RIM4 plays a pathway-specific role in cerebellar Purkinje cells and appears dispensable in hippocampus and cerebral cortex. (A) Representative images of sagittal brain sections stained with fluorescent Nissl green from RIM4 wild-type (WT) (left) and KO_const (right) mice. White lines delineate the measured areas, cortex (CX), cerebellum (CB) and hippocampus (HC). Scale bars = 1 mm. (B) Bar graph showing the quantitative analysis of the area of the analysed brain regions, CX (WT 12.46 ± 0.12 mm², KO_const 11.99 ± 0.39 mm²), CB (WT 7.78 ± 0.36 mm², KO_const 6.47 ± 0.13 mm²) and HC (WT 1.43 ± 0.05 mm², KO_const 1.43 ± 0.05 mm²), normalized to the WT value. n = 4 mice per group (12–15 weeks), unpaired t-test. (C) Example images of calbindin (magenta) and Hoechst (blue) stained cerebellar sections from RIM4 wild-type and KO_const mice. White lines indicate measured subcerebellar areas, molecular layer (ML), granule cell layer (GCL), white matter (WM). Scale bars = 1 mm. (D) Bar graph showing the quantitative analysis of the area of the analysed subcerebellar regions, molecular layer (ML) (WT 4.04 ± 0.25 mm², KO_const 2.99 ± 0.15 mm²), granule cell layer (GCL) (WT 3.22 ± 0.22 mm², KO_const 2.59 ± 0.1 mm²), white matter (WM) (WT 0.95 ± 0.13 mm², KO_const 0.88 ± 0.07 mm²), normalized to the wild-type value. n = 4 mice per group (12–15 weeks), unpaired t-test. (E–H) ML thickness (WT 173.05 ± 4.36 μm, KO_const 151.08 ± 2.83 μm), total Purkinje cell layer (PCL) length, and Purkinje cell density (WT 4.18 ± 0.13 / 100 μm, KO_const 4.61 ± 0.14 / 100 μm) were measured in calbindin-stained cerebellar tissue from RIM4 wild-type and KO_const mice. The estimated number of Purkinje cells was calculated by multiplying the total PCL length by the Purkinje cell density. n = 4 mice per group (12–15 weeks), unpaired t-test. (I) Representative confocal image of Purkinje cells labelled with the red fluorescent dye TMR-Dextran (1 mM) in RIM4 wild-type and KO_const mice. Maximum intensity projections (top) and 3D reconstructed dendritic trees (right) with branch level annotations (fourth = blue; fifth = yellow; sixth = red). Scale bars = 50 µm. (J) Bar graph showing the quantification of the dendritic tree area in RIM4 wild-type and KO_const mice (WT 1.92 ± 0.12 × 10⁻² mm², KO_const 1.38 ± 0.08 × 10⁻² mm²). n mice/n cells: WT 11/22, KO_const 10/30 (>100 days), unpaired t-test. (K) Graph showing Sholl intersections measured from 3D reconstructed Purkinje cells plotted against the radial distance from the cell body in RIM4 wild-type and KO_const mice. WT n = 11, KO_constn = 10 mice (>100 days), Kolmogorov-Smirnov test. (L) Bar graph showing quantification of dendritic length for each branching level (branch level 4: WT 1.6 ± 0.1 mm, KO_const 1.1 ± 0.04 mm; branch level 5: WT 1.4 ± 0.1 mm, KO_const 0.7 ± 0.04 mm; branch level 6: WT 0.9 ± 0.1 mm, KO_const 0.4 ± 0.04 mm). WT n = 11 KO_constn = 10 mice (>100 days), unpaired t-test and with Bonferroni's correction for multiple comparisons. (M) Cartoon of extracellular recordings of spontaneous spikes in Purkinje cells. The recording electrode was placed around the axon hillock of Purkinje cells. CNQX (20 µM) and 10 µM gabazine were bath-applied during recording. Right: Representative traces of spontaneous spikes in RIM4 wild-type and KO_const mice (left). Scale bars = 0.1 s (horizontal) and 0.1 mV (vertical). (N) Quantification of Purkinje cell mean firing in RIM4 wild-type and KO_const mice (WT 53.3 ± 0.04 Hz, KO_const 16.1 ± 0.85 Hz). n mice/n cells: WT 5/48; KO_const 6/56 (mice >100 days), unpaired t-test. (O) Representative traces of spikes before and after 1 mM caffeine application (left) in RIM4 wild-type and KO_const mice (left). Time course graph showing Purkinje cell firing rate before, during and after caffeine bath application. n mice/n cells: WT 4/20; KO_const 3/17 (mice >100 days), one-way ANOVA test. KO = knockout.