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. 2022 Mar 4;15:21. doi: 10.1186/s13041-022-00907-w

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© The Author(s) 2022

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 1 — Animal models for measuring putative orexin neuronal activity. A1 Schematic drawing showing specific expression of GCaMP6 and mCherry in putative orexin neurons by injecting adeno-associated virus (AAV) vectors into the hypothalamus of preproorexin knockout (ORX^−/−) and orexin-tTA transgenic (ORX-tTA) double-mutant mice. We refer to this animal as “model 1” in this study. A2 Schematic drawing showing specific expression of GCaMP6 in putative orexin neurons by crossing double mutants of ORX^−/− and ORX-tTA with TetO-GCaMP knock-in mice. We call this animal “model 2” in this study. B1 Histological confirmation showing overlapping distribution of GCaMP6 (green) and mCherry (red) in the hypothalamus of a model 1 mouse. B2 Histological confirmation showing GCaMP6 (green) in the hypothalamus of a model 2 mouse. Note that no orexin-like immunoreactivity was found, as expected. B2′ Histological confirmation showing overlapping distribution of GCaMP6 (green) and orexin-like immunoreactivity (red) in the hypothalamus of a mouse carrying the orexin-knockout heterozygous allele in model 2. C1 & C2 Confirmation of fiber tracts after fluorescent recordings. The dashed line indicates the location of the inserted optic fibers. The left panel C1 shows a brain from a model 1 mouse and the right panel C2 shows a brain from a model 2 mouse