pattern of subunit immunoreactivity (IR) was analyzed at the cellular and subcellular levels by confocal laser microscopy. To visualize the al, a3, and 'Y2 subunits, polyclonal antisera were used whose specificity had been assessed by Western blotting, immunoprecipitation, and immunohistochemistry (13-15). Since antibodies for single P subunits are not available, the monoclonal antibody bd-17 (16, 17), which recognizes both the P2 and the f3 subunit (18), was used to represent the /-subunit family. Subunits not included in the present study are not of major preponderance (a2, a4, a5, 81, yi, )y3, p) (5,8,9,11,(19)(20)(21) or are largely restricted to a particular population of neuronse.g., cerebellar granule cells (a6, 8) (7,22,23). The present analysis was restricted to the olfactory bulb, basal forebrain, basal ganglia, brainstem, and cerebellum, where the pattern of subunit distribution was most distinctive. Five distinct combinations of subunits were detected denoting different GABAA receptor subtypes. Some of the receptors could be allocated to defined neurons by double staining with neurotransmitter markers. MATERIALS AND METHODSFollowing anesthesia with chloral hydrate, adult male Wistar rats were perfused transcardially with paraformaldehyde/ picric acid (24). Brains were removed, postfixed for 2 hr, and stored overnight in phosphate-buffered saline containing 10%6 dimethyl sulfoxide for cryoprotection. Sections were cut from frozen blocks with a sliding microtome and processed free-floating for double and triple immunofluorescence staining (25). Polyclonal antisera directed against subunit-specific peptides (a,, residues 1-16; a3, 1-15; Y2, 1-15) raised in rabbits (al, a3, Y2) (13-15) and guinea pigs (a3, V2), as well as the mouse monoclonal antibody bd-17 (16, 18) (#2 and 183 subunits), were used as primary antibodies. After overnight incubation of sections with antibodies diluted in phosphatebuffered saline containi...

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABAAReceptor Clustering in Cultured Hippocampal Neurons

“…The ␣ 2 subunit concentrates postsynaptically to terminals of parvalbumin-negative basket cells and in axo-axonic synapses on the axon initial segment of hippocampal CA1 pyramidal cells (Nusser et al, 1996a). Others have also presented data at the light microscopy level consistent with differential distribution of GABA A R ␣ 1 , ␣ 2 , and ␣ 3 subunit isoforms in various synapses (Fritschy et al, 1992;Koulen, 1996;Fletcher et al, 1998). However, it is not entirely proven that the receptor puncta observed with the light microscope correspond to synapses.…”

“…Moreover, in these studies, the possible heterogeneity of the GABA A subunit or isoform composition in the receptor clusters has not been addressed. This is an important issue in light of observations that in the intact brain and retina some GABAergic synapses and puncta show selectivity for certain ␣ subunit-isoform-containing GABA A Rs (Fritschy et al, 1992;Koulen et al, 1996;Nusser et al, 1996a;Fletcher et al, 1998;Nyiri et al, 2001).…”

A Conceptualization of Integrated Actions of Ethanol Contributing to its GABAmimetic Profile: A Commentary

“…Additionally, Harris et al (1997) found that ethanol enhanced muscimol-induced chloride flux in L(tk À ) cells transfected with a 1 b 2/3 g 2L GABA A receptor subunits. From immunoprecipitation (Benke et al, 1991a) and immunohistochemical studies of brain (Fritschy et al, 1992), the a 1 receptor was found to combine with b 2/3 and g 2 GABA A receptor subunits (see review by Mohler et al, 1995). This GABA A receptor combination (a 1 b X g 2 ) reportedly is the most abundant BZD/GABA A receptor in brain Lüddens and Korpi, 1995;Mckernan and Whiting, 1996).…”

“…Thus, with this clarification, ethanol action on Purkinje neurons was consistent with the hypothesis that the ethanol enhancement of the effect of GABA in vivo related to ethanol having a direct effect on GABA A receptors sensitive to zolpidem. Since other GABA A receptor subunits had differing distributions from that of the a 1 subunit forming the type-1-BZD receptor (Fritschy et al, 1992;Wisden et al, 1992), it was proposed that this differing distribution of GABA A receptor subtypes accounted for the regional specificity of ethanol to enhance GABA responsiveness .…”

Properties of GABAAReceptors Underlying Inhibitory Synaptic Currents in Neocortical Pyramidal Neurons

“…The GABA A receptor channel is thought to be a heteropentameric complex assembled from at least 16 potential subunits (Macdonald and Olsen, 1994;Sieghart, 1995), and different neuronal types have been shown to express distinct combinations of GABA A receptor subunits (Fritschy et al, 1992;Wisden et al, 1992;Ruano et al, 1997) (for review, see McKernan and Whiting, 1996). Because subunit composition determines GABA A receptor kinetic properties (Verdoorn et al, 1990), this structural heterogeneity could provide cell-type specificity in the kinetic characteristics of the I PSC s (Puia et al, 1994).…”