Fig. 3: Developing Purkinje neuron calcium imbalance induces a persistent morphological change in cerebellar afferent synapses and a deficit in inter-limb adaptation task in adult.
A Schematic to determine whether dysregulation of developing Purkinje neuron calcium balance leads to long-lasting changes in cerebellar circuit function. B Experimental timeline for developing Purkinje neuron calcium dysregulation. CNO was administered from P9 to P21 to mice expressing hM3D-CTS or RFP in Purkinje neurons, and behavioral testing was conducted 30 days later at P50; an independent cohort was established for confocal analysis of climbing fiber synapses to control for cerebellar plasticity effects induced by motor training. C Representative confocal images (left) of Purkinje neurons expressing hM3D-CTS (co-expressed RFP reporter, red) co-stained with VGluT2 (magenta), and a three-dimensional reconstruction (right) of virally transduced dendrites (Red), neighboring presynaptic terminals (Yellow) and non-adjacent terminals (Blue), from mice that were CNO-treated (top) or vehicle-treated (bottom). Scale bar: 10 μm. D Quantification of VGluT2 staining puncta size and intensity was obtained from n = 4 – 5 mice of each treatment group (both sexes included), showing a significant enlargement in climbing fiber afferent synapses in CNO-treated hM3D-CTS mice during the early postnatal period compared to controls. E CNO-treated hM3D-CTS mice showed impaired adaptation to the long-stride pattern. Individual data points of the last (Day4) training session were graphed and trend across days displayed in an inset. Data are presented as the mean ± SEM.; p(RFP vs. Pcp2::hM3D-CTSveh) = 0.3035, **p(RFP vs. Pcp2::hM3D-CTScno) = 0.0027; Ordinary ANOVA with post-hoc Dunnett’s test.
