K. Kannenberg et al., Clusters of GABA(A) receptors on cultured hippocampal cells correlate onlypartially with functional synapses, EUR J NEURO, 11(4), 1999, pp. 1256-1264
We describe a method to label gamma-aminobutyric acid (GABA)(A) receptors o
n the surface of living hippocampal neurons in primary culture, and we comp
are the distribution of receptors with that of active synapses. To visualiz
e GABA(A) receptors, the affinity-purified antibody beta 3(1-13), recognizi
ng the extracellular N-termini of the GABA(A) receptor beta 2- and beta 3-s
ubunits, was used in combination with fluorescent secondary antibodies. The
beta 2- and beta 3-subunits belong to the predominant GABA(A) receptor sub
units in the hippocampus, As expected for aggregates of GABA(A) receptors i
n the somato-dendritic plasma membrane, a patchy staining pattern similar t
o that seen by labelling neurons after fixation was obtained. An antiserum
recognizing an intracellular epitope of GABA(A) receptor beta 3-subunits di
d 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 wi
th the labelling of active presynaptic terminals with the fluorescent dyes
FM1-43 or FM4-64, consistently resulted in the detection of GABA(A) recepto
r clusters that were not located at active synapses. These amounted to appr
oximate to 50% of all labelled GABA(A) receptor clusters. GABA(A) receptor
clusters that were not associated with active presynaptic terminals partial
ly 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 hippocamp
al neurons. They also indicate that for the maintenance of GABA(A) receptor
aggregates, the release of GABA from an opposing active terminal is not es
sential.