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. 2020 Mar 26;10:5546. doi: 10.1038/s41598-020-62468-z

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© The Author(s) 2020

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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Irs4 expression defines a PVH subpopulation. In situ hybridization was performed on coronal slices from adult IRS4-iCre mice (A) for Irs4 (red) and Cre (green), showing substantial overlap between Cre and Irs4 mRNA in the PVH (B). To identify overlap between IRS4^PVH neurons and other PVH populations, mice were stereotaxically injected in the PVH with a Cre-dependent GFP reporter virus (AAV-Flex-GFP, C) to visualize Cre-expressing neurons in the adult mouse. Immunohistochemistry (IHC) in the PVH demonstrates limited co-localization between GFP-labeled IRS4^PVH neurons (green) and OXT (D), NOS1 (E), or AVP (F) neurons in the PVH. Dashed boxes indicate regions that are digitally enlarged and shown as insets. 3 V = third ventricle.