Supplementary Figure 10: Electrophysiological circuit identification of D1r-specific VS=>VP and DS=>SNr pathways, and in vivo assessment of optogenetic effects.

A. Outward postsynaptic currents were evoked in VP neurones by blue light pulses (10ms) delivered at various frequencies (1,5,10,20 Hz) as well as continuous light trains, to VP neurones targeted by Chr2-expressing terminals from D1r-neurones in VS. Whole-cell voltage-clamp recording was performed at the holding potential of -40mV. The GABA_A receptor antagonist bicuculine (Bic, 50μM) completely abolished the outward currents evoked by blue light, suggesting that optogenetic activation of VS D1r neurones terminals functionally act on (inhibit) VP neurones via GABAergic mechanisms. B. Representative traces showing outward GABAergic postsynaptic current evoked in SNr neurones by blue light (10ms) pulses delivered at various frequencies (1,5,10,20 Hz) to SNr neurones targeted by Chr2-expressing terminals from D1r-neurones in DS. Recordings performed as in A. C-D. Verification of the ability of optogenetic pulses to VS D1-neurones to inhibit DREADD-induced activity in VP in vivo. We proceeded to analyse Fos expression upon unilateral stimulation in VP (as shown in Figure 8 above) concomitantly to DREADD activation by intra-peritoneal infusions of designer drug CNO. C shows that counts of Fos+ cells through slices containing VP regions revealed a significantly greater number of Fos+ cells in the non-stimulated compared to the light-stimulated side (because activation of D1 GABAergic VS terminals should inhibit VP; N=5 mice, paired t-test t[4]=5.06, Bonferroni * p=0.014). D shows that this was nevertheless not the case for preoptic area (POA) in the same animals, as the number of Fos+ counts was similar on both stimulated and non-stimulated sides (t[4]=0.81, p=0.46). The results therefore suggest that POA is unlikely to have had any influence on the results associated with VP manipulation. n.s.= non-statistically significant.

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