Supplementary Figure 2: Optogenetic activation of Purkinje cells in the cerebellar flocculus

(a) Optrode recordings in awake mice verified that illumination with blue light successfully elicited spiking in Purkinje cells of the flocculus, the region of the cerebellum necessary for VOR learning. In vivo, extracellular optrode recording from an awake animal showing Purkinje cell activation with trains of blue light (trains of 5 ms pulses at 50 Hz for 420 ms every 1 second; ≤ 3 mW/mm² intensity). Rasters show the spikes elicited by all the stimulus trains delivered during six 5-min blocks of stimulation (40 seconds between blocks); histogram shows the effect of stimulation over the entire 30-min period (below). Bin size 5 ms. (b) Expanded view of red box in a, and example spontaneous (black) and ChR2-elicted (cyan) Purkinje cell simple spike waveforms (black dot), which differ from the spontaneous complex spike waveform (orange dot) recorded from the same cell. ChR2-mediated depolarization of Purkinje cells elicited action potentials resembling spontaneous simple spikes, rather than the complex spikes elicited by climbing fiber input, in that they occurred at high rates and did not have the Ca²⁺ spikelets characteristic of spontaneous or optogenetically elicited complex spikes. Bin size 5 ms. (c) Histograms of the simple spike inter-spike intervals (ISIs) of two, representative Purkinje cells immediately before the start of stimulation (open bars), and during optogenetic stimulation (cyan bars; trains of 5 ms pulses at 50 Hz for 420 ms every 1 second; ≤ 3 mW/mm² intensity). Optogenetic stimulation increased the mean firing rate of the Purkinje cells, as shown by the leftward shift in the ISI distribution, with no evidence of bursting. In some cells, optogenetic stimulation induced some entrainment of firing at the frequency of stimulation (50 Hz/ 20 ms ISI) (right). Data in the left panel are from the same Purkinje cell as in panel a. Bin size 500 μs. (d) Blue light stimulation elevated Purkinje cell firing rates (Light ON; 100.1 ± 7.8 Hz) above the spontaneous levels recorded in between the stimulus trains (Light OFF; 45.9 ± 2.6 Hz) (t₍₈₎ = 7.322, P < 0.0001, Paired t-test, n = 9 cells from 8 mice; circles, transgenic; diamond, virus injected). The increase in Purkinje cell firing rate achieved with optogenetic stimulation was comparable to what has been reported during visually and vestibularly driven eye movements. Mean ± s.e.m.