Figure 4. The Clstn3 KO decreases inhibitory synapse numbers in the cerebellar cortex as revealed by immunocytochemistry for pre- and post-synaptic markers.

(A) Representative confocal images of cerebellar cortex sections imaged for presynaptic vGAT and for tdTomato. Sections are from mice in which the cerebellar cortex was infected with control AAVs (Ctrl) or AAVs that induce the CRISPR-mediated Clstn3 KO (green, vGAT; red, AAV-encoded tdTomato signal; ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer). Calibration bar applies to all images. (B–D) Quantifications of vGAT-positive synaptic puncta demonstrating that the Clstn3 KO suppresses the number of inhibitory synapses in the cerebellar cortex (B and C, density and size of vGAT-positive puncta in the molecular layer (ML) of the cerebellar cortex (separated into deep (40–80%) and superficial areas (0–14%)); D, vGAT-staining intensity in the Purkinje cell layer (PCL) and granule cell layer (GCL) of the cerebellar cortex as a proxy of synapse density since individual vGAT-positive puncta cannot be resolved in these layers). (E) Representative confocal images of cerebellar cortex sections imaged for postsynaptic GABA-A_α1 receptor subunits and for tdTomato (green, GABA-A_α1 receptor subunit; red, AAV-encoded tdTomato signal; labeling is the same as in A). (F–H) Quantifications of GABA-A_α1 receptor-positive synaptic puncta independently confirmed that the Clstn3 KO suppresses the number of inhibitory synapses in the cerebellar cortex (F-H are the same as B-D, but for GABA-A_α1 receptor staining instead of vGAT staining). Data are means ± SEM (numbers of sections/mice analyzed are indicated in bar graphs). Statistical analyses were performed using two tailed unpaired t-tests, with *p < 0.05, **p < 0.01.