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Desynchronizing the sleep­­–wake cycle from circadian timing to assess their separate contributions to physiology and behaviour and to estimate intrinsic circadian period

Nature Protocols volume 18, pages 579–603 (2023)Cite this article

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Abstract

Circadian clocks drive cyclic variations in many aspects of physiology, but some daily variations are evoked by periodic changes in the environment or sleep–wake state and associated behaviors, such as changes in posture, light levels, fasting or eating, rest or activity and social interactions; thus, it is often important to quantify the relative contributions of these factors. Yet, circadian rhythms and these evoked effects cannot be separated under typical 24-h day conditions, because circadian phase and the length of time awake or asleep co-vary. Nathaniel Kleitman’s forced desynchrony (FD) protocol was designed to assess endogenous circadian rhythmicity and to separate circadian from evoked components of daily rhythms in multiple parameters. Under FD protocol conditions, light intensity is kept low to minimize its impact on the circadian pacemaker, and participants have sleep–wake state and associated behaviors scheduled to an imposed non-24-h cycle. The period of this imposed cycle, Τ, is chosen so that the circadian pacemaker cannot entrain to it and therefore continues to oscillate at its intrinsic period (τ, ~24.15 h), ensuring circadian components are separated from evoked components of daily rhythms. Here we provide detailed instructions and troubleshooting techniques on how to design, implement and analyze the data from an FD protocol. We provide two procedures: one with general guidance for designing an FD study and another with more precise instructions for replicating one of our previous FD studies. We discuss estimating circadian parameters and quantifying the separate contributions of circadian rhythmicity and the sleep–wake cycle, including statistical analysis procedures and an R package for conducting the non-orthogonal spectral analysis method that enables an accurate estimation of period, amplitude and phase.

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Fig. 1: Example schedules and body temperature rhythms for different experimental protocols.
Fig. 2: Schematic of wake-sleep and corresponding light–dark (LD) cycle effects of the FD protocol compared to the free-run protocol and to standard (entrainment) conditions.
Fig. 3: Circadian and homeostatic regulation of performance during a 42.85-h FD protocol.
Fig. 4: Circadian period estimation using FD protocols.
Fig. 5: Sleep efficiency of young participants and older participants during a 28-h T-cycle FD protocol.
Fig. 6: Percentage of recording time with SEMs during wakefulness under a 42.85-h FD protocol.

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Data availability

All original datasets used in the preparation of this paper contain confidential or identifiable human data and are not publicly available due to human subject research regulations. De-identified sample datasets are available upon reasonable request and are subject to internal review and approval. Execution of a materials transfer agreement is required by our institution for transfer of data.

Code availability

SAS codes for data analyses and R codes for the NOSA program can be found at https://github.com/wwang4bwh/FD-NOSA.

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Acknowledgements

The authors acknowledge Drs. J.S. Allan, D.B. Boivin, S.W. Cain, C. Cajochen, A.M. Chang, D. Cohen, S.P. Grady, C. Gronfier, J.T. Hull, M.Y. Münch, D.W. Rimmer, A.W. McHill and K.D. Scheuermaier, each of whom carried out FD studies when they were a trainee in the laboratory. The authors acknowledge Seungyeop Kang for help with R programming. The authors also acknowledge the critical contributions of the late Richard E. Kronauer to the development of the FD protocol and its analyses. The FD studies and data described here were collected in the Environmental Scheduling Facility (ESF) and the Intensive Physiological Monitoring (IPM) Unit at the Brigham and Women’s Hospital. Both facilities were part of the BWH General Clinical Research Center (supported by National Institutes of Health (NIH) Grant M01 RR002635), and the IPM was part of the Harvard Clinical and Translational Science Center (supported by NIH Award UL1 RR025758 and financial contributions from the Brigham and Women’s Hospital and from Harvard University and its affiliated academic health care centers). The development of the FD protocol and NOSA program and carrying out the FD studies described here were supported by NIH Grants P01 AG09975, R01 HL080978, R01 HL52992, R21 AT02571, U01 AG12642 and T32 HL07901; by National Aeronautics and Space Administration Grants NAS9-19435 and NAG 5-3952 and NASA Cooperative Agreement NCC9-58 with the National Space Biomedical Research Institute; and by Air Force Office of Scientific Research Grants F49620-94-1-0398, F49620-95-1-0388, FA9550-06-0080 and F49620-00-1-0266.

