Intact sorting, targeting, and clustering of γ‐aminobutyric acid A receptor subtypes in hippocampal neurons in vitro

Abstract

The cellular and subcellular distribution of four GABA A receptor subtypes, identified by the presence of the α1, α2, α3, or α5 subunit, was investigated immunocytochemically in dissociated cultures of hippocampal neurons. We addressed the questions whether (1) cell‐type specific expression, (2) axonal/somatodendritic targeting, and (3) synaptic/extrasynaptic clustering of GABA A receptor subtypes was retained in vitro. For comparison, the in vivo distribution pattern was assessed in sections from adult rat brain. The differential expression of GABA A receptor subunits allowed to identify five morphologically distinct cell types in culture: the α1 subunit was observed in glutamic acid decarboxylase–positive interneurons, the α2 and α5 subunits marked pyramidal‐like cells, and the α3 subunit labeled three additional cell types, including presumptive hilar cells. All subunits were found in the somatodendritic compartment. In addition, appropriate axonal targeting was evidenced by the intense α2, and sometimes α3 subunit labeling of axon‐initial segments (AIS) of pyramidal cells and hilar cells, respectively. Accordingly, both receptor subtypes were targeted to AIS in vivo, as well. Synaptic receptors were identified by colocalization with gephyrin, a postsynaptic clustering protein, and apposition to presynaptic terminals labeled with synapsin I. In vitro and in vivo, α1‐ and α2‐receptor subtypes formed numerous synaptic clusters, α3‐GABA A receptors were located either synaptically or extrasynaptically depending on the cell type, whereas α5‐GABA A receptors were extrasynaptic. We conclude that receptor targeting to broad subcellular locations does not require specific GABAergic innervation patterns, which are disturbed in vitro, but depends on protein‐protein interactions in the postsynaptic cell that are both subunit‐ and neuron‐specific. J. Comp. Neurol. 443:43–55, 2002. © 2002 Wiley‐Liss, Inc.