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. 2018 Jan 24;9:354. doi: 10.1038/s41467-017-02765-w

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© The Author(s) 2018

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. 2 — Inhibition of dorsolateral pons by vlPAG GABAergic projection suppresses REM sleep and wakefulness while promoting NREM sleep. a Left, schematic depicting ChR2-mediated activation of GABAergic axons projecting from vlPAG to the dorsolateral pons. Right, coronal diagram of mouse brain (top) and fluorescence image (bottom) of dorsolateral pons (black box in diagram) in a GAD2-Cre mouse injected with AAV expressing ChR2–eYFP (green) into the vlPAG. Blue bar, optic fiber. Blue, DAPI. Scale bar, 500 μm. DTg dorsal tegmental nucleus, scp superior cerebellar peduncle. b Top, normalized EEG spectrogram. Bottom, percentage of REM, NREM, or wake states before, during, and after laser stimulation of GABAergic axons (n = 5). Shading, 95% CI. c Schematic showing rabies-mediated trans-synaptic tracing. TC^66T mutant EnvA receptor fused with mCherry, RG rabies glycoprotein, RVdG RG-deleted rabies virus. d Top, fluorescence image of SLD in VGLUT2-Cre mouse. Scale bar, 200 μm. Bottom, enlarged view of white box showing starter cells (yellow, arrowheads), expressing both eGFP and mCherry. Scale bar, 20 μm. e Top, fluorescence image of LDT in ChAT-Cre mouse. Scale bar, 200 μm. Bottom, enlarged view of white box showing starter cells (yellow, arrowheads). Scale bar, 20 μm. f Top, fluorescence image of LC in TH-Cre mouse. Scale bar, 200 μm. Bottom, enlarged view of white box showing starter cells (yellow, arrowheads). Scale bar, 20 μm. g Coronal diagram of mouse brain. Aq aqueduct. h vlPAG GABAergic (GAD2) neurons innervating glutamatergic (VGLUT2) SLD neurons. Top, fluorescence image showing rabies-eGFP-labeled (green) and GAD2-positive neurons in vlPAG (n = 3 mice). Red, GAD2. Blue, DAPI. Scale bar, 200 μm. Bottom, enlarged view of white box showing eGFP-labeled neurons expressing GAD2 (arrowheads). Scale bar, 30 μm. i Similar to h, for vlPAG GABAergic neurons innervating cholinergic (ChAT) LDT neurons (n = 3). j Similar to h, for vlPAG GABAergic neurons innervating noradrenergic (TH) LC neurons (n = 3). k Percentages of rabies-eGFP-labeled neurons in vlPAG innervating glutamatergic SLD neurons, cholinergic LDT neurons, and noradrenergic LC neurons (n = 3). Circles, single mice; error bars, ±s.d. Brain figures in a, c, g were adapted from Allen Mouse Brain Atlas (© 2015 Allen Institute for Brain Science. Allen Brain Atlas API. Available from: http://brain-map.org/api/index.html)