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The study of circadian rhythms has been critically dependent upon analysing mouse home cage activity, typically employing wheel running activity under different lighting conditions. Here we assess a novel method, the Digital Ventilated Cage (DVC ® , Tecniplast SpA, Italy), for circadian phenotyping. Based upon capacitive sensors mounted under black individually ventilated cages with inbuilt LED lighting, each cage becomes an independent light-controlled chamber. Home cage activity in C57BL/6J mice was recorded under a range of lighting conditions, along with circadian clock-deficient cryptochrome-deficient mice (Cry1 -/- , Cry2 -/- double knockout). C57BL/6J mice exhibited a 24 h period under light/dark conditions, with a free-running period of 23.5 h under constant dark, and period lengthening under constant light. Animals displayed expected phase shifting responses to jet-lag and nocturnal light pulses. Sex differences in circadian parameters and phase shifting responses were also observed. Cryptochrome-deficient mice showed subtle changes in activity under light/dark conditions and were arrhythmic under constant dark, as expected. Our results show the suitability of the DVC system for circadian behavioural screens, accurately detecting circadian period, circadian disruption, phase shifts and mice with clock defects. We provide an evaluation of the strengths and limitations of this method, highlighting how the use of the DVC for studying circadian rhythms depends upon the research requirements of the end user., Competing Interests: Declarations. Competing interests: The authors (ST and SNP) have attended meetings funded by Tecniplast. However, Tecniplast had no involvement in the study design, data interpretation, or conclusions drawn from the research findings. As such, the authors declare no conflict of interest regarding the use of the DVC system. Ethics statement: The animal study was reviewed and approved by the Clinical Medicine Animal Welfare and Ethical Review Body (AWERB), University of Oxford., (© 2025. The Author(s).)

Optogenetic therapy is a promising vision restoration method where light-sensitive opsins are introduced to the surviving inner retina following photoreceptor degeneration. The cell type targeted for opsin expression will likely influence the quality of restored vision. However, a like-for-like preclinical comparison of visual responses evoked following equivalent opsin expression in the two major targets, ON bipolar (ON BCs) or retinal ganglion cells (RGCs), is absent. We address this deficit by comparing stimulus-response characteristics at single-unit resolution in the retina and dorsal lateral geniculate nucleus of retinally degenerate mice genetically engineered to express the opsin ReaChR in Grm6- or Brn3c-expressing cells (ON BC vs. RGCs, respectively). For both targeting strategies, we find ReaChR-evoked responses have equivalent sensitivity and can encode contrast across different background irradiances. Compared with ON BCs, targeting RGCs decreased response reproducibility and resulted in more stereotyped responses with reduced diversity in response polarity, contrast sensitivity, and temporal frequency tuning. Recording ReaChR-driven responses in visually intact retinas confirmed that RGC-targeted ReaChR expression disrupts visual feature selectivity of individual RGCs. Our data show that, while both approaches restore visual responses with impressive fidelity, ON BC targeting produces a richer visual code closer to that of wild-type mice., Competing Interests: Declaration of interests R.J.L. and J.R. are named inventors on patent applications for the use of animal opsins in optogenetics. R.J.L. has received investigator-initiated research funding from Kubota Vision Inc. and acted as a consultant for Kubota Vision Inc., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Chronic pain is a hallmark of joint diseases. Although these conditions are often accompanied by negative affective symptoms including depression and anxiety, these comorbidities are rarely studied simultaneously in preclinical models where they are poorly characterised. Moreover, how affective symptoms relate to the more obvious sensory and functional symptoms of joint diseases is not well understood. Here, we have addressed these gaps in knowledge., Methods: We used 2 preclinical models of joint pain in male mice and an array of behavioural and molecular assays to fully characterise functional deficits, mechanical hypersensitivity, affective symptoms, and nociceptive signaling in joint pain, as well as investigate their relationship., Results: Ankle joint inflammation and knee osteoarthritis induced mechanical hypersensitivity that lasted at least 3 months and that was not different between the 2 models on most days. However, the models presented with markedly different weight-bearing deficits, molecular profiles, and affective outcomes. Specifically, only the model of knee osteoarthritis was accompanied by an increase in negative affective behaviors, including early changes in circadian patterns, persistent cognitive impairments, and late development of depressive-like behavior. Importantly, the early weight-bearing deficit strongly correlated with the emotional profiles and the hypersensitivity at 3 months, suggesting that early objective functional measures may be used as predictors of long-term affective symptoms and pain., Conclusion: The predictive value of early weight-bearing deficit could prove useful in the clinical setting for adapted therapeutic approaches for the prevention of emotional comorbidities and better pain management for patients with joint pain., Competing Interests: The authors have no conflict of interest to declare., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The International Association for the Study of Pain.)

Background: The natural light environment is far more complex than that experienced by animals under laboratory conditions. As a burrowing species, wild mice are able to self-modulate their light exposure, a concept known as light environment sampling behaviour. By contrast, under laboratory conditions mice have little opportunity to exhibit this behaviour. To address this issue, here we introduce a simple nestbox paradigm to allow mice to self-modulate their light environment. Dark nestboxes fitted with passive infrared sensors were used to monitor locomotor activity, circadian entrainment, decision making and light environment sampling behaviour., Results: Under these conditions, mice significantly reduce their light exposure to an average of just 0.8 h across a 24 h period. In addition, mice show a distinct pattern of light environment sampling behaviour, with peaks at dawn and dusk under a ramped light dark cycle. Furthermore, we show that the timing of light environment sampling behaviour depends upon endogenous circadian rhythms and is abolished in mice lacking a circadian clock, indicating a feedback loop between light, the circadian clock and behaviour., Conclusions: Our results highlight the important role of behaviour in modifying the light signals available for circadian entrainment under natural conditions., (© 2024. The Author(s).)

Light is recognized as an important component of the environment for laboratory animals. It supports vision, sets the phase of circadian clocks, and drives wide-ranging adjustments in physiological and behavioral state. Manipulating light is meanwhile a key experimental approach in the fields of vision science and chronobiology. Nevertheless, until recently, there has been no consensus on methods for quantifying light as experienced by laboratory animals. Widely adopted practices employ metrics such as illuminance (units = lux) that are designed to quantify light as experienced by human observers. These weight energy across the spectrum according to a spectral sensitivity profile for human vision that is not widely replicated for non-human species. Recently, a Consensus View was published that proposes methods of light measurement and standardization that take account of these species-specific differences in wavelength sensitivity. Here, we draw upon the contents of that consensus to provide simplified advice on light measurement in laboratory mammal experimentation and husbandry and quantitative guidance on what constitutes appropriate lighting for both visual and circadian function.

Monitoring body temperature and energy expenditure in freely-moving laboratory mice remains a powerful methodology used widely across a variety of disciplines-including circadian biology, sleep research, metabolic phenotyping, and the study of body temperature regulation. Some of the most pronounced changes in body temperature are observed when small heterothermic species reduce their body temperature during daily torpor. Daily torpor is an energy saving strategy characterized by dramatic reductions in body temperature employed by mice and other species when challenged to meet energetic demands. Typical measurements used to describe daily torpor are the measurement of core body temperature and energy expenditure. These approaches can have drawbacks and developing alternatives for these techniques provides options that can be beneficial both from an animal-welfare and study-complexity perspective. First, this paper presents and assesses a method to estimate core body temperature based on measurements of subcutaneous body temperature, and second, a separate approach to better estimate energy expenditure during daily torpor based on core body temperature. Third, the effects of light exposure during the habitual dark phase and sleep deprivation during the light period on body temperature dynamics were tested preliminary in fed and fasted mice. Together, the here-published approaches and datasets can be used in the future to assess body temperature and metabolism in freely-moving laboratory mice., (© 2024. The Author(s).)

