Synaptic and extrasynaptic γ-aminobutyric acid type A receptor clusters in rat hippocampal cultures during development

We have simultaneously measured the expression of postsynaptic gamma -aminobutyric acid type A (GABA(A)) receptor clusters and of presynaptic boutons in neonatal rat hippocampal cultures between days 1 and 30. GABA(A) receptors were labeled with antibodies recognizing the extracellular domains of beta2/3 and gamma2 subunits. Boutons were visualized by activity-dependent uptake of the styryl dye FM4-64, or by antibodies against the presynaptic vesicular protein SV2 or the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). GABA(A) receptor clusters could be seen in living neurons already 6 h after culturing, much before presynaptic markers could be identified in nerve terminals. The densities of receptor clusters that contained the beta2/3 subunits were constant between days 10 and 30 in culture, whereas gamma2 subunit-containing clusters fluctuated and reached a maximum on day 20. SV2 and GAD staining could be measured from day 2 onwards. Clustering of GAD in presynaptic terminals and FM4-64 uptake were observed only at day 5 and afterward. SV2 staining and FM4-64 uptake increased in parallel between days 5 and 20 and remained constant thereafter. CAD-stained boutons were fewer than those labeled with other, less specific, presynaptic stains. They reached a maximum on day 20 and fell again toward day 30. Double labeling of GABA(A) receptors and of presynaptic boutons in neurons during differentiation showed that, even after 30 days in culture, large fractions of GABA(A) receptor clusters containing beta2/3 and/or gamma2 subunits remained extrasynaptic.