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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. 5 — a Schematic representation of AAV-RAM-tTA-TRE-hM3Dq vector. b Experimental process of the CNO-evoked CPP experiment. c Representative images of expression of hM3Dq-HA in VTA Sal-Ens and Mor-Ens. Scale bar, 100 μm. d, e Chemoactivation of Mor-Ens drives CPP and promotes anxiolytic behavior. Two-way RM ANOVA, F_{groups × session} (1, 38) = 7.365, P = 0.011, Sal-Ens+hM3Dq vs Mor-Ens+hM3Dq within test, P = 0.006; F_{groups × session} (1, 30) = 4.917, P = 0.034, Mor-Ens+hM3Dq vs Mor-Ens+EGFP within test, P = 0.029; F_{groups × session} (1, 24) = 0.0006, P = 0.9799, Sal-Ens+hM3Dq vs Sal-Ens+EGFP within test, P > 0.999 in (d). Mann–Whitney test, Sal-Ens+hM3Dq vs Mor-Ens+hM3Dq, U = 20, P = 0.0065; Mor-Ens+hM3Dq vs Mor-Ens+EGFP, U = 26, P = 0.0233; Sal-Ens+hM3Dq vs Sal-Ens+EGFP, U = 60, P = 0.9851 in (e). f Schematic representation of virus injection and optic-fiber implantation in the VTA. g Experimental process of the opto- and chemogenetic manipulated behavioral tests. h Representative images of CRH neurons in the CeA expressing hChR2-EYFP (top), and Sal-Ens or Mor-Ens in the VTA labeled with hM3Dq-HA (bottom). Green: EYFP; Red: HA; Blue: DAPI. Scale bars, top: 100 µm; bottom: 20 μm. i, j The effect of chemogenetic activation of Mor-Ens and optogenetic activation of CRH^CeA→VTA terminals on the CPA and EPM tests. Two-way RM ANOVA, F_{groups × session} (1, 24) = 2.257, P = 0.146, hChR2+Sal-Ens vs hChR2+Mor-Ens within test, P = 0.010 in (i); Mann–Whitney test, U = 37, P = 0.014 in (j). k Left: Schematic representation of virus injection and optic-fiber implantation in the VTA for negative reinforcement task. Right: Experimental process of the operant response training. l Representative images of ChR2-EYFP expression in CeA and the projective terminals in the VTA. The dashed white lines indicated the optic fiber trace. Green: EYFP, Scale bars, 100 µm. m Average number of nose pokes in 60-min session on day 4. Unpaired t test, t = 3.026, df = 17, P = 0.0076. In total, 8–11 mice/group. n Left: Schematic representation of virus injection and optic-fiber implantation in the VTA for negative-reinforcement task. Right: Experimental process of the optical and chemogenetic manipulated operant-response training. o Representative images of ChR2-EYFP in CeA, and hM3Dq-HA in VTA. The dashed white lines indicated the optic-fiber trace. Green: EYFP; Red: HA; Blue: DAPI. Scale bars, 100 µm. p Average number of nose pokes in 60-min session on day 4. Unpaired t test, t = 2.215, df = 17, P = 0.0407. In total, 9–10 mice/group. *P < 0.05, **P < 0.001. Data are presented as mean ± SEM.