Vocalisations are increasingly being recognised as an important aspect of normal rodent behaviour yet little is known of how they interact with other spontaneous behaviours such as sleep and torpor, particularly in a social setting. We obtained chronic recordings of the vocal behaviour of adult male and female Djungarian hamsters (Phodopus sungorus) housed under short photoperiod (8 h light, 16 h dark, square wave transitions), in different social contexts. The animals were kept in isolation or in same-sex sibling pairs, separated by a grid which allowed non-physical social interaction. On approximately 20% of days hamsters spontaneously entered torpor, a state of metabolic depression that coincides with the rest phase of many small mammal species in response to actual or predicted energy shortages. Animals produced ultrasonic vocalisations (USVs) with a peak frequency of 57 kHz in both social and asocial conditions and there was a high degree of variability in vocalisation rate between subjects. Vocalisation rate was correlated with locomotor activity across the 24-h light cycle, occurring more frequently during the dark period when the hamsters were more active and peaking around light transitions. Solitary-housed animals did not vocalise whilst torpid and animals remained in torpor despite overlapping with vocalisations in social-housing. Besides a minor decrease in peak USV frequency when isolated hamsters were re-paired with their siblings, changing social contexts did not influence vocalisation behaviour or structure. In rare instances, temporally overlapping USVs occurred when animals were socially-housed and were grouped in such a way that could indicate coordination. We did not observe broadband calls (BBCs) contemporaneous with USVs in this paradigm, corroborating their correlation with physical aggression which was absent from our experiment. Overall, we find little evidence to suggest a direct social function of hamster USVs. We conclude that understanding the effects of vocalisations on spontaneous behaviours, such as sleep and torpor, will inform experimental design of future studies, especially where the role of social interactions is investigated., (© 2024. The Author(s).)

Light enables vision and exerts widespread effects on physiology and behavior, including regulating circadian rhythms, sleep, hormone synthesis, affective state, and cognitive processes. Appropriate lighting in animal facilities may support welfare and ensure that animals enter experiments in an appropriate physiological and behavioral state. Furthermore, proper consideration of light during experimentation is important both when it is explicitly employed as an independent variable and as a general feature of the environment. This Consensus View discusses metrics to use for the quantification of light appropriate for nonhuman mammals and their application to improve animal welfare and the quality of animal research. It provides methods for measuring these metrics, practical guidance for their implementation in husbandry and experimentation, and quantitative guidance on appropriate light exposure for laboratory mammals. The guidance provided has the potential to improve data quality and contribute to reduction and refinement, helping to ensure more ethical animal use., Competing Interests: RJL has received research grant funding from Philips Lighting/Signify and honoraria from Samsung Electronics. GCB has received research grant funding from the Nova Institute, Toshiba Materials Science, Seoul Semiconductor, BIOS, Robern, and PhotoPharmics Inc. He has a current patent (USPTO 7678140 B2) that is licensed by Litebook Company Ltd. He is a paid member of the PhotoPharmics Scientific Advisory Board; has been a paid consultant by Lutron, Inc. and McCullough Hill LLC; and has received honoraria from the Institute for Functional Medicine. BNG has received funding from Tecniplast to travel and present at a symposium. RAH has received research grant funding from Philips Lighting/Signify and is a scientific advisor for the Good Light Group and Chrono@Work. JST is a co-founder and SAB member of Synchronicity Pharma. SNP has received consulting fees from NASA Ames and Sleep Standards., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Rationale: Non-invasive home cage monitoring is emerging as a valuable tool to assess the effects of experimental interventions on mouse behaviour. A field in which these techniques may prove useful is the study of repeated selective serotonin reuptake inhibitor (SSRI) treatment and discontinuation. SSRI discontinuation syndrome is an under-researched condition that includes the emergence of sleep disturbances following treatment cessation., Objectives: We used passive infrared (PIR) monitoring to investigate changes in activity, sleep, and circadian rhythms during repeated treatment with the SSRI paroxetine and its discontinuation in mice., Methods: Male mice received paroxetine (10 mg/kg/day, s.c.) for 12 days, then were swapped to saline injections for a 13 day discontinuation period and compared to mice that received saline injections throughout. Mice were continuously tracked using the Continuous Open Mouse Phenotyping of Activity and Sleep Status (COMPASS) system., Results: Repeated paroxetine treatment reduced activity and increased behaviourally-defined sleep in the dark phase. These effects recovered to saline-control levels within 24 h of paroxetine cessation, yet there was also evidence of a lengthening of sleep bouts in the dark phase for up to a week following discontinuation., Conclusions: This study provides the first example of how continuous non-invasive home cage monitoring can be used to detect objective behavioural changes in activity and sleep during and after drug treatment in mice. These data suggest that effects of paroxetine administration reversed soon after its discontinuation but identified an emergent change in sleep bout duration, which could be used as a biomarker in future preclinical studies to prevent or minimise SSRI discontinuation symptoms., (© 2023. The Author(s).)

Photoreceptor degeneration sufficient to produce severe visual loss often spares the inner retina. This raises hope for vision restoration treatments using optogenetics or electrical stimulation, which generate a replacement light input signal in surviving neurons. The success of these approaches is dependent on the capacity of surviving circuits of the visual system to generate and propagate an appropriate visual code in the face of neuroanatomical remodeling. To determine whether retinally degenerate animals possess this capacity, we generated a transgenic mouse model expressing the optogenetic actuator ReaChR in ON bipolar cells (second-order neurons in the visual projection). After crossing this with the rd1 model of photoreceptor degeneration, we compared ReaChR-derived responses with photoreceptor-driven responses in wild-type (WT) mice at the level of retinal ganglion cells and the visual thalamus. The ReaChR-driven responses in rd1 animals showed low photosensitivity, but in other respects generated a visual code that was very similar to the WT. ReaChR rd1 responses had high trial-to-trial reproducibility and showed sensitivity normalization to code contrast across background intensities. At the single unit level, ReaChR-derived responses exhibited broadly similar variations in response polarity, contrast sensitivity, and temporal frequency tuning as the WT. Units from the WT and ReaChR rd1 mice clustered together when subjected to unsupervised community detection based on stimulus-response properties. Our data reveal an impressive ability for surviving circuitry to recreate a rich visual code following advanced retinal degeneration and are promising for regenerative medicine in the central nervous system., Competing Interests: Declaration of interests R.J.L. and J.R. are named inventors on patent applications for the use of animal opsins in optogenetics. R.J.L. has received investigator-initiated research funding from Kubota Vision and acted as a consultant for Kubota Vision., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

