Bright light increases alertness and not cortisol in healthy men:A forced desynchrony study under dim and bright light (I)

Light-induced improvements in alertness are more prominent during nighttime than during the day, suggesting that alerting effects of light may depend on internal clock time or wake duration. Relative contributions of both factors can be quantified using a forced desynchrony (FD) designs. FD designs have only been conducted under dim light conditions (<10 lux) since light above this amount can induce non-uniform phase progression of the circadian pacemaker (also called relative coordination). This complicates the mathematical separation of circadian clock phase from homeostatic sleep pressure effects. Here we investigate alerting effects of light in a novel 4 × 18 h FD protocol (5 h sleep, 13 h wake) under dim (6 lux) and bright light (1300 lux) conditions. Hourly saliva samples (melatonin and cortisol assessment) and 2-hourly test sessions were used to assess effects of bright light on subjective and objective alertness (electroencephalography and performance). Results reveal (1) stable free-running cortisol rhythms with uniform phase progression under both light conditions, suggesting that FD designs can be conducted under bright light conditions (1300 lux), (2) subjective alerting effects of light depend on elapsed time awake but not circadian clock phase, while (3) light consistently improves objective alertness independent of time awake or circadian clock phase. Reconstructing the daily time course by combining circadian clock phase and wake duration effects indicates that performance is improved during daytime, while subjective alertness remains unchanged. This suggests that high-intensity indoor lighting during the regular day might be beneficial for mental performance, even though this may not be perceived as such

A Computational Study of Sleep and the Hemispheres of the Brain

Sleep and sleep cycles have been studied for over a century, and scientists have worked on modeling sleep for nearly as long as computers have existed. Despite this extensive study, sleep still holds many mysteries. Larger and more extensive sleep-wake models have been developed, and the circadian drive has been depicted in numerous fashions, as well as incorporated into scores of studies. With the ever-growing knowledge of sleep comes the need to find more ways to examine, quantify, and define it in the context of the most complex part of the human anatomy – the brain. Presented here is the development of a computational model that explores the activity of individual neurons, modeled with coupled nonlinear ordinary differential equations, in key sleep-related brain regions. The activity patterns of the individual neurons are studied, as well as their synchronization with other neurons within the same region. The model is expanded into two separate interacting hemispheres, whose activity and synchronization reveal chimera-like activity. Multiple different perspectives on jetlag are presented, exploring the impact of circadian rhythm changes. Unihemispheric sleep, the unusual form of sleep exhibited by some ocean creatures and species of birds, is observed, as well as asymmetric sleep, which occurs in human subjects suffering from sleep apnea. These investigations provide a new perspective on the intricate balance between the neural activity in different brain regions that drives the essential phenomenon that is sleep

A computational study of sleep and the hemispheres of the brain

Sleep and sleep cycles have been studied for over a century, and scientists have worked on modeling sleep for nearly as long as computers have existed. Despite this extensive study, sleep still holds many mysteries. Larger and more extensive sleep-wake models have been developed, and the circadian drive has been depicted in numerous fashions, as well as incorporated into scores of studies. With the ever-growing knowledge of sleep comes the need to find more ways to examine, quantify, and define it in the context of the most complex part of the human anatomy -- the brain. Presented here is the development of a computational model that explores the activity of individual neurons, modeled with coupled nonlinear ordinary differential equations, in key sleep-related brain regions. The activity patterns of the individual neurons are studied, as well as their synchronization with other neurons within the same region. The model is expanded into two separate interacting hemispheres, whose activity and synchronization reveal chimera-like activity. Multiple different perspectives on jetlag are presented, exploring the impact of circadian rhythm changes. Unihemispheric sleep, the unusual form of sleep exhibited by some ocean creatures and species of birds, is observed, as well as asymmetric sleep, which occurs in human subjects suffering from sleep apnea. These investigations provide a new perspective on the intricate balance between the neural activity in different brain regions that drives the essential phenomenon that is sleep --Abstract, page iii

