Fig. 1. Acute heroin exposure causes long-lasting activation of both VTA dopamine neurons and DRN serotonin neurons in a dose-dependent manner.

a Schematic of the experimental setup for fiber photometry of neuronal GCaMP fluorescence change (Ca²⁺ signals) from freely behaving mice that received intravenous drug infusions. b, c Ca²⁺ signals of dopamine neurons (b) and serotonin neurons (c) from two mice individually challenged with 20 intravenous infusions of heroin (small triangles) at the indicated doses. Small triangles indicate 20 repetitive infusions. The vertical dash line indicates the start of the first infusion. Gray, the original trace showing Ca²⁺ signals; black, the overall trend of Ca²⁺ signals. Moreover, red and blue indicate significant increase and decrease from the baseline before drug infusion, respectively (p < 0.05; permutation tests). d Overall Ca²⁺ signal intensities during the infusion phase [n = 6 dopamine mice; F(3, 20) = 24.47, p < 0.0001, one-way ANOVA with Tukey’s post-hoc test; n = 7 serotonin mice; F(3, 24) = 8.381, p = 0.0005, one-way ANOVA with Tukey’s post-hoc test]. e Initiation time required for heroin to activate dopamine neurons and serotonin neurons [n = 6 dopamine mice; F(2, 15) = 1.686, p = 0.2186, one-way ANOVA with Tukey’s post-hoc test]. f Rise time and decay time of the heroin-evoked Ca²⁺ signals [n = 6 dopamine mice, n = 7 serotonin mice; for rise time, F(2, 15) = 0.2508, p = 0.7814, one-way ANOVA with Tukey’s post-hoc test; for decay time, F(2, 15) = 1.180, p = 0.3342, one-way ANOVA with Tukey’s post-hoc test]. Error bars indicate SEM (d–f). *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant, DM dichroic mirror, PMT photomultiplier tube, Amp amplifier, DAQ data acquisition interface