Extended Data Fig. 8: Plcb4+ Purkinje neuron activity is required for associative learning.

a, AAV-Plcb4-tdTomato labels Plcb4+ Purkinje neurons transduced by Purkinje neuron-specific GCaMP (AAV-L7-cre + rAAV-CAG-FLEx-jGCaMP7f). Scale bar, 50 μm. Similar results were found in 2 animals for representative data. b, AAV-Plcb4-ChR2-mCherry representative histology. Scale bars, 100 μm for the top panel and 50 μm for the lower panels. Similar results were found in 2 animals for representative data. c, Quantification analysis of mCherry intensities in Plcb4+ and Aldoc+ Purkinje neurons as in b. Data are presented as standard box and whisker plots. ***p = 1.2e-9, by two-tailed unpaired t-test. n = 301 Plcb4+ and 113 Aldoc+ Purkinje neurons derived from 3 mice. d, (Left panel) Schematic representation of the whole cerebellum, anterior cerebellum and light-spatially-targeted ADCV. (Right panel) Comparison of the ratio of Plcb4+ to all Purkinje neurons extracted in whole cerebellum (0.49 ± 0.005, n = 2 biologically independent experiments), ADCV (0.65 ± 0.02, n = 2 biologically independent experiments) (as shown in Extended Data figure 2d), excited by spatially directed optogenetic light (PcP2-Ai27D targeted light, 0.89 ± 0.04, 12 slices derive from 4 mice) and Plcb4- specific optogenetic experiments (Plcb4-ChR2-mCherry, 0.95 ± 0.01, cell counts from 3 mice). Data are presented as mean ± s.e.m. e, Fraction of trials with a CR during sessions with light-on or light-off optostimulation for animals transduced by Plcb4-ChR2 with L7-cre (n = 7 mice) or L7-Cre alone (controls, n = 4 mice). As evidenced by CR performance during the first five training sessions, daily optostimulation of Plcb4+ Purkinje neurons in lobules IV/V impairs associative learning in mice. During optostimulation light off sessions, Plcb4-ChR2 mice show gradual increase in CR performance similar to L7-control mice. Data are presented as mean ± s.e.m. p = 0.03, 0.005, 0.01 for days 4–6, by two-tailed unpaired t-test. f, Average UR on session 1 (upper left panel). Unlike Plcb4-ChR2 mice, control mice learned to make well-timed CRs by session 5 despite equivalent exposure to light-on optostimulation (lower left panel). Both groups exhibit well-timed CRs with light-off optostimulation by session 10 (lower right panel). g, The correlation of ChR2 expression with CR success rate at training section 5 and 6.