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. 2021 May 4;12:2517. doi: 10.1038/s41467-021-22730-y

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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. 9 — a–c Cat odor exposure activated VMH-aPVT neurons. a Timeline of the cat odor-induced neuronal activity test. b Representative micrographs showing VMH-aPVT neurons (green) expressing immunoreactivity to cFos (red) in both neutral odor (left) or cat odor (right) groups. Inset: white arrows showing examples of co-labeled cells. c Cat odor exposure (brown bars, n = 5) significantly increased (left) the percentage of VMH-aPVT cells that were cFos-positive and (right) the percentage of cFos-positive cells that were aPVT projecting, when compared to neutral odor controls (white bars, n = 6; unpaired Student’s t test; left, P < 0.001, t = 7.64; right, P < 0.001, t = 5.78). d–f Photoactivation of VMH afferents induces monosynaptic EPSCs in NAc-projecting aPVT neurons in vitro. d Schematics showing CTB infusion in NAc, viral vector AAV-ChR2-eYFP infusion in VMH, and slice recordings from NAc-projecting aPVT neurons. e Action potentials evoked by a depolarizing current step in a NAc-projecting aPVT neuron. f For the same cell, optically evoked EPSC (black) was completely blocked following bath application of the AMPA receptor antagonist NBQX (red). Similar findings were made in nine additional neurons (two rats). g (Top) Timeline of the conflict test during chemogenetic inhibition of VMH-aPVT neurons. (Bottom left) Representative micrograph showing the unilateral expression of hM4Di in VMH. (Bottom left-center) Red areas represent the minimum (dark) and the maximum (light) viral expression. (Bottom right-center) Representative micrograph showing the expression of retrograde AAV-Cre-GFP in aPVT. (Bottom right) Green areas represent the minimum (dark) and the maximum (light) viral expression. h–m Chemogenetic inhibition of VMH-aPVT neurons (blue bars, n = 7) reduced the percentage of time rats spent exhibiting h freezing (F_{(1, 13)} = 7.35, P = 0.017), i avoidance (F_{(1, 13)} = 4.59, P = 0.051 with Bonferroni planned comparison test P = 0.005) and j head-out responses (F_{(1, 13)} = 18.64, P < 0.001). No changes were observed in k food-approach time (F_{(1, 13)} = 0.92, P = 0.35), l lever press (F_{(1, 13)} = 0.18, P = 0.67) and m latency to press (F_{(1, 13)} = 1.26, P = 0.28) during the conflict test, when compared to mCherry controls (gray bars, n = 8). 3V third ventricle, f fornix, sm stria medullaris, cc corpus callosum, MD mediodorsal thalamus, CA3 hippocampal CA3 subregion. Scale bars: 200 μm; inset scale bar: 25 μm. Two-way repeated-measures ANOVA followed by Bonferroni post hoc test. Data are shown as mean ± SEM. *P < 0.05. See also Supplementary Movie 9.