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. 2021 Oct 12;26(11):6170–6186. doi: 10.1038/s41380-021-01321-9

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a–d Representative traces (a, c) and quantification of the IPSC frequency (b, d) in Mor-Ens following optical stimulation in the presence of antalarmin or antisavagine-30. Paired t test, Baseline vs Opto, t = 3.493, df = 13, P = 0.0044; Opto vs antalarmin, t = 4.482, df = 13, P = 0.0006 in (b). Baseline vs Opto, t = 2.909, df = 6, P = 0.0207; Opto vs antisavagine-30, t = 0.5294, df = 6, P = 0.6155 in (d). e, f Representative traces (e) and quantification of O-IPSC amplitudes (f) in Mor-Ens in the presence of antalarmin or antisavagine-30. Paired t test, Opto vs antalarmin, t = 2.920, df = 6, P = 0.0266; Opto vs antisavagine-30, t = 1.973, df = 8, P = 0.0840. g Schematic representation of the virus injection. h Quantification of the fluorescent intensity of Crhr1 mRNA in mCherry⁺Cre⁺ cells. sgCrhr1: 419 cells from 4 mice; scramble: 596 cells from 5 mice. Unpaired t test, t = 9.632, df = 7, P < 0.0001; Two-sample KS test, P < 0.0001. i Representative images of Crhr1, Cre, and mCherry ISH probes in sgCrhr1 or scramble groups. Blue: DAPI; Green: Cre; Red: mCherry; Gray: Crhr1. Scale bar, 20 μm. j–p The effect of deletion of Crhr1 in Mor-Ens on negative effect during morphine withdrawal. Experimental process of the behavioral assays (j). Morphine-withdrawal-induced CPA test (k), locomotor activity (l), EPM (m), SPT (n), TST (o), and social-interaction test (p). Two-way RM ANOVA, F_{groups × session} (1, 41) = 1.677, P = 0.2025, scramble vs sgCrhr1 within test, P = 0.203 in (k). Mann–Whitney test or unpaired t test, scramble vs sgCrhr1, (l): U = 51, P = 0.2415; (m): U = 126, P = 0.0460; (n): U = 47, P = 0.1600; (o): t = 6.212, df = 23, P < 0.0001; (p): U = 36, P = 0.0387. q Schematic representation of optical stimulation and ensemble recordings. r Representative traces of PPR in response to optical stimulation (473 nm, 2-ms pulse width at 100-ms interval) and quantification of PPR values in sgCrhr1 and scramble groups. Unpaired t test, t = 0.5813, df = 30, P = 0.5654. s Representative traces and quantification of the amplitudes of OIPSCs in Mor-Ens in response to different laser intensities (n = 16 neurons from five mice in sgCrhr1 group, n = 16 neurons from six mice in scramble group). Two-way ANOVA, F_{(3, 124)} = 3.967, P = 0.0097. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are presented as mean ± SEM.