Fig. 4: Operant experimental procedure demonstrates behavioral drug-cue reactivity.

a Schematic of experimental setup during training (top) and recording (bottom) sessions in Experiment 2. Boxes equipped with infusion pump (a), swivel (b), speaker (c), trial light (d), feeder lights I, feeders (f), retractable lever (g), dippers (h), commutator (i), and recording computer (j). b Experimental design. Gray shading indicates omitted reward in block two of recording trials. c Behavior training results for opioid-exposed animals (n = 5) = 5). Circles indicate % correct responses, Xs indicate % incorrect, and diamonds indicate % not responded to. Error bars = SEM. d Similar to 4c for opioid-naive rats (n = 6). e Total number of trials completed per recording session for opioid-exposed and opioid-naive rats (All bars = mean + SEMn = 106 sessions from N = 11 rats,two-tailed Mann-Whitney test, U = 1135, n = 106 sessions from N = 113 sessions from N = 11 rats, = 11 rats, p = 0.0975). f Number of correct responses per session to Cue A and Cue B (2-way RM ANOVA, Cue factor: F(1,114) = 4.786, n = 113 sessions from N = 11 rats, p = 0.0307. g Trial accuracy response index for opioid-exposed and opioid-naive animals (two-tailed one-sample t tests, H_o: μ = 1. Opioid-exposed: t(= 64, 47) = 2.168, n = 48, p = 0.0353; Opioid-naive: t(63) = 0.8954, n = 64, p = 0.3740). h Probability that first feeder entry after cue onset is congruent with cue identity (2-way RM ANOVA, opioid exposure factor: n = 100 sessions from Nn = 47, = 11 rats, F(1,101) = 5.032, p = 0.027; interaction F(1,101) = 8.621, p = 0.0041). i Ratio of total congruent feeder entries for Cue B/Cue A in both groups (two-tailed one-sample t tests. Opioid-exposed: t(46) = 3.343, n = 47, p = 0.0017; Opioid-naive: t(5 = 110 sessions from N = 11 rats, 4) = 0.6371, n = 55, p = 0.5268). j Cumulative congruent feeder entries following auditory cue onset (F-test for difference of slopes, F(3,156) = 5.482, n = 10 p = 0.0023; rat 8: t2 sessions from = 1.730, N = 11 rats, p = 0.0013). k Left axis: Session-mean of lever press hazard rate. 2-way Mixed effects model, Opioid-exposure factor: F(1,108) = 24.04, n = 110 sessions from N = 11 rats, n = 49 sessions from N = p < 0.0001). Right axis: cumulative % of sessions with lever press at time t is shifted to the left in opioid-exposed rats. All traces show mean ± SEM. l–n As in g–i respectively, but for rat 8 (red) and all other opioid-exposed animals: l (Opioid-exposed: t(36) = 3.284, n = 37, p = 0.0023; rat 8: t(11) = 1.730, n = 12, p = 0.1115); m (interaction F(1,47) = 24.4, n = 49 sessions from N = 5 rats, p = <0.0001); n (Opioid-exposed: t(34) = 5.352, n = 35, p < 0.0001; rat 8: t(10) = 5.000, n = 11, p = 0.0005). o Mean hazard rate for lever press during 0-2 s after lever extension in rat 8 vs. other opioid-exposed rats (two-tailed Mann-Whitney test, n = 49, U = 91, p = 0.0038). p Left: First two principal components (PCs) extracted from composite behavior measures for opioid-naive rats (purple), rat 8 (red filled), and all other opioid-exposed rats (teal). Crosses represent corresponding centroids. Right: Mean difference of rat 8 centroid under true labels (red dashed line) compared to bootstrapped distribution under shuffled labeling shows greater similarity of rat 8 to the opioid-naive group. Black dashed lines indicate two-tailed 95% threshold of null distribution. Source data are provided as a Source Data file.