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Authors and Affiliations

  1. Division of Sleep and Circadian Disorders, Departments of Medicine and Neurology, Brigham and Women’s Hospital, Boston, MA, USA

    Wei Wang, Robin K. Yuan, Kirsi-Marja Zitting, Melissa A. St. Hilaire, Frank A. J. L. Scheer, Joseph M. Ronda, Elizabeth B. Klerman, Jeanne F. Duffy & Charles A. Czeisler

  2. Division of Sleep Medicine and Department of Medicine, Harvard Medical School, Boston, MA, USA

    Wei Wang, Robin K. Yuan, Kirsi-Marja Zitting, Melissa A. St. Hilaire, Frank A. J. L. Scheer, Joseph M. Ronda, Elizabeth B. Klerman, Jeanne F. Duffy & Charles A. Czeisler

  3. Department of Brain and Cognitive Sciences, University of Rochester, Rochester, NY, USA

    Jude F. Mitchell

  4. Department of Psychiatry and Behavioral Sciences, Rush University Medical Center, Chicago, IL, USA

    James K. Wyatt

  5. Broad Institute, Cambridge, MA, USA

    Frank A. J. L. Scheer

  6. Sleep and Chronobiology Laboratory, Department of Integrative Physiology, University of Colorado Boulder, Boulder, CO, USA

    Kenneth P. Wright Jr

  7. Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, MA, USA

    Emery N. Brown

  8. Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA, USA

    Emery N. Brown

  9. Institute for Medical Engineering and Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA

    Emery N. Brown

  10. Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA, USA

    Emery N. Brown & Elizabeth B. Klerman

  11. Institute for Data Systems and Society, Massachusetts Institute of Technology, Cambridge, MA, USA

    Emery N. Brown

  12. Department of Neurology, Massachusetts General Hospital, Boston, MA, USA

    Elizabeth B. Klerman

  13. Surrey Sleep Research Centre, Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK

    Derk-Jan Dijk

  14. UK Dementia Research Institute Care Research and Technology Centre, Imperial College London and the University of Surrey, Guildford, UK

    Derk-Jan Dijk

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  1. Wei Wang
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Contributions

C.A.C. developed the concept and designed the FD protocol as implemented in our laboratory. J.M.R. designed and built the data-collection hardware and software, the protocol-scheduling software and the data-management system used in our laboratory. E.N.B. designed the statistical model, analysis and computational framework for the NOSA procedure. E.N.B., J.F.M., D.-J.D., and C.A.C. contributed to the implementation of the computational framework for the NOSA procedure. C.A.C., D.-J.D., E.B.K., and M.A.S.H. carried out simulations to determine the impact of light levels on period estimates from FD protocols and free runs. D.-J.D. developed the analyses framework for the separation of circadian and sleep–wake contributions to sleep and performance. R.K.Y., K.-M.Z., J.K.W., F.A.J.L.S., K.P.W., E.B.K., J.F.D. and D.-J.D. carried out FD studies and analyses. W.W. re-analyzed FD data presented here and wrote and updated the NOSA program in R for sharing. W.W., R.K.Y. and K.-M.Z. created figures for the manuscript. All authors contributed to drafting, writing and/or editing the manuscript.

Corresponding author

Correspondence to Wei Wang.