For nearly a century, we have known that brain photoreceptors regulate avian seasonal biology. Two photopigments, vertebrate ancient opsin (VA) and neuropsin (OPN5), provide possible molecular substrates for these photoreceptor pathways. VA fulfills many criteria for providing light input to the reproductive response, but a functional link has yet to be demonstrated. This study examined the role of VA and OPN5 in the avian photoperiodic response of Japanese quail (Coturnix japonica). Non-breeding male quail were housed under short days (6L:18D) and received an intracerebroventricular infusion of adeno-associated viral vectors with shRNAi that selectively inhibited either VA or OPN5. An empty viral vector acted as a control. Quail were then photostimulated (16L:8D) to stimulate gonadal growth. Two long days significantly increased pituitary thyrotrophin-stimulating hormone β-subunit (TSHβ) and luteinizing hormone β-subunit (LHβ) mRNA of VA shRNAi treated quail compared to controls. Furthermore, at one week there was a significant increase, compared to controls, in both hypothalamic gonadotrophin releasing hormone-I (GnRH-I) mRNA and paired testicular mass in VA shRNAi birds. Opn5 shRNAi facilitated the photoinduced increase in TSHβ mRNA at 2 days, but no other differences were identified compared to controls. Contrary to our expectations, the silencing of deep brain photoreceptors enhanced the response of the reproductive axis to photostimulation rather than preventing it. In addition, we show that VA opsin plays a dominant role in the light-dependent neuroendocrine control of seasonal reproduction in birds. Together our findings suggest the photoperiodic response involves at least two photoreceptor types and populations working together with VA opsin playing a dominant role., Competing Interests: Declaration of competing interest The authors have no relevant competing financial or non-financial interests to disclose., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Bats, quoted as sleeping for up to 20 h a day, are an often used example of extreme sleep duration amongst mammals. Given that duration has historically been one of the primary metrics featured in comparative studies of sleep, it is important that species specific sleep durations are well founded. Here, we re-examined the evidence for the characterization of bats as extreme sleepers and discuss whether it provides a useful representation of the sleep behavior of Chiroptera. Although there are a wealth of activity data to suggest that the diurnal cycle of bats is dominated by rest, estimates of sleep time generated from electrophysiological analyses suggest considerable interspecific variation, ranging from 83% to a more moderate 61% of the 24 h day spent asleep. Temperature-dependent changes in the duration and electroencephalographic profile of sleep suggest that bats represent a unique model for investigating the relationship between sleep and torpor. Further sources of intra-specific variation in sleep duration, including the impact of artificial laboratory environments and sleep intensity, remain unexplored. Future studies conducted in naturalistic environments, using larger sample sizes and relying on a pre-determined set of defining criteria will undoubtedly provide novel insights into sleep in bats and other species., (© Sleep Research Society 2022. Published by Oxford University Press on behalf of the Sleep Research Society.)

Mistimed exposure to light has been demonstrated to negatively affect multiple aspects of physiology and behavior. Here we analyzed the effects of chronic exposure to abnormal lighting conditions in mice. We exposed mice for 1 year to either: a standard light/dark cycle, a "light-pollution" condition in which low levels of light were present in the dark phase of the circadian cycle (dim light at night, DLAN), or altered light cycles in which the length of the weekday and weekend light phase differed by 6 h ("social jetlag"). Mice exhibited several circadian activity phenotypes, as well as changes in motor function, associated particularly with the DLAN condition. Our data suggest that these phenotypes might be due to changes outside the core clock. Dendritic spine changes in other brain regions raise the possibility that these phenotypes are mediated by changes in neuronal coordination outside of the clock. Given the prevalence of artificial light exposure in the modern world, further work is required to establish whether these negative effects are observed in humans as well., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Delorme, Srikanta, Fisk, Cloutier, Sato, Pothecary, Merz, Foster, Brown, Peirson, Cermakian and Banks.)

