Supplementary Figure 9: Analysis of the effects in vivo of optogenetically stimulating the striatal outputs Substantia Nigra, pars reticulata (SNr) and ventral pallidum (VP).
From: Separate circuitries encode the hedonic and nutritional values of sugar
Ai32 mice, which express a channelrhodopsin-2/EYFP fusion protein following exposure to Cre recombinase, were used for ChR2 expression in VP and SNr. The construct AAV-CMV-Cre was injected bilaterally. A. EYFP expression SNr as shown on a coronal brain slice. The image shows that ChR2 expression was contained to SNr boundaries. B. To demonstrate that light pulses increased neuronal activity in vivo in SNr, mice were unilaterally stimulated with light (represented by the fibre tip in blue) whereas the other hemisphere was not stimulated. Animals were in their home cages during this protocol and stimulation side was chosen arbitrarily across animals. Mice were perfused 90 minutes after stimulation period for Fos immunohistochemical analyses. Note Fos-positive cells under blue fibre tip. C. Counts of Fos-positive (Fos+) cells through slices containing SNr regions revealed a significantly greater number of Fos+ cells in the stimulated compared to non-stimulated side (N=5 mice, paired t-test t[4]=6.2, * p=0.003). D-G. Same protocol as above, but for VP. As we show in D, unfortunately, the injections encompassed the VP and neighbouring pre-optic area (POA) in some animals. E. We then proceeded to analyse Fos expression upon unilateral stimulation in both POA and VP. Because fibre tips were localized just above the VP, unilateral light stimulation resulted in ipsilateral increases in Fos expression in VP but not POA, although some neurons expressed ChR2 in POA. F. shows that counts of Fos+ cells through slices containing VP regions revealed a significantly greater number of Fos+ cells in the stimulated compared to non-stimulated side (N=5 mice, paired t-test t[4]=6.1, Bonferroni * p=0.008). G shows that this was not the case for POA in the same animals, as the number of Fos+ counts was similar on both stimulated and non-stimulated sides (t[4]=1.19, p=0.29). n.s.= non-statistically significant. acp = posterior limb of the anterior commissure.
