Fig. 3: Learning evoked by optogenetic Purkinje cell stimulation is temporally coupled to stimulation onset and not evoked blinks or SSpk modulation.

a,e,i, Schemes for Pkj-ChR2-US experiments in which stimulation onset timing, duration and intensity were varied systematically to dissociate candidate instructive signals (Extended Data Fig. 2). a, US onset shifts to obtain CS + US ISIs of 200 ms (yellow) or 400 ms (orange). b, CS + US trials before training, showing evoked blinks occurring at US offset in the two conditions (N = 4 mice for each ISI, ±s.e.m. in shadows). c, CS-only trials after training, showing the dependence of timing of learned eyelid closures on timing of US onset. d, Timing of peak eyelid closures occurring later for the longer ISI. Peak time: 200-ms versus 400-ms ISI, ^**P = 0.009, two-sample Student’s t-test (4 versus 4 mice). Shaded rectangles indicate laser US duration and dashed lines blink onset. Each dot is one mouse; the box plots indicate median (center bar) and 25th to 75th percentiles (bottom and top borders), with whiskers extending to data extremes. e, US duration adjusted so that CS + US onset times were identical, but US offset (and blink) timing varied with respect to the CS. f, US-evoked blinks on CS + US trials occurring at stimulus offset (note temporal correspondence with blinks in b, ±s.e.m.) (Extended Data Fig. 2b–e). g,h, Learned CRs (g), and timing (h), showing timing dependence not on stimulus offset or the evoked blink, but, rather, stimulation onset (peak time: 100-ms versus 300-ms duration, P = 0.87 NS, two-sample Student’s t-test, 4 versus 2 mice). i,j, Laser intensity adjusted (i) to evoke a blink (j) (associated with a decrease in SSpks; Extended Data Fig. 2f–h) either at laser offset (orange ±s.e.m., as above) or, with higher intensities, at laser onset (lime green ±s.e.m., N = 3 mice). Laser US timings and durations were identical in the two conditions. k,l, Learned CRs (k) showing timing dependent only on time of stimulation onset and not varying with the timing of the evoked blink (l) (peak time: high versus medium laser power, P = 0.67 NS, two-sample Student’s t-test, 4 versus 3 mice) or the direction of SSpk modulation (Extended Data Fig. 2f–h).