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Additional file 2: Figure S1. Cells expressing GFP by the mGAD65 (delE1) were co-immunolabeled for GABA. A mouse received an intravenous infusion of AAV-PHP.eB expressing GABA by the mGAD65 (delE1). Three weeks after the injection, sagittal brain sections were produced and double-immunolabeled for GFP and GABA. Note that all GFP-expressing cells expressed GABA, suggesting that the mGAD65 (delE1) serves as an inhibitory neuron-specific promoter. Scale bar; 50 μm. Figure S2. Significant labeling of chandelier-cell cartridges by the mGAD65 promoter over the mDlx enhancer. (A) A viral mixture (equal viral titer) comprising of AAV-PHP.B expressing GFP under the control of the mGAD65 promoter and that expressing tdTomato under the control of the mDlx enhancer was intravenously injected to adult mice. (B) A representative fluorescent image of the chandelier-cell cartridge double-labeled with GFP and tdTomato. Brain sections were immunostained 3 weeks after the viral injection for ankyrin-G (blue). (C) Graph showing percentage of GFP-labeled chandelier-cell cartridges co-labeled with tdTomato and vice versa. To determine the ratio, we identified GFP-labeled cartridges along with the AIS by the GFP channel and determined whether those were co-labeled with tdTomato by the RFP channel (magenta box) (39 GFP-labeled cartridges from 4 mice were examined, in which 14 cartridges were modestly co-labeled with tdTomato) (magenta box). The green box was obtained by the opposite procedure. Namely, tdTomato-labeled cartridges were identified by the RFP channel and it was determined if they were co-labeled with GFP by the GFP channel (22 tdTomato-labeled cartridges from 4 mice were examined, in which 16 cartridges were robustly co-labeled with tdTomato). See the Materials & Methods section for more details. Floating bars indicate upper, median, and lower quartiles (boxes) and value from each mouse (dots). p = 0.0041 by unpaired t-test. Scale bar; 5 μm. Figure S3. Transduction of Golgi cells by the mGAD65 promoter. (A) Diagram illustrating the experimental procedure. Mice received intravenous infusions of AAV-PHP.B expressing GFP under the control of the mGAD65 promoter. Three weeks after the viral injection, the mice were sacrificed, and the cerebellar sections were immunostained for mGluR2, a marker for Golgi cells. (B) A native GFP (green) fluorescent image of the cerebellar cortex immunolabeled for mGluR2 (magenta). (C) Magnification of the box in A. Arrowheads indicate GFP-expressing Golgi cells, which were proved by mGluR2 immunolabeling. Scale bars; 50 μm (B) and 20 μm (C). IHC; immunohistochemistry. Figure S4. Cerebellar interneuron-specific transduction by direct parenchymal injection of AAV-PHP.B carrying the mGAD65 promoter. (A) Schematic showing the experimental procedure. (B) Immunohistochemistry of the cerebellar slice from mice 3 weeks after the viral injection. Cerebellar sections were immunostained for calbindin (magenta), a marker for Purkinje cell. Scale bar; 50 μm. IHC; immunohistochemistry. Figure S5. Transduction of GABAergic neurons in various brain regions by the mGAD65 promoter. Adult VGAT-tdTomato mice received intravenous infusions of AAV-PHP.B expressing GFP under the control of the mGAD65 promoter. Three weeks after the viral injection, mice were sacrificed. Confocal microscopy showed overall co-labeling of tdTomato-expressing cells with GFP in various brain regions such as the striatum (caudate-putamen), the hippocampus (CA1), the anterior interposed nucleus (IntA), the superior vestibular nucleus (SuVe), and the retina. Scale bars; 50 μm (right) and 20 μm (left).

Additional file 1: Table S1. Antibodies used for immunohistochemistry.

Figure S1. ICaST system for optogenetic control and simultaneous calcium imaging in freely moving animals. The light paths are indicated by colored arrows. Details are described in the Materials and Methods and a previous report [27]. Figure S2. R-CaMP2 imaging in RME. (A) Representative images of a transgenic animal expressing both R-CaMP2 and EGFP in RME neurons in forward (top panels) and backward (bottom panels) movements. Transmitted-light images (TD), raw fluorescent images of R-CaMP2 and EGFP, fluorescent merged images, and pseudocolor ratio images (R-CaMP2/EGFP) are shown. (B) Fluorescent intensity ratio values (R=R-CaMP2/EGFP) of RME in a freely moving animal are plotted as a function of time. (C) Quantitative analysis of mean fluorescent ratio changes of RME during forward (gray) and backward (red) locomotion. The mean value of R during forward locomotion was normalized as 100%. Figure S3. tbh-1 mutants exhibit normal calcium responses in RME during backward locomotion. A representative calcium trace of RME in tbh-1(ok1193) mutants. Figure S4. tph-1 and cat-2 mutants exhibit normal calcium responses in RME during backward locomotion. (A) Biosynthetic pathways of serotonin and dopamine. Genes encoding synthetic enzymes are shown under the arrows. (B, C) Calcium dynamics of RME in tph-1 (mg280) (B) and cat-2 (jq6) (C) mutants during spontaneous locomotion. Table S1. C. elegans strains used in this study. Table S2. Transgenic lines generated in this study. Table S3. Mutations and primers for genotyping. Table S4. Primers for molecular biology. (PDF 359 kb)