(A) Left: photometry setup. Light path for fluorescence excitation and emission is through a single 400µm fiberoptic implanted in VTA. Right: viral targeting of GCaMP5 to VTA-DA neurons. (B) Photometry traces from mice expressing eYFP (bottom) and GCaMP5.0 (top) in VTA during the sucrose lickometer test, showing robust increases in GCaMP fluorescence correlated with sucrose licking epochs (red dashes). dF/F represents change in fluorescence from median of the entire time-series. (C) Top: example trace of VTA-DA activity in social behavior. Red dashes: interaction bouts. Bottom: zoom-in of dashed interval relating VTA-DA GCaMP signal and social interaction (colored boxes). (D) Example heatmaps (top) and peri-event plots (bottom) aligned to start of interaction for mice expressing GCaMP (left) or eYFP (right). Heatmaps: warmer colors indicate higher fluorescence signal; peri-event plots: warmer colors represent earlier interaction bouts. (E) VTA-DA activity in novel object investigation. Red dashes: interaction bouts. (F) Average peak fluorescence over first ten interaction bouts (16.4% dF/F: social; 13.7% dF/F: novel object; n=10, Wilcoxon signed-rank test, p=0.5). (G) Signal changes across bouts: social (blue) and novel object (green). (H) Signal changes within bouts; novel object peak responses occur closer to interaction-bout end than do social peak responses (n=10 individual animals plotted, gray lines; difference of peaks over 0.5 s from bout-end and bout-start: −1.7% dF/F social vs. 9.7% dF/F novel object; Wilcoxon signed-rank test: p=0.0051). (I) Specific behaviors during 1 sec behavioral video clips centered around peak fluorescence within each bout. Peak during novel object investigation occurs predominantly in withdrawing from object (92%), while peak fluorescence during social interaction occurs in approach (14%) or active investigation (81%) (n=10 animals, 15 bouts/animal). (J) Directed-graph model of causal mediation analysis (Methods); while time-elapsed partially mediates effects of VTA-DA neuron activity on latency to social interaction only, the majority of effect was direct rather than mediated (74.0% average direct effect, 26.0% average indirect effect mediated by time elapsed; both p<0.0001). See also Figure S1.