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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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Identification of monosynaptic inputs to NTS-projecting or PBN-projecting IRS4^PVH neurons using modified rabies virus. A Cre-dependent helper virus construct (AAV-Flex-TVA-B19G) is used to insert rabies B19 glycoprotein (B19G) and the TVA receptor in IRS4^PVH cell bodies and terminals. Modified rabies virus expresses a fluorescent tag (mCherry, B; GFP, C) instead of B19G. After initial infection with helper virus, rabies-mCherry is injected at one projection site (NTS), whereas rabies-GFP is injected at another (PBN) in the same mouse (D). IHC for mCherry and GFP identify largely non-overlapping NTS-projecting and PBN-projecting IRS4^PVH neurons, respectively (E–G). Sites upstream of both NTS and PBN-projecting IRS4^PVH neurons include the lateral hypothalamic area (LHA, H), supraoptic nucleus (SON, K), amygdala (K), bed nucleus of the stria terminalis (BNST, L), and the preoptic area (POA, L). The ventromedial hypothalamus (VMH) is upstream of PBN-projecting IRS4^PVH neurons (H^II, green), whereas both the arcuate nucleus (ARC, H^II, red) and PBN (I, red) are upstream of IRS4^PVH neurons projecting to the NTS. Glial damage represents injection site in the PBN (I, green) and NTS (J, red). 3 V = third ventricle, oc = optic chiasm, MeA = medial amygdala, BMA = basomedial amygdala, vBNST = ventral BNST, vlPOA = ventral lateral POA, MPOA = medial POA, MnPO = median preoptic nucleus, aca = anterior part of anterior commissure, DMH = dorsomedial hypothalamus, aq = aqueduct, 4 V = fourth ventricle.