Synaptic and extrasynaptic gamma -aminobutyric acid type A receptor clusters in rat hippocampal cultures during development - PubMed (original) (raw)

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

A L Scotti et al. Proc Natl Acad Sci U S A. 2001.

Abstract

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. 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 beta2/3 and/or gamma2 subunits remained extrasynaptic.

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Figures

Figure 1

Figure 1

HEK cells (A) transfected with recombinant α1β2γ2 (B) or α1β3γ2 (C) GABAA receptors. In living cells, the antibody recognized both β2- and β3-containing receptors. They appeared as clusters on the cell surface (red). Expression of cotransfected GFP accounts for the cytoplasmic green fluorescence. (D) Live stain for β2/3 subunits 6 h after plating (green label on DIC image). (E–G) β2/3-containing receptors in 20-day-old cultures. Synaptic terminals (red label) were either loaded in living cultures with FM4-64 (E), or labeled with antibodies against the synaptic vesicle marker SV2 (F) or against the GABA-synthesizing enzyme GAD (G). Arrowheads point to some doubly stained, synaptically located β2/3 clusters, and arrows to extrasynaptic receptors. (Bars equal 10 μm; bar in_C_ also applies to B, bar in_D_ also to E–G.) (H) Double labeling of β2/3 (with bd-17 antibody, red) and γ2 subunits (green) in living 20-day-old cultures. Arrowheads point to GABAA clusters, where the β2/3 and γ2 subunits colocalize (yellow spots). β2/3 clusters appear more numerous (arrows) than γ2 clusters. (I) On the second day, γ2-labeled neurites (green) failed to take up FM4-64 during stimulation, but SV2 (red) labeling (J) was detectable. (K) GAD was homogeneously distributed on day 2 in all processes of GABAergic neurons (red). γ2-immunoreactive GABAA clusters (green) and FM4-64 (L), SV2 (M), and GAD (N) labeled punctate synaptic terminals (all red) have increased by 20 days of culture. Arrows point to extrasynaptic γ2 clusters, whereas arrowheads indicate synaptic γ2 clusters. (Bars equal 10 μm; the bar in_H_ applies for H–J and_L–M_ .)

Figure 2

Figure 2

(A–C) Relative densities of β2/3 subunit-containing clusters with presynaptic contacts. Synaptically located receptors increase with increasing age of the cultures. However, the majority of all β2/3 clusters are lacking presynaptic partners even in well-differentiated cultures. Colocalizations with FM4-64 or SV2-positive synapses are maximal at day 30 (A and_B_). The highest density of synaptic β2/3 clusters at GABAergic terminals was measured on day 20 (C). *, P < 0.05; ***,P < 0.001 vs. day 10. †, P < 0.05 vs. day 30. (D–F) Relative densities of γ2 subunit-containing clusters with presynaptic contacts. Synaptic receptor clusters containing γ2 increase with increasing age of the cultures, although the majority of the clusters remain without a presynaptic partner, even in mature cultures on day 30. With GAD-stained synapses, the density is highest on day 20 (F). ***, P < 0.0001 vs. day 5; †, P < 0.002 vs. day 10 and_P_ < 0.05 vs. day 20; **,P < 0.02 vs. day 10; §, P < 0.0001 vs. days 5 and 10.

Figure 3

Figure 3

(A–C) Densities of γ2 subunit-containing GABAA receptor clusters vary with age. γ2 clusters were counted in three independent sets of cells with double labelings (n = 15–47 dendrites per time point). §§,P < 0.0001 vs. days 2 and 10,P < 0.01 vs. day 5; §†, P < 0.001 vs. day 2; P < 0.05 vs. day 10; §,P < 0.001 vs. day 2. **,P < 0.0001 vs. days 2–10; *,P < 0.01 day 5 vs. day 2, and day 30 vs. days 2 and 10. ¶¶, P < 0.0001 vs. days 5, 10, and 30; ¶, P < 0.05 vs. day 10. (D) Values of densities of γ2 clusters in A–C were pooled and plotted together with pooled densities of β2/3 clusters. Numbers in parentheses represent the pooled numbers of dendrites. γ2 density varies with age in culture and peaks on day 20 (**,P < 0.0001 vs. days 2, 10, and 30; *†,P < 0.0001 vs. days 2 and 10; *,P < 0.001 vs. days 2 and 10), whereas β2/3 clusters remain constant between days 10 and 30. GABAA receptors containing the γ2 subunit were significantly less than β2/3-labeled receptors (P < 0.001). (E) Densities of presynaptic boutons increased with age of cells in culture. Presynaptic labelings (FM4-64 and SV2) were low between 2 and 5 days, but all presynaptic boutons, including those labeled with GAD antibody, doubled between 5 and 10 days in vitro, and reached maximal values on day 20. Each time point is the mean ± SEM of the counts in 15–47 dendrites; (*,P < 0.001 vs. day 2 or 5 for FM4-64 and SV2;P < 0.05 for GAD. §, P < 0.001 vs. days 2, 5, 10). The density of GAD-labeled boutons declined significantly between days 20 and 30 (P < 0.05). GAD-positive boutons were significantly less than FM4-64- or SV2-labeled ones at the same age (P < 0.001).

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