RIM4 plays a pathway specific role in cerebellar Purkinje cells that is distinct from the function of the long RIM isoforms. (A) β-Galactosidase staining of brain sections from RIM4 knockout (KO) first mice, in which LacZ is expressed under the control of the endogenous RIM4 promoter. Cerebellum (CB), inferior olive (IO), cortex (CX) and hippocampus (HC). Scale bar, sagittal section = 1 mm, CB and IO = 50 mm. n = 5 mice per group. (B and C) Cartoons showing the major regions in which RIM4 was ablated in RIM4 KO_CRF (IO neurons) and RIM4 KO_NEX (excitatory neurons in the cortex and hippocampus). (D) Representative in situ hybridization (RNAScope) images showing the density of RIM4 mRNA (green) and Nissl counterstain (red) in the CB and IO of RIM4 wild-type (WT) and KO_CRF mice. Scale bar = 20 µm. Right: Quantification of RIM4 mRNA density in the CB and IO of wild-type and RIM4 KO_CRF mice. n = 4 mice per group, unpaired t-test. (E) Representative in situ hybridization (RNAScope) images showing the density of RIM4 mRNA (green) in the and Nissl counterstain (red) CB and CX of wild-type and RIM4 KO_NEX mice. Scale bar = 20 µm. Right: Quantification of RIM4 mRNA density in the CB and IO of RIM4 wild-type and KO_CRF mice. n = 4 mice per group, unpaired t-test. (F) Representative images of brain sections stained with fluorescent Nissl from RIM4 wild-type and KO_CRF mice (left). Bar graph showing the quantitative analysis of the size of the analysed brain regions, CX, CB and HC, normalized to the wild-type value. n = 3 mice per group, unpaired t-test. (G) Representative images of brain sections stained with fluorescent Nissl from RIM4 wild-type and KO_NEX mice (left). Bar graph showing the quantitative analysis of the size of the analysed brain regions, CX, CB and HC, normalized to the wild-type value. n = 3 mice per group, unpaired t-test. (H) Raster plots showing the onset and duration of spontaneously occurring episodes of motor impairment (solid black line) during a continuous 5-day observation period. RIM4 KO_constn = 10, KO_PCP2n = 6, KO_CRF, KO_NEX and RIM1/2 KO_PCP2n = 3. (I) Diagram showing the susceptibility of RIM4 wild-type and KO_const, KO_PCP2, KO_CRF, KO_NEX and RIM1/2 KO_PCP2 to the induction of an episode of motor impairment by intraperitoneal administration of saline (NaCl), ethanol (EtOH, 2 g/kg mouse), or caffeine (Caff, 25 mg/kg mouse). WT n = 3–10, KO n = 5–10 mice. (J) Bar graph showing the latency to fall off an accelerating rotarod (4 to 40 rpm in 300 s) for RIM4 wild-type and KO_const, KO_PCP2, KO_CRF, KO_NEX and RIM1/2 KO_PCP2 mice. RIM4 WT n = 6, KO_CRFn = 9, RIM4 WT n = 4, KO_NEXn = 3 and RIM1/2 WT n = 10 and KO_PCP2n = 10, unpaired t-test. (K) RIM1/2 conditional mice were crossed with the Purkinje cell-specific PCP2 Cre driver line. Juxtacellular recordings of Purkinje cells were performed in the presence of blockers for excitatory (20 μM CNQX) and inhibitory (10 μM gabazine) postsynaptic currents. Example traces of Purkinje cell firing in RIM1/2 WT (black) and KO_PCP2 (grey) mice. Right: Quantitative analysis of the mean spontaneous firing rate during baseline recordings. WT n = 3 mice, 56 cells, RIM1/2 KO_PCP2n = 3 mice, 55 cells. Bar graph, unpaired t-test. GCL = granule cell layer; ML = molecular layer; PCL = Purkinje cell layer.