Ocular light exposure has important influences on human health and well-being through modulation of circadian rhythms and sleep, as well as neuroendocrine and cognitive functions. Prevailing patterns of light exposure do not optimally engage these actions for many individuals, but advances in our understanding of the underpinning mechanisms and emerging lighting technologies now present opportunities to adjust lighting to promote optimal physical and mental health and performance. A newly developed, international standard provides a SI-compliant way of quantifying the influence of light on the intrinsically photosensitive, melanopsin-expressing, retinal neurons that mediate these effects. The present report provides recommendations for lighting, based on an expert scientific consensus and expressed in an easily measured quantity (melanopic equivalent daylight illuminance (melaponic EDI)) defined within this standard. The recommendations are supported by detailed analysis of the sensitivity of human circadian, neuroendocrine, and alerting responses to ocular light and provide a straightforward framework to inform lighting design and practice., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: GCB was supported, in part, by DOE grant DE- EE0008207, NAS Award #HR 05-23 UNIT 905; NASA grant NNX15AC14G; the NSF ERC LESA Center; BIOS, Toshiba Materials Science, The Nova Institute (The Institute for Integrative Health) and the Philadelphia Section of the Illuminating Engineering Society. GCB reports having issued (USPTO 7,678,140 and 8,366,755) and pending (USPTO 16/252,300, 15/085,522 and 14/273,971) patents related to the photoreceptor system for melatonin regulation. That intellectual property has been licensed by Litebook Company Ltd. He has been a paid consultant by Lutron, Inc., and McCullough Hill LLC. In addition, he and his research program have received financial, material and travel support from a range of federal, industrial, legal and philanthropic organizations in the past and present. CC has received consulting, honoraria, travel, accommodation and/or subsistence fees for invited keynote lectures, conference presentations or teaching from Toshiba Materials, Velux, Firalux, Lighting Europe, Electrosuisse, Novartis, Roche, Elite, Servier, and WIR Bank. CC is a member of the Daylight Academy. CAC reports grants to BWH from FAA, NHLBI, NIA, NIOSH, NASA, and DOD; is/was a paid consultant to AARP (2018), American Academy of Dental Sleep Medicine (2017, 2018), Eisenhower Medical Center (2018), Emory University (2019), Ganésco, Inc. (2017), Inselspital Bern (2019), Institute of Digital Media and Child Development (2017, 2018, 2019), Klarman Family Foundation (2017, 2018, 2019), M. Davis and Co (2018), Physician’s Seal (2019), Samsung (2016), Sleep Research Society Foundation (2019), State of Washington Board of Pilotage Commissioners (2018), Tencent Holdings Ltd (2019), Teva Pharma Australia (2019, 2020), UC San Diego (2018), University of Michigan (2017), University of Washington (2017, 2018), and Vanda Pharmaceuticals Inc (2017, 2018, 2019, 2020), in which CAC also holds an equity interest; received travel support from Annenberg Center for Health Sciences at Eisenhower (2018), Aspen Brain Institute (2018), Bloomage International Investment Group, Inc. (2018, 2019), UK Biotechnology and Biological Sciences Research Council (2019), Bouley Botanical (2017, 2018, 2019), Dr. Stanley Ho Medical Development Foundation (2019), European Biological Rhythms Society (2017, 2019), German National Academy of Sciences (Leopoldina) (2019), Illuminating Engineering Society (2018), National Safety Council (2017, 2018, 2019), National Sleep Foundation (2017, 2018, 2019), Society for Research on Biological Rhythms (2018), Sleep Research Society Foundation (2018), Stanford Medical School Alumni Association (2019), Tencent Holdings Ltd (2019), University of Zurich (2018), and Vanda Pharmaceuticals Inc (2017, 2018, 2019), Ludwig-Maximilians-Universität München (2018), National Highway Transportation Safety Administration (2018), Office of Naval Research (2018), Salk Institute for Biological Studies/Fondation Ipsen (2018), The National Academy of Sciences, Engineering, and Medicine (2017), The Wonderful Company (2017), Department of Defense (2017); receives research/education support through BWH from Cephalon, Mary Ann & Stanley Snider via Combined Jewish Philanthropies, Harmony Biosciences LLC (2019, 2020), Jazz Pharmaceuticals PLC Inc (2017, 2018, 2019, 2020), Johnson & Johnson (2019), NeuroCare, Inc. (2019), Philips Respironics Inc/Philips Homecare Solutions (2017, 2 018, 2019, 2020), Regeneron Pharmaceuticals (2018, 2019, 2020), Regional Home Care (2019), Teva Pharmaceuticals Industries Ltd, Sanofi SA, Optum, ResMed, San Francisco Bar Pilots, Sanofi, Schneider, Simmons, Sysco, Philips, Vanda Pharmaceuticals; is/was an expert witness in legal cases, including those involving Advanced Power Technologies, Aegis Chemical Solutions LLC (2019), Amtrak (2019); Casper Sleep Inc (2019), C&J Energy Services (2019), Complete General Construction Co (2017), Dallas Police Association (2019), Enterprise Rent-A-Car (2019), Espinal Trucking/Eagle Transport Group LLC/Steel Warehouse Inc (2017, 2018, 2019), FedEx, Greyhound Lines Inc/Motor Coach Industries/FirstGroup America (2017, 2018, 2019), Pomerado Hospital/Palomar Health District (2017, 2018), PAR Electrical Contractors Inc (2019), Product & Logistics Services LLC/Schlumberger Technology Corp/Gelco Fleet Trust (2019), Puckett Emergency Medical Services LLC (2019), South Carolina Central Railroad Company LLC (2017, 2018), Union Pacific Railroad (2019), United Parcel Service/UPS Ground Freight Inc (2017, 2018), and Vanda Pharmaceuticals (2019, 2020); serves as the incumbent of an endowed professorship provided to Harvard University by Cephalon, Inc.; and receives royalties from McGraw Hill, and Philips Respironics (2017, 2018, 2019, 2020) for the Actiwatch-2 and Actiwatch Spectrum devices. CACs interests were reviewed and are managed by the Brigham and Women’s Hospital and Mass General Brigham in accordance with their conflict of interest policies. SWL has received consulting fees from Atlanta Falcons, Atlanta Hawks, BHP Billiton, Delos Living LLC, EyeJust Inc., McCullough Hill Leary PS, Noble Insights, OpTerra Energy Services Inc., Pegasus Capital Advisors, Phillips Lytle LLP, Plan LED, Rec Room, Serrado Capital, Slingshot Insights, Stantec and Team C Racing. He has current consulting contracts with Akili Interactive, Apex 2100 Ltd, Consumer Sleep Solutions, Headwaters Inc., Hintsa Performance AG, KBR Wyle Services, Light Cognitive, Lighting Science Group Corporation/HealthE, Look Optic, Mental Workout/Timeshifter, Paul Weiss Rifkind Wharton & Garrison LLP, Six Senses, and View Inc. SWL has received unrestricted equipment gifts from Bionetics Corporation and F.LUX Software LLC; a fellowship gift from Stockgrand Ltd; has equity in iSLEEP, Pty; royalties from Oxford University Press; honoraria plus travel, accommodation and/or meals for invited seminars, conference presentations or teaching from Estée Lauder, Ineos, Informa Exhibitions, MIT, Roxbury Latin School, and Teague; travel, accommodation and/or meals only (no honoraria) for invited seminars, conference presentations, teaching or editorial duties from DIN, Emory University, Lightfair, SLTBR, Solemma and Wiley. SWL has an ongoing investigator-initiated grant from F. Lux Software LLC and a Clinical Research Support Agreement from Vanda Pharmaceuticals Inc. SWL holds a process patent for ‘Systems and methods for determining and/or controlling sleep quality’, which is assigned to the Brigham and Women’s Hospital per Hospital policy, and ‘Method and system for generating and providing notifications for a circadian shift protocol’ held by Mental Workout Inc. SWL has served as a paid expert in legal proceedings related to light, health and work patterns. SWL was the Program Leader for ‘Safety and Productivity Improvements’ in the CRC for Alertness, Safety and Productivity from 2015-2019, through a part-time Adjunct Professor appointment at Monash University, Australia. SWL’s interests are reviewed and managed by Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict of interest policies. RJL and TMB have received research funding from Signify (formerly Philips Lighting). RJL has received speaker fees from Samsung. RJL, LP, LJMS and MS have served as members of the CIE Joint Technical Committee 9 on the definition of CIE S 026:2018. These were unpaid roles. JPH was supported, in part, by DOE grant DE- EE0008207, NAS Award #HR 05-23 UNIT 905; NASA grant NNX15AC14G; NSF Award #2037357; Rensselaer Polytechnic Institute; BIOS, Toshiba Materials Science, The Institute for Integrative Health; and the Philadelphia Section of the Illuminating Engineering Society. He has been a paid consultant by Lutron, Inc. and McCullough Hill LLC. MM is an unpaid founding member of the Daylight Academy (DLA). SNP reports no conflicting interests. JOH, In addition to paid employment with Public Health England, is Vice-President Standards of the CIE, a member of the Scientific Expert Group of the International Commission on Non-Ionizing Radiation Protection and a number of their Project Groups, Co-Convenor of an ISO committee on Integrative Lighting, a member of two committees of the Illuminating Engineering Society of North America, Convenor of an IEC committee and a member of a Core Group for the World Health Organization, all as unpaid roles. LP served as the CIE reporter to CIE TN 003:2015 on the first Manchester Workshop in 2013, is currently serving as Director and as Secretary of the CIE Division “Photobiology and Photochemistry”, as the CIE reporter on the CIE S 026 Toolbox (doi.org/10.25039/S026.2018.TB), as a member of CIE Joint Technical Committee 14 (working with ISO Joint Working Group 4) on Integrative Lighting, and as the CIE reporter on the second Manchester Workshop 2019 (attended by all the authors), all as unpaid roles LJMS’s full time position at Eindhoven University has been partially funded by Signify, he is also active in various unpaid roles within the International Commission on Illumination (CIE). DJS is a co-inventor on issued patents (EP1614441A1 and WO2015052207A1), a Founding member of the Daylight Academy and reports receiving research support from BBSRC and EU H2020-SC1-2020-Two-Stage-RTD, ENLIGHTENme, Innovative policies for improving citizens’ health and wellbeing addressing indoor and outdoor lighting (No. 945238). CV is an unpaid member of the Circadian Light Therapy (Inc.) and the Chronsulting Scientific Advisory Boards. In addition, CV’s research and scholarship is funded by the University of Colorado Boulder, the Colorado Clinical and Translational Sciences Institute, the National Institutes of Health, and the Department of Energy. MS is currently an unpaid member of CIE Technical Committee TC 1-98 ("A Roadmap Toward Basing CIE Colorimetry on Cone Fundamentals"). MS was an unpaid advisor to the Division Reportership DR 6-45 of Division 3 ("Publication and maintenance of the CIE S026 Toolbox"). Between 2017 and 2020, MS was elected Chair of the Color Technical Group within the Optical Society. Since 2020, MS is an elected member of the Daylight Academy, an unpaid member of the Board of Advisors of the Center for Environmental Therapeutics, and MS is a member of the Technical Advisory Board of Faurecia IRYStec Inc. Over the past two years (2019-2020), MS has received industrial research support from f.lux software LLC, Ocean Insight, and BIOS Lighting. MS reports funding from the Wellcome Trust, the Royal Society, EPSRC, Bioscientifica Trust, Fight for Sight, Freie Akademische Gesellschaft Basel, and the University of Oxford. PCZ reports funding from National Institutes of Health, Eisai, Philips, Jazz Pharmaceuticals, Technogel, Harmony Biosciences, Apnimed, X – a Division of Alphabet, Inc., Merck and Sanofi. In addition, PCZ has patent applications pending (62/038700; 15/517458). KPW reports during the conduct of the 2nd International Workshop and preparation of this manuscript being a board member of the Sleep Research Society; chair of the American Academy of Sleep Medicine Clinical Practice Guideline for the Treatment of Adults with Shift Work Disorder and Jet Lag Disorder Workgroup; receiving research support from the NIH, the Office of Naval Research, the PAC-12 conference, and consulting for Circadian Therapeutics, LTD., Circadian Biotherapies, Inc. Philips Respironics, U.S. Army Medical Research and Materiel Command-Walter Reed Army Institute of Research outside the submitted work.

