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. 2019 Feb 1;10:542. doi: 10.1038/s41467-019-08427-3

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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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Fig. 4 — VIP results in CRE activation and re-programmes the TTFL of the SCN. a Representative CRE-Luc bioluminescence rhythms of SCN slices treated with vehicle (left) or 1 µM VIP (right; treatments marked by plus). A detrended trace is included with the VIP-treated slice to aid visualisation of the oscillation. b, c Group data for acute induction of CRE-Luc bioluminescence (b) and period change (c) following treatment with vehicle or VIP (mean ± SEM, veh, n = 3; VIP, n = 4, unpaired t-test, *P < 0.05). d Representative CRE-Luc bioluminescence and RCaMP Ca²⁺ rhythms from SCN slice treated with VIP ( + ). e Detrended CRE-Luc bioluminescence and RCaMP Ca²⁺ rhythms before and after VIP (slice as in d). Note altered phase relationship following VIP. Rhythms were detrended by 24 h rolling average subtraction, and have been offset on the y-axis to aid visualisation. f, g Group data for phase of CRE-Luc rhythm relative to RCaMP rhythm (f negative values where CRE-Luc peaks later than RCaMP, mean ± SEM, n = 3) and period comparison between CRE-Luc and RCaMP, before and after VIP treatment (g two-way ANOVA with Sidak’s multiple comparisons test, **P < 0.01). h, i Group data for amplitude changes of CRE-Luc (h) and RCaMP (i) rhythms before and after VIP treatment (mean ± SEM, n = 3, paired t-test, *P < 0.05, **P < 0.01). Data were normalised to the highest value in the recording. j Representative PER2::LUC, Per1-Luc and Cry1-Luc bioluminescence rhythms of SCN slices treated with 1 µM VIP (at CT10, marked by plus). Bioluminescence was normalised to the first peak of the recording (not shown). k, l Group data for phase shift (k) and period change (l) responses (mean ± SEM) to VIP applied to either PER2::LUC (n = 4), Per1-Luc (n = 9) or Cry1-Luc (n = 7) slices, with vehicle controls (PER2::LUC: n = 6; Per1-Luc: n = 8; Cry1-Luc: n = 7; two-way ANOVA with Sidak’s multiple comparisons test, *P < 0.05, ***P < 0.001)