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

Abstract

We have simultaneously measured the expression of postsynaptic ␥-aminobutyric acid type A (GABAA) 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 ␤2͞3 and ␥2 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 ␤2͞3 subunits were constant between days 10 and 30 in culture, whereas ␥2 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. GAD-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 ␤2͞3 and͞or ␥2 subunits remained extrasynaptic. T he type A receptors for ␥-aminobutyric acid (GABA A receptors) are ligand-gated ion channels that mediate synaptic inhibition. These chloride channels are heteropentameric structures and can be assembled from a multitude of subunits (␣1-6, ␤1-3, ␥1-3, ␦, , , ). Some subunit combinations are very common and widely distributed in the nervous system, whereas others are restricted to specific neuronal populations (1-4). The GABA A receptor subunit combinations that are most frequently observed in hippocampal granular and pyramidal cells are ␣2␤2͞3␥2 and ␣1␤2͞3␥2. The ␤1 subunit, although less frequent, has a similar distribution as ␤3. The ␣1 and the ␤2 subunits are particularly enriched in interneurons of the hippocampus, where they colocalize with the ␥2 subunit (1, 5, 6). In the rat hippocampus, the ␤2͞3 subunits are present at early stages of embryonal development, whereas expression of ␥2 increases only after birth to moderate levels (7).There is increasing evidence for extrasynaptic GABA A receptors in neurons of the adult brain (8-11), and it was recently found (12) that, in hippocampal cultures, only about 50% of GABA A receptor clusters correlated with apposed presynaptic boutons. Furthermore, recent electrophysiological results from brain slices have shown that ion conductances and kinetics of currents through synaptic and extrasynaptic GABA-activated channels are quite different, which may be important for neuronal function in vivo (8,9).In the present study, GABA A receptor clu...

Clustering of Extrasynaptic GABAA Receptors Modulates Tonic Inhibition in Cultured Hippocampal Neurons

“…Consistent with previous reports (20,25,26,39,53), our immunocytochemical data clearly demonstrate that, in addition to synaptic receptors, also extrasynaptic ones are arranged in clusters and that they become uniformly distributed throughout the cell surface after nocodazole treatment. In agreement with other groups (39, 54 -56), we have found that extrasynaptic receptors in the hippocampus include the ␦ and ␥ 2 subunits.…”

Distribution of α1, α4, γ2, and δ subunits of GABAA receptors in hippocampal granule cells

“…Using the same criteria used to define colocalization of α1 and γ2 subunits with synaptic markers, we found that 1.58±0.13% (n=5) of all α4 subunit clusters colocalized with GAD65 clusters (Fig. 5A), which was significantly lower than the degree of colocalization of α1 subunit clusters (n=6), 26…”

“…Hippocampal sections were stained for GAD65, the synthetic enzyme for GABA, which is known to be concentrated in the synaptic terminals of GABAergic neurons [4,23,26]. Punctate GAD65 immunostaining formed a dense network in the dentate granule cell layer and inner molecular layer, and less staining in the outer molecular layer of the dentate gyrus.…”

Gephyrin Regulates the Cell Surface Dynamics of Synaptic GABAAReceptors

“…We analyzed the synaptic targeting of pHluorin-tagged ␤3 ( pHGFP ␤3) and ␥2 ( pHGFP ␥2) subunits expressed under the CMV promoter in live neurons using FM4-64 to selectively stain active synapses (Lagnado et al, 1996;Mammen et al, 1997;Scotti and Reuter, 2001). This methodology produced robust staining of active presynaptic terminals, and using this marker, it was evident pHGFP ␤3 subunits exhibited both synaptic and extrasynaptic localization (Fig.…”