During inherited retinal degenerations (IRDs), vision is lost due to photoreceptor cell death; however, a range of optogenetic tools have been shown to restore light responses in animal models. Restored response characteristics vary between tools and the neuronal cell population to which they are delivered: the interplay between these is complex, but targeting upstream neurons (such as retinal bipolar cells) may provide functional benefit by retaining intraretinal signal processing. In this study, our aim was to compare two optogenetic tools: mammalian melanopsin (hOPN4) and microbial red-shifted channelrhodopsin (ReaChR) expressed within two subpopulations of surviving cells in a degenerate retina. Intravitreal adeno-associated viral vectors and mouse models utilising the Cre/lox system restricted expression to populations dominated by bipolar cells or retinal ganglion cells and was compared with non-targeted delivery using the chicken beta actin (CBA) promoter. In summary, we found bipolar-targeted optogenetic tools produced faster kinetics and flatter intensity-response relationships compared with non-targeted or retinal-ganglion-cell-targeted hOPN4. Hence, optogenetic tools of both mammalian and microbial origins show advantages when targeted to bipolar cells. This demonstrates the advantage of bipolar-cell-targeted optogenetics for vision restoration in IRDs. We therefore developed a bipolar-cell-specific gene delivery system employing a compressed promoter with the potential for clinical translation., Competing Interests: M.L. has received financial support from Heidelberg Engineering, Optos, and Genentech and is an equity owner of Fresenius Medical Care. The other authors declare no competing interests., (© 2022 The Author(s).)

Light is known to exert powerful effects on behavior and physiology, including upon the amount and distribution of activity across the day/night cycle. Here we use home cage activity monitoring to measure the effect of differences in home cage light spectrum and intensity on key circadian activity parameters in mice. Due to the relative positioning of any individually ventilated cage (IVC) with regard to the animal facility lighting, notable differences in light intensity occur across the IVC rack. Although all mice were found to be entrained, significant differences in the timing of activity onset and differences in activity levels were found between mice housed in standard versus red filtering cages. Furthermore, by calculating the effective irradiance based upon the known mouse photopigments, a significant relationship between light intensity and key circadian parameters are shown. Perhaps unsurprisingly given the important role of the circadian photopigment melanopsin in circadian entrainment, melanopic illuminance is shown to correlate more strongly with key circadian activity parameters than photopic lux. Collectively, our results suggest that differences in light intensity may reflect an uncharacterized source of variation in laboratory rodent research, with potential consequences for reproducibility. Room design and layout vary within and between facilities, and caging design and lighting location relative to cage position can be highly variable. We suggest that cage position should be factored into experimental design, and wherever possible, experimental lighting conditions should be characterized as a way of accounting for this source of variation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Steel, Tir, Tam, Bussell, Spitschan, Foster and Peirson.)

Over the last decades remarkable advances have been made in the understanding of the photobiology of circadian rhythms. The identification of a third photoreceptive system in the mammalian eye, in addition to the rods and cones that mediate vision, has transformed our appreciation of the role of light in regulating physiology and behavior. These photosensitive retinal ganglion cells (pRGCs) express the blue-light sensitive photopigment melanopsin and project to the suprachiasmatic nuclei (SCN)-the master circadian pacemaker-as well as many other brain regions. Much of our understanding of the fundamental mechanisms of the pRGCs, and the processes that they regulate, comes from mouse and other rodent models. Here we describe the contribution of rodent models to circadian photobiology, including both their strengths and limitations. In addition, we discuss how an appreciation of both rodent and human data is important for translational circadian photobiology. Such an approach enables a bi-directional flow of information whereby an understanding of basic mechanisms derived from mice can be integrated with studies from humans. Progress in this field is being driven forward at several levels of analysis, not least by the use of personalized light measurements and photoreceptor specific stimuli in human studies, and by studying the impact of environmental, rather than laboratory, lighting on different rodent models., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Dysfunction of the glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA1 subunit and deficits in synaptic plasticity are implicated in schizophrenia and sleep and circadian rhythm disruption. To investigate the role of GluA1 in circadian and sleep behaviour, we used wheel-running, passive-infrared, and video-based home-cage activity monitoring to assess daily rest-activity profiles of GluA1-knockout mice (Gria1 -/- ). We showed that these mice displayed various circadian abnormalities, including misaligned, fragmented, and more variable rest-activity patterns. In addition, they showed heightened, but transient, behavioural arousal to light→dark and dark→light transitions, as well as attenuated nocturnal-light-induced activity suppression (negative masking). In the hypothalamic suprachiasmatic nuclei (SCN), nocturnal-light-induced cFos signals (a molecular marker of neuronal activity in the preceding ~1-2 h) were attenuated, indicating reduced light sensitivity in the SCN. However, there was no change in the neuroanatomical distribution of expression levels of two neuropeptides-vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP)-differentially expressed in the core (ventromedial) vs. shell (dorsolateral) SCN subregions and both are known to be important for neuronal synchronisation within the SCN and circadian rhythmicity. In the motor cortex (area M1/M2), there was increased inter-individual variability in cFos levels during the evening period, mirroring the increased inter-individual variability in locomotor activity under nocturnal light. Finally, in the spontaneous odour recognition task GluA1 knockouts' short-term memory was impaired due to enhanced attention to the recently encountered familiar odour. These abnormalities due to altered AMPA-receptor-mediated signalling resemble and may contribute to sleep and circadian rhythm disruption and attentional deficits in different modalities in schizophrenia., (© 2021. The Author(s).)

Light provides the primary signal for entraining circadian rhythms to the day/night cycle. In addition to rods and cones, the retina contains a small population of photosensitive retinal ganglion cells (pRGCs) expressing the photopigment melanopsin (OPN4). Concerns have been raised that exposure to dim artificial lighting in the evening (DLE) may perturb circadian rhythms and sleep patterns, and OPN4 is presumed to mediate these effects. Here, we examine the effects of 4-h, 20-lux DLE on circadian physiology and behavior in mice and the role of OPN4 in these responses. We show that 2 wk of DLE induces a phase delay of ∼2 to 3 h in mice, comparable to that reported in humans. DLE-induced phase shifts are unaffected in Opn4 -/- mice, indicating that rods and cones are capable of driving these responses in the absence of melanopsin. DLE delays molecular clock rhythms in the heart, liver, adrenal gland, and dorsal hippocampus. It also reverses short-term recognition memory performance, which is associated with changes in preceding sleep history. In addition, DLE modifies patterns of hypothalamic and cortical cFos signals, a molecular correlate of recent neuronal activity. Together, our data show that DLE causes coordinated realignment of circadian rhythms, sleep patterns, and short-term memory process in mice. These effects are particularly relevant as DLE conditions-due to artificial light exposure-are experienced by the majority of the populace on a daily basis., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Light plays a critical role in regulating physiology and behavior, including both visual and non-visual responses. In mammals, loss of both eyes abolishes all of these responses, demonstrating that the photoreceptors involved are exclusively ocular. By contrast, many non-mammalian species possess extra-ocular photoreceptors located in the pineal complex and deep brain. Whilst there have been suggestions of extra-ocular photoreception in mammals, including man, evidence for these photoreceptors is limited. One approach to objectively determine the presence of such receptors is to measure brain responses to light using functional magnetic resonance imaging (fMRI). Moreover, by using participants who are clinically anophthalmic (congenital and acquired), it is possible to investigate potential light detection in the absence of the retina. Here we scanned participants with anophthalmia and sighted participants in 4 different conditions; the first 3 conditions had a bright light source applied to the following locations: behind the right ear ("ear"), just below the nasal bridge and between the eyes ("head"), and at the right popliteal fossa ("knee"). In the fourth and final scan, the light source was switched off so that there was no light stimulus. All participants were scanned in a completely dark room. No consistent brain activity was detected during any of the light conditions in either sighted controls or anophthalmic participants. Thus, we do not provide any evidence for the presence of extraocular photoreceptors modulating human brain activity, despite recent evidence for gene transcription that may occur as a result of these photoreceptors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bridge, Morjaria, Peirson, Coullon, Warnaby, Pothecary, Leatherbarrow, Foster and Downes.)

