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Fig. S2
(A) Overview of the cerebellum of a Tg(elavl3:Hsa.H2B-GCaMP6s) embryo expressing SwiChR-YFP. Box indicates the nuclei whose calcium traces are given in (B). Anatomical features are indicated. Scale bar: 25 μm. (B) Example calcium F/F0 traces of SwiChR-expressing Tg(elavl3:Hsa.H2B-GCaMP6s) embryos. Note that the bottom trace was obtained from a cell that expressed only H2B-GCaMP6s and serves as negative control. Fourier transformed traces of the same examples are given on the right. None of the traces show a peak except for frequencies in the range of the endogenous oscillations. The blue light illumination of SwiChR would correspond to a 1.67 mHz frequency (5 nuclei expressed both markers in 2 embryos). (C) Overview of the cerebellum of a Tg(atoh1a:KalTA) embryo co-expressing NLS-GCaMP6s/H2B-tagRFP and ChR2-YFP. Box indicates the nucleus whose calcium traces are given in (D) and (E) as nucleus 1. Anatomical features are indicated. Scale bar: 25 μm. (D) Examples of calcium F/F0 traces of ChR2 or SwiChR-positive THNs co-expressing NLS-GCaMP6s. Only the nuclei with ChR2 show strong regular peaks (arrows; 9 nuclei out of 36 measured including controls from 9 embryos). In SwiChR, occasionally some nuclei respond to endogenous signals (top). These events are too rare to be noted in the Fourier transformation (see (E); 13 nuclei from 13 embryos expressed both markers). Note that the bottom trace in SwiChR is derived from a cell that expressed only NLS-GCaMP6s, but no SwiChR, and serves as negative control. (E) Fourier transformed traces of the examples given in (D). Nucleus 1 of the ChR2 traces shows a clear peak at the expected frequency of 16.7 mHz. SwiChR-derived traces do not show such a peak except for frequencies in the range of the endogenous oscillations. The blue light illumination of SwiChR would correspond to a 1.67 mHz frequency. (F) Example traces from Tg(elavl3:Hsa.H2B-GCaMP6s) embryos treated with hexamethonium demonstrate that calcium signal amplitudes are decreased (left). FFTs of these traces are given on the right. (G) Example traces from Tg(elavl3:Hsa.H2B-GCaMP6s) embryos treated with glycine demonstrate that calcium signal amplitudes are not affected by the hyperpolarizing agent (left). Note that these examples are chosen from the minority of nuclei that still exhibited calcium transients. FFTs of these traces are given on the right. ChR2, channelrhodopsin; F/F0, fluorescence over background; FFT, fast Fourier transform; GCaMP6, circular permutated green florescent protein-Calmodulin-M13 peptide 6; H2B, Histone 2B; NLS, nuclear localization sequence; SwiChR, mutated channelrhodopsin; YFP, yellow fluorescent protein.