Figure 3.

Distribution of astrocyte volume at GABAergic synapses compared to glutamatergic synapses. (A) Example of a visualization of an EGFP-expressing astrocyte (green), presynaptic GABAergic terminals (VGAT, red) and postsynaptic gephyrin clusters (Geph, cyan) using expansion microscopy. Left panel: overview. Right panel: zoomed in region outlined in the left panel. The astrocytic volume distribution was analyzed around GABAergic synapses with 1 gephyrin cluster by quantifying the average EGFP fluorescence intensity in concentric three-dimensional shells with increasing diameter centered on the middle of the assumed synaptic cleft between the VGAT and gephyrin cluster (not all shells shown). EGFP fluorescence profiles were averaged and normalized per astrocyte (see main text and methods). (B) EGFP fluorescence increases as a function of distance and reaches near maximum levels at 0.6–1.0 µm from the synapse (filled circles: analysis centered on synapses, empty circles: random location). Repeated measures one-way ANOVA of synapse-centered analysis: distance p < 0.001, n = 9 average profiles (86 synapses in 9 astrocyte territories from 3 independent expansion microscopy experiments, 3 animals, filled circles). Analysis of 100 randomly placed volumes of interest from the same data set (empty circles, repeated measures one-way ANOVA p = 1.0). (C) Comparison of astrocytic EGFP distribution at GABAergic synapses (same data as in (B) with a re-analysis of the EGFP distribution at glutamatergic synapses from [13] (121 synapses in 6 astrocyte territories from 3 independent expansion microscopy experiments). Repeated measures two-way ANOVA: distance p < 0.001, interaction of distance and synapse type p = 0.0128.