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. 2018 Jun 27;8:9732. doi: 10.1038/s41598-018-28095-5

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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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Mechanical stimulation increases RIS activity most strongly during lethargus. Sleep deprivation was applied using dish tapping. (A) Worms that do not get stimulated mechanically show two main phases during which RIS transients occur, the first during the onset of lethargus and one at the end of lethargus. Plot modified from Urmersbach et al., with permission (Wiley)⁴⁰. (B) Sleep deprivation at the onset of sleep behavior leads to an additional activation phase of RIS, mostly during continuous tapping. (C) Extraction of the first response to mechanical stimulation shows a simultaneous increase in RIS activity and mobilization. (D) Sleep deprivation 30 minutes after the onset of sleeping behavior also led to an increase in RIS activation. (E) A comparable mechanical stimulation protocol outside of lethargus also leads to a small increase in RIS activation. (F) Quantification of RIS activation upon by measuring RIS GCaMP3 activity over 30 minutes before lethargus (“wake”), at the onset of lethargus (“sleep onset”, non-pumping phase start), early lethargus (“early sleep”), 30 minutes into lethargus (“mid sleep”), and after lethargus (“L2 wake”). **Denotes statistical significance with p < 0.01, and ***denotes statistical significance with p < 0.001, Welch test.