Figure 3. Plcb4+ Purkinje neuron activity is required for associative motor learning.

(a) Schematic of viral delivery for imaging. (b) Representative window with epifluorescent (left panel) and two-photon images (middle and right panels) showing Plcb4-specific tdTomato and Purkinje neuron-specific GCaMP expression. Scale bar, 0.5 mm for left panel and 50 μm for other panels. (c) Session correlations Aldoc+ or Plcb4+ Purkinje neurons. (d) Heat map and average traces showing trial-averaged GCaMP responses of Plcb4+ and Aldoc+ populations. Mean behavior in response to an isolated CS (CR) or paired CS and US (CR+UR). Data for 38 Plcb4+ cells and n=24 Aldoc+ were obtained from 4 mice. (e) Mean amplitude of responses of Plcb4+ and Aldoc+ neurons from d across sessions 100-200 ms after conditioned stimulus (CS) onset. p=0.049, 8.2e-4 for day3 and day5. (f) Mean amplitude of responses of Plcb4+ and Aldoc+ neurons across sessions 0-100 ms after unconditioned stimulus (US) onset. p=0.01, 0.02, 0.03 for day1, 3, 5. (g) Mid-sagittal cerebellar sections from Ai27-PC mice stained with anti-Aldoc or anti-Plcb4. Purkinje neurons expressing ChR2 were labeled with tdTomato. Scale bars, 0.5 mm for the whole cerebellum image. (h) Measurement of optostimulated areas and number of Aldoc+ and Plcb4+ Purkinje neurons within the area. Data from 12 sections in 4 mice. p=2.0e-6 (right panel). (i) Fraction of trials with a CR during sessions with light-on or light-off optostimulation. Data from 4 animals in each cohort. p=0.043, 0.005, 0.006, 0.019, 0.022 for days4-8. (j) Average UR on session 1 (upper left panel), CR by session 5 (lower left panel) and CR with light-off optostimulation by session 10 (lower right panel). Average traces are presented as mean ± s.e.m. All pairwise comparisons were made by two-tailed t-test. n.s. indicates not significant, *p < 0.05, **p < 0.01, ***p < 0.001.