Electrophysiological alterations induced by RIM4 deficiency are reversible, but the reduced complexity of the dendritic tree is not. (A) Cartoon of the viral vectors that were injected into the vermis (lobules IV–VI), RIM4 expression (rAAV2/1-CMV-mRFP-T2A-Flag-RIM4) and control (rAAV2/1-CMV-mRFP) (left). Representative fluorescence images of acute sections showing strong expression of mRFP in Purkinje cells (right). Scale bars = 500 μm and 50 μm. (B and D) AAV particles were injected into the cerebellar vermis at either P0 (B) or 12 weeks (Adult, D), scale bar = 30 μm. Experiments were performed 8 weeks after injection for the P0 group (B) and 4–6 weeks after injection for the adult treatment group (D). Control animals wild-type (WT) and RIM4 knockout (KO) were injected with virus expressing mRFP and treatment animals, P0 and Adult, were injected with virus expressing RIM4 and mRFP (treatment). Right: Representative maximum intensity projections of Purkinje cells labelled with Alexa 488 (green) for all experimental groups. (C and E) Bar graphs showing the quantitative analysis of the dendritic tree area for P0 (WT 2.2 ± 0.1 × 10⁴ μm², KO_const 1.2 ± 0.1 × 10⁴ μm², RIM4_P0 1.8 ± 0.2 × 10⁴ μm²) and adult (WT 2.0 ± 0.1 × 10⁴ μm², KO_const 1.2 ± 0.1 × 10⁴ μm², RIM4_P0 1.3 ± 0.1 × 10⁴ μm²) treatment groups. P0 treatment, n = 3 mice per group, n = cells, WT n = 10, KO_constn = 14, RIM4_P0n = 11; Adult treatment, n mice/n cells, WT 8/23 cells, KO_PCP2 10 mice/42, RIM4_Adult 6/37, unpaired t-test. (F) Representative traces from recordings of spontaneously firing Purkinje cells from wild-type, RIM4 KO_PCP2, P0 treatment and adult treatment. Right: Bar graph showing the mean firing rate for the four groups (WT 33.7 ± 2.4 Hz, KO 25.0 ± 2.4 Hz, RIM4_P0 35.0 ± 3.5 Hz, RIM4_Adult 37.9 ± 5.3 Hz). n mice/n cells, WT n = 3/10, KO_PCP2n= 4/13, RIM4_Adultn = 4/5, RIM4_Adultn = 6/28, unpaired t-test. Scale bars = 0.1 s (horizontal ) and 0.5 mV (vertical). (G) Time course of PC firing rate of Purkinje cells from the four experimental groups before and during caffeine application (1 mM, 10 min) (left). Bar graph showing the mean firing rate during caffeine application relative to baseline (right) (WT 91.5 ± 1.5%, KO 59.4 ± 12.0%, RIM4_P0 102.7 ± 11.5%, RIM4_Adult 89.2 ± 2.9%). n mice/n cells, WT 3/9, KO_PCP2 4/13, RIM4_Adult 3/7, RIM4_Adult 6/16, unpaired t-test. Legend of F also applies to G.