Study Objectives: Torpor is a regulated and reversible state of metabolic suppression used by many mammalian species to conserve energy. Whereas the relationship between torpor and sleep has been well-studied in seasonal hibernators, less is known about the effects of fasting-induced torpor on states of vigilance and brain activity in laboratory mice., Methods: Continuous monitoring of electroencephalogram (EEG), electromyogram (EMG), and surface body temperature was undertaken in adult, male C57BL/6 mice over consecutive days of scheduled restricted feeding., Results: All animals showed bouts of hypothermia that became progressively deeper and longer as fasting progressed. EEG and EMG were markedly affected by hypothermia, although the typical electrophysiological signatures of non-rapid eye movement (NREM) sleep, rapid eye movement (REM) sleep, and wakefulness enabled us to perform vigilance-state classification in all cases. Consistent with previous studies, hypothermic bouts were initiated from a state indistinguishable from NREM sleep, with EEG power decreasing gradually in parallel with decreasing surface body temperature. During deep hypothermia, REM sleep was largely abolished, and we observed shivering-associated intense bursts of muscle activity., Conclusions: Our study highlights important similarities between EEG signatures of fasting-induced torpor in mice, daily torpor in Djungarian hamsters and hibernation in seasonally hibernating species. Future studies are necessary to clarify the effects on fasting-induced torpor on subsequent sleep., (© Sleep Research Society 2021. Published by Oxford University Press on behalf of the Sleep Research Society.)

Acute exposure to light exerts widespread effects on physiology, in addition to its key role in photoentrainment. Although the modulatory effect of light on physiological arousal is well demonstrated in mice, its effect on memory performance is inconclusive, as the direction of the effect depends on the nature of the behavioural task employed and/or the type of stimulus utilised. Moreover, in all rodent studies that reported significant effects of light on performance, brain activity was not assessed during the task and thus it is unclear how brain activity was modulated by light or the exact relationship between light-modulated brain activity and performance. Here we examine the modulatory effects of light of varying intensities on recognition memory performance and frontoparietal waking electroencephalography (EEG) in mice using the spontaneous recognition memory task. We report a light-intensity-dependent disruptive effect on recognition memory performance at the group level, but inspection of individual-level data indicates that light-intensity-dependent facilitation is observed in some cases. Using linear mixed-effects models, we then demonstrate that EEG fast theta (θ) activity at the time of encoding negatively predicts recognition memory performance, whereas slow gamma (γ) activity at the time of retrieval positively predicts performance. These relationships between θ/γ activity and performance are strengthened by increasing light intensity. Thus, light modulates θ and γ band activities involved in attentional and mnemonic processes, thereby affecting recognition memory performance. However, extraneous factors including the phase of the internal clock at which light is presented and homeostatic sleep pressure may determine how photic input is translated into behavioural performance., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Bipolar disorder is a chronic neuropsychiatric condition associated with mood instability, where patients present significant sleep and circadian rhythm abnormalities. Currently, the pathophysiology of bipolar disorder remains elusive, but treatment with lithium continues as the benchmark pharmacotherapy, functioning as a potent mood stabilizer in most, but not all patients. Lithium is well documented to induce period lengthening and amplitude enhancement of the circadian clock. Based on this, we sought to investigate whether lithium differentially impacts circadian rhythms in bipolar patient cell lines and crucially if lithium's effect on the clock is fundamental to its mood-stabilizing effects. We analyzed the circadian rhythms of bipolar patient-derived fibroblasts (n = 39) and their responses to lithium and three further chronomodulators. Here we show, relative to controls (n = 23), patients exhibited a wider distribution of circadian period (p < 0.05), and that patients with longer periods were medicated with a wider range of drugs, suggesting lower effectiveness of lithium. In agreement, patient fibroblasts with longer periods displayed muted circadian responses to lithium as well as to other chronomodulators that phenocopy lithium. These results show that lithium differentially impacts the circadian system in a patient-specific manner and its effect is dependent on the patient's circadian phenotype. We also found that lithium-induced behavioral changes in mice were phenocopied by modulation of the circadian system with drugs that target the clock, and that a dysfunctional clock ablates this response. Thus, chronomodulatory compounds offer a promising route to a novel treatment paradigm. These findings, upon larger-scale validation, could facilitate the implementation of a personalized approach for mood stabilization., (© 2020. The Author(s).)

Cortical and subcortical circuitry are thought to play distinct roles in the generation of sleep oscillations and global state control, respectively. Here we silenced a subset of neocortical layer 5 pyramidal and archicortical dentate gyrus granule cells in male mice by ablating SNAP25. This markedly increased wakefulness and reduced rebound of electroencephalographic slow-wave activity after sleep deprivation, suggesting a role for the cortex in both vigilance state control and sleep homeostasis., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Mutations in transcription factors often exhibit pleiotropic effects related to their complex expression patterns and multiple regulatory targets. One such mutation in the zinc finger homeobox 3 (ZFHX3) transcription factor, short circuit (Sci, Zfhx3 Sci/+ ), is associated with significant circadian deficits in mice. However, given evidence of its retinal expression, we set out to establish the effects of the mutation on retinal function using molecular, cellular, behavioral and electrophysiological measures. Immunohistochemistry confirms the expression of ZFHX3 in multiple retinal cell types, including GABAergic amacrine cells and retinal ganglion cells including intrinsically photosensitive retinal ganglion cells (ipRGCs). Zfhx3 Sci/+ mutants display reduced light responsiveness in locomotor activity and circadian entrainment, relatively normal electroretinogram and optomotor responses but exhibit an unexpected pupillary reflex phenotype with markedly increased sensitivity. Furthermore, multiple electrode array recordings of Zfhx3 Sci/+ retina show an increased sensitivity of ipRGC light responses., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Purpose: Retinal bipolar cells survive even in the later stages of inherited retinal degenerations (IRDs) and so are attractive targets for optogenetic approaches to vision restoration. However, it is not known to what extent the remodelling that these cells undergo during degeneration affects their function. Specifically, it is unclear if they are free from metabolic stress, receptive to adeno-associated viral vectors, suitable for opsin-based optogenetic tools and able to propagate signals by releasing neurotransmitter., Methods: Fluorescence activated cell sorting (FACS) was performed to isolate labelled bipolar cells from dissociated retinae of litter-mates with or without the IRD mutation Pde6b rd1/rd1 selectively expressing an enhanced yellow fluorescent protein (EYFP) as a marker in ON-bipolar cells. Subsequent mRNA extraction allowed Illumina® microarray comparison of gene expression in bipolar cells from degenerate to those of wild type retinae. Changes in four candidate genes were further investigated at the protein level using retinal immunohistochemistry over the course of degeneration., Results: A total of sixty differentially expressed transcripts reached statistical significance: these did not include any genes directly associated with native primary bipolar cell signalling, nor changes consistent with metabolic stress. Four significantly altered genes (Srm2, Slf2, Anxa7 & Cntn1), implicated in synaptic remodelling, neurotransmitter release and viral vector entry had immunohistochemical staining colocalising with ON-bipolar cell markers and varying over the course of degeneration., Conclusion: Our findings suggest relatively few gene expression changes in the context of degeneration: that despite remodelling, bipolar cells are likely to remain viable targets for optogenetic vision restoration. In addition, several genes where changes were seen could provide a basis for investigations to enhance the efficacy of optogenetic therapies., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The accumulation of adenosine is strongly correlated with the need for sleep and the detection of sleep pressure is antagonised by caffeine. Caffeine also affects the circadian timing system directly and independently of sleep physiology, but how caffeine mediates these effects upon the circadian clock is unclear. Here we identify an adenosine-based regulatory mechanism that allows sleep and circadian processes to interact for the optimisation of sleep/wake timing in mice. Adenosine encodes sleep history and this signal modulates circadian entrainment by light. Pharmacological and genetic approaches demonstrate that adenosine acts upon the circadian clockwork via adenosine A 1 /A 2A receptor signalling through the activation of the Ca 2+  -ERK-AP-1 and CREB/CRTC1-CRE pathways to regulate the clock genes Per1 and Per2. We show that these signalling pathways converge upon and inhibit the same pathways activated by light. Thus, circadian entrainment by light is systematically modulated on a daily basis by sleep history. These findings contribute to our understanding of how adenosine integrates signalling from both light and sleep to regulate circadian timing in mice.

