Fig. 1: Electrical PF stimulation produces a localized calcium response in PC dendrites of awake mice.

a AAV vectors were injected into Crus I of the cerebellar cortex to express Cre-dependent GCaMP6f specifically in PCs. b, c Schematic illustrations of the experimental setup of PF stimulation and calcium recording. A microelectrode was used to apply electrical stimuli to a bundle of PFs in Crus I while calcium responses in PC dendrites were recorded using a two-photon microscope. The bottom (c) shows a representative field of view during cerebellar recording and the resulting calcium response in PCs to the PF stimulus. Calcium images were obtained with a 31 Hz frame rate, 3–4× digital zoom, and dimensions of 380-669 μm width and 306–539 μm height. Images were collected during periods without animal movement. d Two-photon images demonstrating calcium signals in PCs over time. Test pulses were applied at 0 ms. e Schematic of imaging approach with a linescan plane of focus (left, in blue), which forms a cross-section field of view (FOV) of a single representative PC (right). f–h Calcium signals of the example cell in (e) are shown as a spatiotemporal calcium map with the y-axis indicating the pixel locations along the linescan in the right (e). Calcium distribution of a single trial (f) and the average of 11 trials (g) during a 10 s recording period. f The triangles with solid black line denote global calcium responses, while the triangles with dashed lines denote local calcium responses. The orange triangle indicates the PF response. h The quantified fluorescence signals in different time windows, which are color-coded to correspond with lines and filled arrows shown in (d, g), respectively. Scale bars are 100 μm (c–d) and 50 μm (e).