CSMD3-deficient mice manifest increased neuronal activity of cerebellar PCs in response to movement activity. A, Schematic for virally expressed GCaMP6 in cerebellar GABAergic neurons or PCs in the Crus I/II lobules and the in vivo fiber photometry Ca²⁺ signal recording in freely moving mice during rotarod test. B, Representative images of immunofluorescence staining with GCaMP6s (green) and calbindin (red)/DAPI (blue), illustrating GCaMP6s expression in cerebellar PCs at the placement of the tip of the fiber. Pie chart shows that 16.8% of the transfected cells by the AAV mix of VGAT1-Cre with DIO-GCamp6s are PCs. Scale bars: L7, left, 1 mm; L7, right, 300 μm; VGAT1, 100 μm. C–H, Ca²⁺ fluorescent signals recorded from cerebellar GABAergic neurons in the Crus I/II lobules. I–N, Ca²⁺ fluorescent signals recorded from cerebellar PCs in the Crus I/II lobules. C, F, I, L, Heat map depicting changes in the Ca²⁺ signals of cerebellar GABAergic neurons (C, F) and PCs (I, L) related to movement activity, respectively. C, I, from rest to run. F, L, from run to rest. Each row shows one trial for one mouse, and five traces are illustrated. Color scale at the right indicates ΔF/F. D, G, J, M, Perievent plot of the averaged Ca²⁺ transients recorded from cerebellar GABAergic neurons (D,G) and PCs (J,M) in the wild-type (wathet blue) and CSMD3^−/− (red) mice during rotarod test. D, J, From rest to run. G, M, From run to rest. Red (or wathet blue) thick lines indicate the mean, and shaded areas represent the SEM (n = 5 mice/group). Dashed line indicates the transition point of the event. E, H, K, N, Comparison of the normalized calcium responses under rest and run conditions across the group of mice recorded (n = 5 mice/group; F_(1,16) = 34.00, E; F_(1,16) = 166.80, H; F_(1,16) = 38.81, K; and F_(1,16) = 37.81, N; two-way ANOVA with Sidak's post hoc test. ΔF/F represents the deviation in Ca²⁺ fluorescence from the baseline, which is the averaged ΔF/F between t of −15 and 0 s. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.