Optogenetic strategies to restore vision in patients blind from end-stage retinal degenerations aim to render remaining retinal neurons light-sensitive. We present an innovative combination of multi-electrode array recordings together with a complex pattern-generating light source as a toolset to determine the extent to which neural retinal responses to complex light stimuli can be restored following viral delivery of red-shifted channelrhodopsin in the retinally degenerated mouse. Our data indicate that retinal output level spatiotemporal response characteristics achieved by optogenetic gene therapy closely parallel those observed for normal mice but equally reveal important limitations, some of which could be mitigated using bipolar-cell targeted gene-delivery approaches. As clinical trials are commencing, these data provide important new information on the capacity and limitations of channelrhodopsin-based gene therapies. The toolset we established enables comparing optogenetic constructs and stem-cell-based techniques, thereby providing an efficient and sensitive starting point to identify future approaches for vision restoration.

Circadian rhythms are 24-h cycles in physiology and behavior that occur in virtually all organisms. These processes are not simply driven by changes in the external environment as they persist under constant conditions, providing evidence for an internal biological clock. In mammals, this clock is located in the hypothalamic suprachiasmatic nuclei (SCN) and is based upon an intracellular mechanism composed of a transcriptional-translational feedback loop composed of a number of core clock genes. However, a clock is of no use unless it can be set to the correct time. The primary time cue for the molecular clock in the SCN is light detected by the eye. The photoreceptors involved in this process include the rods and cones that mediate vision, as well as the recently identified melanopsin-expressing photosensitive retinal ganglion cells (pRGCs). Light information is conveyed to the SCN via the retinohypothalamic tract, resulting in an intracellular signaling cascade which converges on cAMP-response elements in the promoters of several key clock genes. Over the last two decades a number of studies have investigated the transcriptional response of the SCN to light stimuli with the aim of further understanding these molecular signaling pathways. Here we provide an overview of these studies and provide protocols for studying the molecular responses to light in the SCN clock.

Body temperature is an important physiological parameter in many studies of laboratory mice. Continuous assessment of body temperature has traditionally required surgical implantation of a telemeter, but this invasive procedure adversely impacts animal welfare. Near-infrared thermography provides a non-invasive alternative by continuously measuring the highest temperature on the outside of the body (T skin ), but the reliability of these recordings as a proxy for continuous core body temperature (T core ) measurements has not been assessed. Here, T core (30 s resolution) and T skin (1 s resolution) were continuously measured for three days in mice exposed to ad libitum and restricted feeding conditions. We subsequently developed an algorithm that optimised the reliability of a T skin -derived estimate of T core . This identified the average of the maximum T skin per minute over a 30-min interval as the optimal way to estimate T core . Subsequent validation analyses did however demonstrate that this T skin -derived proxy did not provide a reliable estimate of the absolute T core due to the high between-animal variability in the relationship between T skin and T core . Conversely, validation showed that T skin -derived estimates of T core reliably describe temporal patterns in physiologically-relevant T core changes and provide an excellent measure to perform within-animal comparisons of relative changes in T core .

The suprachiasmatic nucleus (SCN) is a complex structure dependent upon multiple mechanisms to ensure rhythmic electrical activity that varies between day and night, to determine circadian adaptation and behaviours. SCN neurons are exposed to glutamate from multiple sources including from the retino-hypothalamic tract and from astrocytes. However, the mechanism preventing inappropriate post-synaptic glutamatergic effects is unexplored and unknown. Unexpectedly we discovered that TRESK, a calcium regulated two-pore potassium channel, plays a crucial role in this system. We propose that glutamate activates TRESK through NMDA and AMPA mediated calcium influx and calcineurin activation to then oppose further membrane depolarisation and rising intracellular calcium. Hence, in the absence of TRESK, glutamatergic activity is unregulated leading to membrane depolarisation, increased nocturnal SCN firing, inverted basal calcium levels and impaired sensitivity in light induced phase delays. Our data reveals TRESK plays an essential part in SCN regulatory mechanisms and light induced adaptive behaviours.

The 24-hr cycle of activity and sleep provides perhaps the most familiar example of circadian rhythms. In mammals, circadian activity rhythms are generated by a master biological clock located in the hypothalamic suprachiasmatic nuclei (SCN). This clock is synchronized (entrained) to the external light environment via light input from retinal photoreceptors. However, sleep is not a simple circadian output and also is regulated by a homeostatic process whereby extended wakefulness increases the need for subsequent sleep. As such, the amount and distribution of sleep depends upon the interaction between both circadian and homeostatic processes. Moreover, the study of circadian activity and sleep is not confined only to these specialized fields. Sleep and circadian rhythm disruption is common in many conditions, ranging from neurological and metabolic disorders to aging. Such disruption is associated with a range of negative consequences including cognitive impairment and mood disorders, as well as immune and metabolic dysfunction. As circadian activity and sleep are hallmarks of normal healthy physiology, they also provide valuable welfare indicators. However, traditional methods for the monitoring of circadian rhythms and sleep in mice can require separate specialized resources as well as significant expertise. Here, we outline a low-cost, non-invasive, and open-source method for the simultaneous assessment of circadian activity and sleep in mice. This protocol describes both the assembly of the hardware used and the capture and analysis of data without the need for expertise in electronics or data processing. © 2020 Wiley Periodicals LLC. Basic Protocol: Assembly of a PIR system for basic activity and sleep recordings Alternate Protocol: Data collection using Raspberry Pi Support Protocol: Circadian analysis using PIR sensors., (© 2020 Wiley Periodicals LLC.)

