Constitutive RIM4 KO mice exhibit ataxia and inducible severe and long-lasting dystonia. (A) Scheme showing (from top to bottom) the structure of the mouse Rims4 gene (exons shown as black boxes), an expanded map around exon 2, the targeting vector (red triangles = loxP sites, grey ovals = FRT recombination sites, light blue box = splice acceptor site, dark blue box = LacZ cassette, N = neomycin resistance cassette, DT = diphtheria toxin cassette), KO (knockout) first allele (LacZ cassette is spliced in after exon 1 resulting in LacZ expression and RIM4 KO), RIM4^fl/fl allele (neomycin resistance cassette was removed by flp recombination), KO allele (Cre recombination deleted exon 2). (B) Representative immunoblot and quantitative analysis (C) of lysates from hippocampus (HC), cortex (CX) and cerebellum (CB) of RIM4 wild-type (WT) (+/+), heterozygous (+/−) and KO_const (−/−) mice. OE = HEK293 T cell lysate expressing RIM4. β-Actin was used as loading control. n = 10 mice per group, two-way ANOVA test with Bonferroni post hoc correction. (D) Graph showing body weight development of RIM4 wild-type (+/+, black), heterozygous (+/−, grey) and KO_const (−/−, white) mice over 35 days. n = 5 mice per group, two-way ANOVA test with Bonferroni post hoc correction. (E) Representative images of different phases of motor impairment in RIM4 KO_const mice: phases I, II and III. (F) Duration of the three phases of motor impairment in individual mice during the episode. The grey colour indicates the phase. Note the broken x-axis. (G) Raster plots showing the onset and duration of spontaneously occurring episodes of motor impairment (solid black line) during a 5-day observation period. WT n = 6, KO_constn = 10 mice (13–17 weeks). (H) Diagram showing the susceptibility of RIM4 WT and KO_const mice to the induction of an episode of motor impairment by the intraperitoneal administration of saline (NaCl), ethanol (EtOH, 2 g/kg mouse) or caffeine (Caff, 25 mg/kg mouse). WT n = 8, KO_constn = 10 mice. (I) Example image of paw prints (left) and footfall/gait pattern (right) of adult RIM4 WT and KO_const mice on the CatWalk. (J) Quantification of the regularity index of the footfall/gait pattern (number of normal step sequences × 4 / number of paw placements), n = 3 per group, unpaired t-test. (K) Bar graph showing the latency to fall off an accelerating rotarod (4 to 40 rpm in 300 s) for RIM4 WT and KO_const mice, n = 10 per group, unpaired t-test. (L) RIM4 WT and KO_const mice were placed in a cage with a running wheel and monitored for four consecutive days: regular wheel (RW) consisting of all 38 rungs for 2 days and complex wheel (CX) consisting of 22 rungs (16 rungs were selected and removed) for 2 days. Black boxes indicate time spent running in the wheel, black lines represent episodes of motor impairment. WT n = 7, KO_constn = 6 mice. (M) Graph of the average instantaneous running speed of the mice shown in L. One-way ANOVA tests with Sidak's post hoc correction.