Reduced amplitude and synchronicity of calcium spikes in Purkinje cells of RIM4 KO_const. (A) For in vivo two-photon calcium imaging, AAV particles encoding GCaMP6f were injected into the cerebellar vermis (lobules V and VI) and the cranial windows were positioned over the left lateral cerebellum (left). Fluorescence image showing selective expression of GCaMP6f (green) and Hoechst staining (blue) in Purkinje cells (right). Scale bar = 1 mm. (B) Representative two-photon non-descanned scans showing GCaMP6f expressing dendrites of Purkinje cells. Different coloured lines delineate the dendritic contours of individual Purkinje cells (PCs) and the corresponding example ΔF/F traces for RIM4 wild-type (WT, left) and KO_const (knockout, right). Line scan is indicated by dashed white line. Scale bars = 20 µm in scans, 10 s (horizontal) and 0.4 ΔF/F (vertical) in traces. (C) Quantitative analysis of the mean amplitude of spontaneous calcium signals in RIM4 wild-type and KO_const mice (WT 0.14 ± 0.02 ΔF/F, KO_const 0.06 ± 0.012 ΔF/F). n = 5 mice per group, unpaired t-test. (D) Histogram showing the data from (C) fitted with a Poisson distribution (dashed lines) to compare estimates of the numbers of inferior olive (IO) axon action potentials underlying the calcium signals on average. See the ‘Discussion’ section for details. Data were fitted with a modified Poisson function to match DF/F data: scale e^−lambdalambda^k / k! x­­–data were transformed onto natural numbers. We obtained lambda = 6.4 and scale = 61.4 for wild-type and lambda = 3.9 and scale = 87.4 for KO mice. (E) Bar graph showing the mean event rate of spontaneous calcium signals in RIM4 wild-type and KO_const mice (WT 0.19 ± 0.03 Hz, KO_const 0.17 ± 0.02 Hz). n = 5 mice per group, unpaired t-test. (F) Histogram showing the distribution of calcium event intervals in RIM4 wild-type and KO_const mice. Black dots indicate distinct peaks in the wild-type population. The KO_const data were fitted with an exponential distribution (grey dashed line, left). Representative ΔF/F traces of calcium spikes (marked in red). Scale bars = 5 s (horizontal), 0.1 ΔF/F (vertical). (G and H) Plots of the synchronicity of spontaneous calcium spikes in neighbouring Purkinje cells as a function of the mediolateral distance between cells for RIM4 wild-type and KO_const mice. The colour scale indicates the frequency of measured data-points (0 white to 1.0 dark blue). The mean synchronicity (black circles connected by a black line) was measured in each bin and the KO_const values were corrected according to the interdendritic interval (see the ‘Materials and methods’ section), two-way ANOVA test. Right: Raster plot of the temporal distribution of calcium events in RIM4 wild-type and KO_const. Black bars indicate individual calcium events in each dendrite and the temporal summation of events across neighbouring dendrites is colour-coded below (from 0 dark blue to 9 spikes bright yellow). Cross-correlation coefficient values are colour-coded and shown for each dendrite (0 white to 1 black). (I) For in vitro calcium imaging, Purkinje cells (PCs) were current-clamped with a recording electrode filled with the calcium indicator OGB-1 (200 μM) and imaged after climbing fibre (CF) stimulation. (J) Representative ΔF/F images showing CF-evoked postsynaptic calcium transients in Purkinje cell dendrites (J, ΔF at peak colour coded, from 0, black, to 1.5 × 10⁴, light green) superimposed on averaged baseline scans (F, white). (K) Representative traces of calcium transients after CF-stimulation (left) and of currents from somatic current-clamp recordings (cells brought to −70 mV to suppress spiking and improve comparability) showing complex spikes (right) from RIM4 wild-type (black) and KO_const (grey) mice. Scale bars = calcium transients 1 s (horizontal), 0.1 ΔF/F (vertical), currents 5 ms (horizontal), 20 mV (vertical). (L) Bar graph showing the mean amplitude of CF stimulation-induced calcium transients in proximal (Prox.) and distal (Dist.) dendrites and in the whole dendritic tree (Tree) of RIM4 wild-type and KO_const mice (4–5 weeks) (WT: proximal 0.13 ± 0.01 ΔF/F, distal 0.34 ± 0.04 ΔF/F, whole tree 0.25 ± 0.03 ΔF/F; KO_const: proximal 0.16 ± 0.02 ΔF/F, distal 0.43 ± 0.06 ΔF/F, whole tree 0.34 ± 0.04 ΔF/F). n mice/n cells, WT 3/5; KO_const 5/8, unpaired t-test. (M) Individual (grey) and mean (black) traces of calcium transients after multiple CF stimuli (stimulus indicated by red box) and corresponding colour-coded profile of ΔF/F values (0 dark blue to 0.6 yellow) for RIM4 wild-type and KO_const mice. Scale bars = 0.5 s (horizontal), 0.1 ΔF/F (vertical). (N) Quantitative analysis of the relative amplitude in response to the first, second and third stimulus in RIM4 wild-type and KO_const mice (4–5 weeks) (WT, 2nd 0.69 ± 0.02, 3rd 0.75 ± 0.02; KO_const, 2nd 0.54 ± 0.02, 3rd 0.56 ± 0.02). n mice/n cells, WT 3/6; KO_constn = 5/10, unpaired t-test. (O) Evoked electrical response in Purkinje cells were recorded in voltage clamp mode (−70 mV holding potential) after climbing fibre stimulation (inter stimulation interval 50 ms). Middle: Mean excitatory postsynaptic potential (EPSC) traces from PCs in response to the first and second CF stimulus. Right: Quantification of the mean EPSC amplitude and paired-pulse ratio. n mice/n cells, WT 8/5; KO_const 5/9, unpaired t-test. Scale bars = 20 ms (horizontal), 0.2 nA (vertical).