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. 2021 Oct 1;12:5763. doi: 10.1038/s41467-021-25959-9

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© The Author(s) 2021

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. 6 — a Schematic for simultaneous SCN^VIP neuron manipulation and PVN^CRH neuron calcium imaging. b Representative (n = 4 replicates) images from a Vip^Flp/+; Crh^Cre/+ mouse showing concurrent Flp-dependent ChR2 (cyan) and Cre-dependent jRCaMP1b (magenta) expression. 3V third ventricle. Scale bar = 100 µm. c The change in calcium fluorescence over each minute of recording in an individual ex vivo PVN slice before and after stimulation (blue bar; 470 nm, 8 Hz, 10 ms pulse width, 2 min duration) around subjective dusk (circadian time (CT) 11-12). Warmer colors represent a greater change in calcium fluorescence. d Top, representative calcium traces (in ΔF/F) from two PVN^CRH neurons recorded before and after stimulation. Bottom, raster plot depicting PVN^CRH neuron calcium activity in individual neurons before and after SCN^VIP stimulation (n = 102 cells, 4 slices). Raster plot was thresholded to show the upper 50% ΔF/F of each trace for visualization. e Calcium event frequency and integrated calcium levels in PVN^CRH neurons in each PVN slice (green lines, mean ± SEM) before and after SCN^VIP stimulation (blue line). Two-way repeated-measures ANOVA, p < 0.001 for event frequency and integrated calcium levels. f The average calcium fluorescence over each minute of recording in an individual ex vivo PVN slice before and during clozapine-N-oxide (CNO) treatment (purple shading, 1 µl of 10 µM CNO) around subjective afternoon (CT 6-8). Warmer colors represent increased calcium fluorescence. g Top, representative calcium traces from two PVN^CRH neurons recorded before and during CNO treatment. Bottom, raster plot depicting PVN^CRH neuron calcium activity in individual neurons before and after CNO treatment (n = 151 cells, 3 slices). h Calcium event frequency and integrated calcium levels in PVN^CRH neurons in each PVN slice (green lines, mean ± SEM) before and during SCN^VIP inhibition (purple shading). Two-way repeated-measures ANOVA, p < 0.001 for integrated calcium levels, p = 0.345 for event frequency. i (Left) Raw and (right) detrended PER2::LUC bioluminescence traces from the PVN of Vip^Cre/+; PER2^LUC/+ (black, n = 6) and Vip^Cre/+; Ai32^fl/+; PER2^LUC/+ (blue, n = 4) mice stimulated for 1 h per day for 3 d (blue bars, 8 Hz, 470 nm, 10 ms). j Peak times of PER2::LUC bioluminescence in PVN (solid circles and lines) and SCN (open circles, dashed lines) slices from Vip-ChR2 (blue) and Vip-Cre (black) mice before, during, and after daily optogenetic stimulation (blue squares). Peak times between ChR2 and control PVN were significantly different on days 4–8 (Two-way circular ANOVA, p < 0.001 on each day). Source data are provided as a Source Data file.