Received for publication September 20, 2004.
Revised November 15, 2004.
Accepted for publication December 21, 2004.

CULTURED HIPPOCAMPAL PYRAMIDAL NEURONS EXPRESS TWO KINDS OF GABA_A RECEPTORS

Abstract

We combined a study of subcellular distribution of the 1,2, 4, 1, 2/3, 2, and subunits of the GABA_A receptor with an electrophysiological analysis of GABA_A receptor currents to determine the types of receptors expressed on cultured hippocampal pyramidal neurons. The immunocytochemistry study demonstrated that 1, 2, 2/3, and 2 subunits formed distinct clusters of various sizes, which were colocalizaed with clusters of glutamate decarboxylase (GAD) immunoreactivity at rates ranging from 22-58%. In contrast, 4, 1, and subunits were distributed diffusely over the cell soma and neuronal processes of cultured neurons and did not colocalize with the synaptic marker GAD. Whole-cell GABA_A receptors currents were moderately sensitive to GABA and were modulated by diazepam. The whole cell currents were also enhanced by the neurosteroid allopregnanolone (10 nM). Tonic currents, measured as changes in baseline current and noise, were sensitive to Zn²⁺, furosemide and loreclezole; and insensitive to diazepam. These studies suggest that two kinds of GABA_A receptors are expressed on cultured hippocampal neurons. One kind of receptors formed clusters, which were present at GABAergic synapses and in the extrasynaptic membrane. The 1, 2, 2/3, and 2 subunits were contained in clustered receptors. The second kind was distributed diffusely in the extrasynaptic membrane. The 4, 1, and subunits were contained in these diffusely distributed receptors. The properties of tonic currents recorded from these neurons were similar to those from recombinant receptors containing 4, 1, and subunits.

Key words: GABAA, GABAC, Fluorescence techniques, Immunocytochemistry

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