(A) (left) Visualization of fiber photometry implant placed above FSIs expressing ChrimsonR (red) and MSNs expressing jGCaMP7s (green; ac, anterior commissure; scale bar, 100 μm). (right) Magnified image of FSI-MSN juxtaposition (scale bar, 10 μm). (B) Setup for simultaneous red-shifted optogenetic manipulation (pulsed for ChrimsonR, constant for eNpHR3.0) and fiber photometry monitoring in vivo. (C) (left) Viral co-injection of Cre-dependent ChrimsonR to excite FSIs with Cre-inactivated jGCaMP7s to monitor MSNs. Insets show photometry signal on a single representative trial. (right) MSN signal decreases from baseline during FSI excitation (green, n=20 trials/mouse), compared to no stimulation trials (black, n=20 trials/mouse) (n=5 male mice). (D) (left) Viral co-injection of Cre-dependent NpHR3.0 to inhibit FSIs with CaMKII-promoted GCaMP6f to monitor MSNs. Insets show photometry signal on a single representative trial. (right) MSN signal increases from baseline during FSI inhibition (green, n=20 trials/mouse), compared to no stimulation trials (black, n=20 trials/mouse) (n=3 male mice). (E) Significant difference in MSN signal between FSI excitation and inhibition. *p<0.05, main effect of group. (F) Viral co-injection of Cre-dependent ChIEF, with or without Cre-dependent hM4Di. Inset shows an optogenetically-evoked inhibitory postsynaptic current (oIPSC, black) blocked by picrotoxin (100 μM, gray). (G) oIPSCs in slices co-expressing hM4Di were reduced by bath application of clozapine-N-oxide (CNO, 10μM, red bar) (ChIEF only, n=4 male cells; ChIEF+hM4Di, n=7 cells; 3 female, 4 male). Inset shows an example trace before (black) and after (red) CNO, from a slice expressing ChIEF+hM4Di. (H) oIPSCs were significantly reduced by chemogenetic inhibition (red) or picrotoxin (PTX, gray; n=3 male cells). *p<0.05, main effect of group.