Supplementary Figure 6: Optogenetic activation of climbing fibers in the cerebellar flocculus
From: Cerebellar Purkinje cell activity drives motor learning
The dependence of learning on which cells expressed the ChR2, the Purkinje cells vs. climbing fibers (compare Figures 1 and 2) rules out any non-specific effects of the experimental procedures, such as the presence of the blue light or general activation of the circuit. (a) Coronal diagram of the cerebellum and brainstem illustrating the site of virus injection and subsequent optogenetic activation of climbing fibers (green) with 473nm light (cyan). ChR2 was expressed in climbing fibers by injecting AAV-CaMKIIα-hChR2(H134R)-EYFP into the dorsal cap of Kooy, the subnucleus of the inferior olive that provides the climbing fiber input to the flocculus. Right, coronal sections showing ChR2-EYFP expression, visualized using immunostaining against EYFP (green), in the climbing fibers of the flocculus (top) and the corresponding injection site in the dorsal cap of Kooy (bottom). Fl, flocculus; CF, climbing fiber; IO, inferior olive; ML, molecular layer; GCL, granule cell layer; Blue, cell bodies stained with DAPI. (b) Representative example of an in vivo extracellular optrode recording from a Purkinje cell in the flocculus showing the complex spikes elicited by optogenetic activation of its climbing fiber input (left). Climbing fiber activation was elicited using 250 ms trains of three pulses of blue light (cyan; 2 ms duration, 0.3 mW/mm2) repeated at 1 s intervals, and delivered to the cerebellar flocculus. Individual waveforms show the optogenetically elicited complex spikes with the stimulus artifact subtracted. Right, overlay of all the optogenetically elicited complex spike waveforms recorded in this cell during a ∼10 minute period of stimulation (1,718 waveforms). Climbing fiber activity has been hypothesized to recruit plasticity in the cerebellar cortex or induce plasticity downstream by causing pauses in Purkinje cell simple spike firing that disinhibit their targets. (c) Histogram from a representative Purkinje cell, showing simple spike rate aligned on the time of complex spikes (t=0) elicited optogenetically using 2ms pulses of light. A typical 10–20 ms pause in simple spike firing occurred after each optogenetically elicited complex spike. Bin size, 2 ms.