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Competing interests

W.W. serves as a consultant for the National Sleep Foundation. E.B.K. receives support from the Gordon Research Conference, the Sleep Research Society, the Santa Fe Institute and DGSM (German Sleep Society); she has consultancies for Circadian Therapeutics, the National Sleep Foundation, the Puerto Rico Science Technology Trust and Sanofi-Genzyme; her partner owns Chronsulting. C.A.C. reports grants/contracts to BWH from Dayzz Live Well, Ltd., Delta Airlines, FAA, Jazz Pharmaceuticals, NHLBI, NIA, NIOSH, NASA, Puget Sound Pilots, Regeneron Pharmaceuticals/Sanofi and DOD; reports grants/gifts to Monash University from the CDC Foundation, with funding from BNY Mellon, and WHOOP; is/was a paid consultant or received lecture fees from Emory University, Inselspital Bern, UCLA, the Institute of Digital Media and Child Development, the Klarman Family Foundation, the National Council for Mental Wellbeing, the National Sleep Foundation, Physician’s Seal, the SRS Foundation, Tencent Holdings, Teva Pharma Australia, With Deep and Vanda Pharmaceuticals Inc., in which he holds an equity interest; received travel support from the Aspen Brain Institute, the Bloomage International Investment Group, UK Biotechnology and Biological Sciences Research Council, Bouley Botanicals, the Dr. Stanley Ho Medical Development Foundation, EBRS, the German National Academy of Sciences (Leopoldina), the National Safety Council, the National Sleep Foundation, Stanford Medical School, Tencent Holdings and Vanda Pharmaceuticals Inc.; receives research/education support through BWH from Arbor Pharmaceuticals, Avadel Pharmaceuticals, Beijing Zhaode Healthcare Management Consulting Co., Bryte, Alexandra Drane, Eisai, Harmony Biosciences, Jazz Pharmaceuticals, Johnson & Johnson, Mary Ann & Stanley Snider via Combined Jewish Philanthropies, NeuroCare, Inc., Optum, Philips Respironics, Regeneron Pharmaceuticals, Regional Home Care, ResMed, San Francisco Bar Pilots, Sanofi, Schneider, Simmons, Sleep Cycle, Sleep Number, Sysco, Teva Pharmaceuticals Industries and Vanda Pharmaceuticals Inc.; is/was an expert witness in legal cases, including those involving Advanced Power Technologies, Aegis Chemical Solutions LLC, Amtrak, Casper Sleep Inc., C&J Energy Services, Catapult Energy Services Group, Covenant Testing Technologies, the Dallas Police Association, Enterprise Rent-A-Car, Espinal Trucking/Eagle Transport Group/Steel Warehouse Inc., FedEx, Greyhound Lines Inc./Motor Coach Industries/FirstGroup America, PAR Electrical Contractors Inc., Product & Logistics Services LLC/Schlumberger Technology Corp/Gelco Fleet Trust, Puckett Emergency Medical Services LLC, Puget Sound Pilots, Union Pacific Railroad, United Parcel Service and Vanda Pharmaceuticals Inc.; serves as the incumbent of an endowed professorship provided to Harvard University by Cephalon, Inc.; and receives royalties from McGraw Hill and from Philips Respironics for the Actiwatch-2 and Actiwatch Spectrum devices. C.A.C.’s interests were reviewed and are managed by the BWH and MGB in accordance with their conflict of interest policies. D.-J.D. was a paid consultant to F. Hoffmann-La Roche Ltd., Pfizer Inc., Eli Lilly and Company, Novo Nordisk A/S and Ono Pharma UK Ltd. and has received research support from Janssen Research & Development LLC, Eli Lilly and Company and GW Pharma. M.A.S.H. has provided paid limited consulting for The MathWorks, Inc. K.P.W. reports research support/donated materials: DuPont Nutrition & Biosciences, Grain Processing Corporation, and Friesland Campina Innovation Centre and being a consultant to and/or receiving personal fees from Circadian Therapeutics, Inc., Circadian Biotherapies, Inc., Philips, Inc, and U.S. Army Medical Research and Materiel Command - Walter Reed Army Institute of Research, outside the submitted work.

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Related links

Key references using this protocol

Czeisler, C. A. et al. Science 284, 2177–2181 (1999): https://doi.org/10.1126/science.284.5423.2177

Dijk, D. J. et al. J. Neurosci. 15, 3526–3538 (1995): https://doi.org/10.1523/JNEUROSCI.15-05-03526.1995

Scheer, F. A. et al. PLoS One 2, e721 (2007): https://doi.org/10.1371/journal.pone.0000721

Cohen, D. A. et al. Sci. Transl. Med. 2, 14ra13 (2010): https://doi.org/10.1126/scitranslmed.3000458

Duffy, J. F. et al. Proc. Natl Acad. Sci. USA 108, 15602–15608 (2011): https://doi.org/10.1073/pnas.1010666108

Key data used in this protocol

Dijk, D. J. et al. J. Physiol. 516, 611–627 (1999): https://doi.org/10.1111/j.1469-7793.1999.0611v.x

Duffy, J. F. et al. Proc. Natl Acad. Sci. USA 108, 15602–15608 (2011): https://doi.org/10.1073/pnas.1010666108

Cohen, D. A. et al. Sci. Transl. Med. 2, 14ra13 (2010): https://doi.org/10.1126/scitranslmed.3000458

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Wang, W., Yuan, R.K., Mitchell, J.F. et al. Desynchronizing the sleep­­–wake cycle from circadian timing to assess their separate contributions to physiology and behaviour and to estimate intrinsic circadian period. Nat Protoc 18, 579–603 (2023). https://doi.org/10.1038/s41596-022-00746-y

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