Figure 2. Heroin increases DA levels in the NAc via enhanced VTA DA neuron activity.

(a) Fluorescence in response to five neuromodulators in HEK293 cells expressing dLight1 (DYN: dynorphin; GLU: glutamate; 5HT: serotonin; NE: norepinephrine). Data are presented as median with 25/75 percentile (box) and min-max (whiskers).( b), Schematic of the experiment for c-f; (c), Left, medial NAc shell of DAT-Cre⁺ mice were bilaterally injected with the DRD1-based DA sensor (dLight). Right, the amber light–drivable channelrhodopsin Chrimson was injected unilaterally in the VTA. (d), D-light-mediated fluorescence change following optogenetic activation of VTA DA neurons by Chrimson (mean of n = 3 animals). (e), Example trace from single animal, showing dLight-mediated fluorescence change in the NAc following intraperitoneal heroin (8 mg/kg) or saline injections. Tick mark indicates injection. (f), Average fluorescence after saline, heroin (8 mg/kg), citalopram (10 mg/kg), reboxetine (20 mg/kg) or cocaine (20 mg/kg) treatment compared to pre-infusion baseline (n = 4–7). Intraperitoneal injection of heroin or cocaine significantly increased fluorescence signals (as compared to control injections for heroin, paired t test, t₆ = 4.117, **p<0.01; for citalopram, reboxetine and cocaine, RM one-way ANOVA: F_(3,15) = 42.48, p<0.01; Bonferroni post hoc analysis: *p<0.05).( g), Schematic of the experiment for h-j; h, Left, VTA of DAT-Cre⁺ mice was bilaterally injected with the floxed version of the calcium indicator GCAMP6m. Right, coronal confocal images of infected VTA. (i), Average GCaMP6m fluorescence in VTA DA neurons following first intravenous infusion of heroin (100 μg/kg/inf) or saline. Red tick marks indicate injection onset. (j), Average fluorescence after heroin or saline treatment compared to pre-infusion baseline (n = 11). Calcium transients significantly increased after heroin infusions (dF/F for saline versus heroin, Lilliefors test for normality, Wilcoxon matched-pairs signed rank test, ***p<0.001). Error bars, SEM.