Expression pattern and specificity of Ca8:eGFP in the Purkinje cells of valvula cerebelli. (a) Sagittal and coronal sections show the specificity of the eGFP expression in all Purkinje cells (green) of the adult zebrafish cerebellum. (b) All eGFP-expressing Purkinje cells (green) are ZebrinII, PV, and GABA positive (magenta). Arrowheads indicate double-labeled cells. (c) Regionalized quantification of the Purkinje cell soma size in the adult zebrafish cerebellum. CCe corpus cerebelli, CDF cumulative distribution frequencies, GABA γ-aminobutyric acid, LCa lobus caudalis cerebelli, GFP green fluorescent protein, PC Purkinje cell, PV parvalbumin, Va valvular cerebelli, Val lateral part of valvular cerebelli, Vam medial part of valvular cerebelli. Data are presented as violin plots showing the median with quartiles. ****P < 0.0001; ns, not significant. For detailed statistics, see Supplementary Table S1.

Variable firing, cellular, and spontaneous activity properties of the adult zebrafish valvular Purkinje cells. (a) Ex-vivo setup of an isolated intact brain from the Tg(Ca8:eGFP) line allows whole-cell patch-clamp recordings of valvular Purkinje cells. Arrow indicates a recorded cell. (b) The Purkinje cells show distinct firing patterns. Black trace shows the response at the rheobase. (c) Differential representation of the Purkinje cell firing classes in the adult zebrafish valvula cerebelli. (d) Valvula cerebelli Purkinje cells have different soma sizes. (e) Sample traces and quantification showing that part of firing class A and B Purkinje cells in valvula cerebelli fail to generate calcium-based spikes or repetitive calcium firing (oscillations). (f) Normalized mean values of the electrical properties detected for the valvular Purkinje cells are detailed in Supplementary Fig. S1. Normalizations were performed for each property to the highest obtained value. (g) PCA and hierarchical clustering plots depicting clusters of the valvular Purkinje cells (top) or valvular and corpus cerebelli Purkinje cells (bottom) based on physiological properties (as in (f)). Cells and data are colored by the assigned cell firing classification. (h,i) Sample traces showing the intense and variable spontaneous activity of the different Purkinje cell firing classes recorded in the adult zebrafish valvula cerebelli. Analysis of the presence, frequency and amplitude of simple and complex spikes between the Purkinje cells. CCe corpus cerebelli, CDF cumulative distribution frequencies, DIC differential interference contrast, Rheo rheobase, RMP resting membrane potential, Rinput input resistance, Val lateral part of valvular cerebelli. Data are presented as violin plots showing the median with quartiles and as box plots showing the median with 25/75 percentile (box and line) and minimum–maximum (whiskers). *P < 0.05; ***P < 0.001; ****P < 0.0001; ns not significant. For detailed statistics, see Supplementary Table S1.

Direct electrical stimulation of inferior olive generates large-amplitude events in a proportion of valvular Purkinje cells. (a) Ex-vivo setup of an isolated intact brain from the Tg(Ca8:eGFP) line allows simultaneous whole-cell patch-clamp recordings of Purkinje cells and electrical stimulation of the inferior olive nucleus. (b) Voltage-clamp recordings of Purkinje cells showing the differential responses of the Purkinje cells located in corpus and valvula cerebelli. (c) Corpus cerebelli Purkinje cells responds to the inferior olive electrical stimulation, generating larger amplitude responses than the ones detected in the valvula cerebelli Purkinje cells. (d) Sample voltage- and current-clamp recordings show the responses of the adult valvular Purkinje cells to inferior olive stimulation pulse (Black arrow). Gray traces from a Purkinje cell that do not respond to inferior olive stimulation. (e) Quantification of the proportion of the valvular Purkinje cells that respond to the inferior olive stimulation. (f) Analysis of the recorded amplitude and duration between the Purkinje cells that respond to the inferior olive stimulation. IO inferior olive, PC Purkinje cell. Data are presented as box plots showing the median with 25/75 percentile (box and line) and minimum–maximum (whiskers). ns not significant. For detailed statistics, see Supplementary Table S1.

Adult zebrafish valvular Purkinje cell do not discharge during fictive locomotion. (a) Ex-vivo setup of the brain-spinal cord allows simultaneous recordings of Purkinje cells and ipsilateral motor nerves. Sample recordings from each valvular Purkinje cell firing class during locomotion. (b) Analysis of membrane potential depolarization and the amplitude of the recorded EPSPs during locomotion between the different classes of Purkinje cells. Data are presented as box plots showing the median with 25/75 percentile (box and line) and minimum–maximum (whiskers). **P < 0.01; ****P < 0.0001; ns not significant. For detailed statistics, see Supplementary Table S1.