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. 2019 Feb 26;9:2830. doi: 10.1038/s41598-019-39063-y

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© The Author(s) 2019

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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Confocal microscope images of quadruple fluorescence staining. Images show the appearance of CTb-labeled neurons (green) immunoreactive for ORX (red) and Synaptophysin (cyan) after injection of BDA (magenta) into the LPB and CTb into the VTA (A) DR (B) PPT (C) LDT (D) or LC (E) Two types of merged images, BDA/ORX/CTb and BDA/ORX/synaptophysin, were obtained from the same area. The arrowheads indicate BDA-labeled axons (magenta), which contact to ORX (red) and CTb (green) double-IR neurons. The arrows indicate synaptophysin (cyan) in BDA-labeled axons (magenta), which contact to ORX (red)-IR neurons. Scale bars, 10 μm. LPB: lateral parabrachial nuclei; ORX: orexin; VTA: ventral tegmental area; IR: immunoreactive; CTb: cholera toxin B subunit; BDA: biotinylated dextranamine; PPT: pedunculopontine tegmental nucleus; VTA: ventral tegmental area; DR: dorsal raphe; LC: locus coeruleus.