Fig. 4: NOPLight detects chemogenetically evoked endogenous N/OFQ release in vivo.

a Schematic of fiber photometry setup. Coronal brain cartoon of fiber implant and viral co-injection of FLEX-NOPLight with either DIO-hM3D(Gq) or mCherry in the VTA of PNOC-Cre mice. b Left: Representative traces of FLEX-NOPLight fluorescence after systemic (i.p.) injection of 5 mg/kg clozapine-N-oxide (CNO) in hM3D(Gq) (light green) or mCherry control (dark green) animals. Right: Mean FLEX-NOPLight fluorescence 40–45 min after CNO injection is significantly elevated relative to the pre-injection baseline period (BL) in hM3D(Gq) (two-tailed Wilcoxon test, *p = 0.0391, n = 8 mice) but not control animals (two-tailed Wilcoxon test, p > 0.9999, ns not significant, n = 3 mice). Z-scores for each individual animal averaged at baseline, and 50–55 min are shown (gray lines). Data represented as mean ± SEM. c Left: FLEX-NOPLight fluorescence (green) averaged before injection of 5 mg/kg CNO (0–10 min), and in 5 min bins following injection. 10 mg/kg of selective NOPR antagonist LY2940094 (LY) was administered (o.g.) to the LY + CNO (purple) group 30 min prior to photometry recording (two-way repeated-measures ANOVA with Bonferroni’s post hoc test, *p = 0.0207 (40–45 min), **p = 0.0094 (45–50 min) or 0.0024 (50–55 min), n = 8 mice, CNO; three mice, LY + CNO). Right: Area under the curve (AUC) of FLEX-NOPLight signal after CNO injection (cumulative, 45 min). Pretreatment with NOPR antagonist LY prevents CNO-induced increases in FLEX-NOPLight fluorescence (two-tailed Mann–Whitney test, ^#p = 0.0485, n = 8 mice, CNO; three mice, LY + CNO). Data represented as mean ± SEM.