Clusters of GABAA receptors on cultured hippocampal cells correlate only partially with functional synapses

We describe a method to label gamma-aminobutyric acid (GABA)(A) receptors on the surface of living hippocampal neurons in primary culture, and we compare the distribution of receptors with that of active synapses. To visualize GABA(A) receptors, the affinity-purified antibody beta 3(1-13), recognizing the extracellular N-termini of the GABA(A) receptor beta 2- and beta 3-subunits, was used in combination with fluorescent secondary antibodies. The beta 2- and beta 3-subunits belong to the predominant GABA(A) receptor subunits in the hippocampus, As expected for aggregates of GABA(A) receptors in the somato-dendritic plasma membrane, a patchy staining pattern similar to that seen by labelling neurons after fixation was obtained. An antiserum recognizing an intracellular epitope of GABA(A) receptor beta 3-subunits did not label the receptors in living neurons. Whole-cell recordings of GABA-evoked Cl (-) currents were not affected after decorating GABA(A) receptors with antibody beta 3(1-13). Combining the staining of GABA(A) receptors with the labelling of active presynaptic terminals with the fluorescent dyes FM1-43 or FM4-64, consistently resulted in the detection of GABA(A) receptor clusters that were not located at active synapses. These amounted to approximate to 50% of all labelled GABA(A) receptor clusters. GABA(A) receptor clusters that were not associated with active presynaptic terminals partially colocalized with the synaptic vesicle marker protein sv2, while another fraction had no presynaptic counterpart at all. These findings suggest the presence of presynaptically silent GABAergic synapses in cultured hippocampal neurons. They also indicate that for the maintenance of GABA(A) receptor aggregates, the release of GABA from an opposing active terminal is not essential.