Fig. 6: NOPLight detection of endogenous VTA N/OFQ release during high effort reward-seeking.

a. Fiber photometry schematic. Cartoon of NOPLight viral injection and fiber implant in the VTA of WT mice (n = 4 mice). Representative image showing expression of DAPI (blue) and NOPLight (green) with fiber placement in VTA. b. Left: Cartoon depicting operant box setup for the progressive ratio (PR) test. Right: Trial structure for reward delivery during the paradigm. c. Top: Averaged trace of NOPLight signal for all mice (n = 4), aligned to rewarded active nose pokes (ANPs) made during the PR test. Epoch includes the 5 s light cue (yellow, shaded) that precedes reward delivery. Time to pellet retrieval and duration of consumption period averaged across all trials (brown, shaded). Data represented as mean ± SEM. Bottom: Heat map, each row corresponds to a single, rewarded ANP epoch. d Top: Averaged trace of NOPLight signal for all mice (n = 4), aligned to non-rewarded ANPs made during the PR test. Data represented as mean ± SEM. Bottom: Heat map, each row corresponds to a single, non-rewarded ANP epoch. e NOPLight fluorescence averaged over 5-s intervals for rewarded (green) and non-rewarded (blue) ANP epochs. Time to pellet retrieval and duration of consumption period averaged across all rewarded trials (brown, shaded). Across rewarded ANP trials, NOPLight signal decreases during reward consumption then immediately increases post-consumption relative to NOPLight signal measured during non-rewarded ANP epochs (two-way repeated-measures ANOVA with Bonferroni’s post hoc test, *p = 0.0122, **p = 0.0033, ***p = 0.0003, n = 4 mice). Data represented as mean ± SEM. f, g Area under the curve (AUC) for photometry traces from c and d, respectively, calculated over 5-s intervals surrounding (f) rewarded ANPs (two-tailed Wilcoxon test, *p = 0.0125 (25–30 s) or 0.031 (30–35 s); **p = 0.007 (5–10 s) or 0.0042 (15–20 s); ***p = 0.0008 (10–15 s); n = 4 mice) and (g) non-rewarded ANPs (two-tailed Wilcoxon test, *p = 0.0128 (5–10 s), ****p < 0.0001 (0–5 s), n = 4 mice). Data represented as mean ± SEM.