Switches between global sleep and wakefulness states are believed to be dictated by top-down influences arising from subcortical nuclei. Using forward genetics and in vivo electrophysiology, we identified a recessive mouse mutant line characterized by a substantially reduced propensity to transition between wake and sleep states with an especially pronounced deficit in initiating rapid eye movement (REM) sleep episodes. The causative mutation, an Ile102Asn substitution in the synaptic vesicular protein, VAMP2, was associated with morphological synaptic changes and specific behavioral deficits, while in vitro electrophysiological investigations with fluorescence imaging revealed a markedly diminished probability of vesicular release in mutants. Our data show that global shifts in the synaptic efficiency across brain-wide networks leads to an altered probability of vigilance state transitions, possibly as a result of an altered excitability balance within local circuits controlling sleep-wake architecture., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Light around twilight provides the primary entrainment signal for circadian rhythms. Here we review the mechanisms and responses of the mouse and human circadian systems to light. Both utilize a network of photosensitive retinal ganglion cells (pRGCs) expressing the photopigment melanopsin (OPN4). In both species action spectra and functional expression of OPN4 in vitro show that melanopsin has a λ max close to 480 nm. Anatomical findings demonstrate that there are multiple pRGC sub-types, with some evidence in mice, but little in humans, regarding their roles in regulating physiology and behavior. Studies in mice, non-human primates and humans, show that rods and cones project to and can modulate the light responses of pRGCs. Such an integration of signals enables the rods to detect dim light, the cones to detect higher light intensities and the integration of intermittent light exposure, whilst melanopsin measures bright light over extended periods of time. Although photoreceptor mechanisms are similar, sensitivity thresholds differ markedly between mice and humans. Mice can entrain to light at approximately 1 lux for a few minutes, whilst humans require light at high irradiance (>100's lux) and of a long duration (>30 min). The basis for this difference remains unclear. As our retinal light exposure is highly dynamic, and because photoreceptor interactions are complex and difficult to model, attempts to develop evidence-based lighting to enhance human circadian entrainment are very challenging. A way forward will be to define human circadian responses to artificial and natural light in the "real world" where light intensity, duration, spectral quality, time of day, light history and age can each be assessed.

Twenty-four hour rhythms of physiology and behavior are driven by the environment and an internal endogenous timing system. Daily restricted feeding (RF) in nocturnal rodents during their inactive phase initiates food anticipatory activity (FAA) and a reorganization of the typical 24-hour sleep-wake structure. Here, we investigate the effects of daytime feeding, where food access was restricted to 4 hours during the light period ZT4-8 (Zeitgeber time; ZT0 is lights on), on sleep-wake architecture and sleep homeostasis in mice. Following 10 days of RF, mice were returned to ad libitum feeding. To mimic the spontaneous wakefulness associated with FAA and daytime feeding, mice were then sleep deprived between ZT3-6. Although the amount of wake increased during FAA and subsequent feeding, total wake time over 24 hours remained stable as the loss of sleep in the light phase was compensated for by an increase in sleep in the dark phase. Interestingly, sleep that followed spontaneous wake episodes during the dark period and the extended period of wake associated with FAA, exhibited lower levels of slow-wave activity (SWA) when compared to baseline or after sleep deprivation, despite a similar duration of waking. This suggests an evolutionary mechanism of reducing sleep drive during negative energy balance to enable greater arousal for food-seeking behaviors. However, the total amount of sleep and SWA accumulated during the 24 hours was similar between baseline and RF. In summary, our study suggests that despite substantial changes in the daily distribution and quality of wake induced by RF, sleep homeostasis is maintained., (© Sleep Research Society 2019. Published by Oxford University Press [on behalf of the Sleep Research Society].)

Purpose: The role of light exposure in accelerating retinitis pigmentosa (RP) remains controversial. Faster degeneration has however been observed in the inferior than superior retina in several forms ("sector" RP), including those caused by the rhodopsin P23H mutation, suggesting a modifying role of incident light exposure in such cases. Rearing of equivalent animal models in complete darkness has been shown to slow the degeneration. Here we investigate the use of red filters as a potential treatment strategy, with the hypothesis that minimizing retinal exposure to light <600 nm to which rods are maximally sensitive may provide therapeutic benefit., Methods: Knockin mice heterozygous for the P23H dominant rhodopsin mutation (RhoP23H/+) housed in red-tinted plastic cages were divided at weaning into either untinted or red-tinted cages. Subsequently, photoreceptor layer (PRL) thickness was measured by spectral-domain ocular coherence tomography, retinal function quantified by ERG, and cone morphology determined by immunohistochemical analysis (IHC) of retinal flatmounts., Results: Mice remaining in red-tinted cages had a significantly greater PRL thickness than those housed in untinted cages at all time points. Red housing also led to a highly significant rescue of retinal function as determined by both dark- and light-adapted ERG responses. IHC further revealed a dramatic benefit on cone morphology and number in the red- as compared with the clear-housed group., Conclusions: Limitation of short-wavelength light exposure significantly slows degeneration in the RhoP23H/+ mouse model. Red filters may represent a cost-effective and low-risk treatment for patients with rod-cone dystrophy in whom a sectoral phenotype is noted.

Circadian rhythms are approximately 24 h cycles in physiology and behaviour that enable organisms to anticipate predictable rhythmic changes in their environment. These rhythms are a hallmark of normal healthy physiology, and disruption of circadian rhythms has implications for cognitive, metabolic, cardiovascular and immune function. Circadian disruption is of increasing concern, and may occur as a result of the pressures of our modern 24/7 society-including artificial light exposure, shift-work and jet-lag. In addition, circadian disruption is a common comorbidity in many different conditions, ranging from aging to neurological disorders. A key feature of circadian disruption is the breakdown of robust, reproducible rhythms with increasing fragmentation between activity and rest. Circadian researchers have developed a range of methods for estimating the period of time series, typically based upon periodogram analysis. However, the methods used to quantify circadian disruption across the literature are not consistent. Here we describe a range of different measures that have been used to measure circadian disruption, with a particular focus on laboratory rodent data. These methods include periodogram power, variability in activity onset, light phase activity, activity bouts, interdaily stability, intradaily variability and relative amplitude. The strengths and limitations of these methods are described, as well as their normal ranges and interrelationships. Whilst there is an increasing appreciation of circadian disruption as both a risk to health and a potential therapeutic target, greater consistency in the quantification of disrupted rhythms is needed.

Circadian rhythms and sleep-wake history determine sleep duration and intensity, and influence subsequent waking. Previous studies have shown that T cycles - light-dark (LD) cycles differing from 24 h - lead to acute changes in the daily amount and distribution of waking and sleep. However, little is known about the long-term effects of T cycles. Here we performed continuous 10 day recording of electroencephalography (EEG), locomotor activity and core body temperature in C57BL/6 mice under a T20 cycle, to investigate spontaneous sleep and waking at baseline compared with when the circadian clock was misaligned and then re-aligned with respect to the external LD cycle. We found that the rhythmic distribution of sleep was abolished during misalignment, while the time course of EEG slow wave activity (1-4 Hz) was inverted compared to baseline. Although the typical light-dark distribution of NREM sleep was re-instated when animals were re-aligned, slow wave activity during NREM sleep showed an atypical increase in the dark phase, suggesting a long-term effect of T cycles on sleep intensity. Our data show that circadian misalignment results in previously uncharacterised long-term effects on sleep, which may have important consequences for behaviour.

Melanopsin is a blue light-sensitive opsin photopigment involved in a range of non-image forming behaviours, including circadian photoentrainment and the pupil light response. Many naturally occurring genetic variants exist within the human melanopsin gene (OPN4), yet it remains unclear how these variants affect melanopsin protein function and downstream physiological responses to light. Here, we have used bioinformatic analysis and in vitro expression systems to determine the functional phenotypes of missense human OPN4 variants. From 1242 human OPN4 variants collated in the NCBI Short Genetic Variation database (dbSNP), we identified 96 that lead to non-synonymous amino acid substitutions. These 96 missense mutations were screened using sequence alignment and comparative approaches to select 16 potentially deleterious variants for functional characterisation using calcium imaging of melanopsin-driven light responses in HEK293T cells. We identify several previously uncharacterised OPN4 mutations with altered functional properties, including attenuated or abolished light responses, as well as variants demonstrating abnormal response kinetics. These data provide valuable insight into the structure-function relationships of human melanopsin, including several key functional residues of the melanopsin protein. The identification of melanopsin variants with significantly altered function may serve to detect individuals with disrupted melanopsin-based light perception, and potentially highlight those at increased risk of sleep disturbance, circadian dysfunction, and visual abnormalities.