Avian Sleep Homeostasis: Electrophysiological, Molecular and Evolutionary Approaches

The function of slow wave sleep (SWS) and rapid eye movement (REM) sleep in mammals is an unanswered question in neuroscience. Aside from mammals, only birds engage in these states. Because birds independently evolved SWS and REM sleep, the study of sleeping birds may help identify shared traits related to the function of these states. Throughout this dissertation, we apply such a bird’s perspective to the sleeping brain. We begin with a review on knowledge gained through the study of sleep in animals (Chapter 1). Next, we present results from the first electrophysiological study of sleep in the most basal group of living birds by studying ostriches (Chapter 2). Although ostriches engage in unequivocal SWS, their REM sleep electrophysiology is unique and resembles features of REM sleep present only in basal mammals. Thus, the evolution REM sleep may have followed a recurring sequence of steps in mammals and birds. The remaining chapters deal with the regulation of sleep (or sleep homeostasis). Sleep homeostasis refers to an increase in the intensity of sleep (typically quantified as slow wave activity, SWA) following an extended period of wakefulness. Although such a response has long been known to occur in mammals, it has been unclear whether birds are capable of similar changes in SWA following sleep loss. We provide the first experimental evidence for a mammalian-like increase in SWA following enforced wakefulness in birds (Chapter 3). In mammals, SWA increases locally in brain regions used more during prior wakefulness. To see if SWS is regulated locally in birds, we stimulated one part of the pigeon brain during enforced wakefulness and observed a local increase in SWA during subsequent sleep (Chapter 4). Brain regions not stimulated asymmetrically during wakefulness showed a symmetric increase in SWA. These patterns of a/symmetry may reflect changes in the strength of synapses, as they do in mammals, because they are mirrored by changes in the slope of slow waves during SWS – a purported marker of synaptic strength. Lastly, we investigate whether local increases in SWA in birds are mediated by similar molecular mechanisms to those of mammals (Chapter 5). Surprisingly, mRNA levels of such proteins did not respond to unilateral visual stimulation during enforced wakefulness in the manner predicted based on work derived from mammals, but further study is needed to resolve the meaning of this difference. Overall, this dissertation presents several novel findings on the evolution and regulation of avian sleep

From disease genes to behavioural screen in zebrafish: early onset Alzheimer’s as case study

To design prevention strategies and disease-modifying therapies in Alzheimer’s disease, we must discover biological processes which contribute to disease. Genomic studies can point to such causal processes, but their findings are rarely exploited in a systematic, hypothesis-free manner. In this thesis, we present a strategy in zebrafish to link disease-associated genes to likely causal processes. The first step is to inactivate each gene in zebrafish larvae. For this purpose, we developed a rapid CRISPR-Cas9 method capable of converting wild-type eggs directly into knockout larvae for any gene of interest. The method effectively cuts the experimental time from gene to knockout zebrafish from months to one day. The second step is to monitor the behaviour of the mutated larvae. As a case study, we targeted the three genes associated with early-onset Alzheimer’s disease. We found, for example, that larvae with loss-of-function mutations in presenilin 2 are less active during the day. The third step will be to use predictive pharmacology to identify drugs which cause the same phenotype in wild- type animals, thereby pointing to the defective process. This strategy is both scalable thanks to the knockout method and generalisable beyond Alzheimer’s disease. It can now be employed to screen tens or hundreds of genes associated with other conditions, such as schizophrenia or epilepsy

Deciphering Chronometabolic Dynamics Through Metabolomics, Stable Isotope Tracers, And Genome-Scale Reaction Modeling

Synchrony across environmental cues, endogenous genetic clocks, sleep/wake cycles, and metabolism evoke physiological harmony for organismal health. Perturbation of this synchrony has been recently correlated with a growing list of pathologies, which is alarming given the ubiquity of sleep deprivation, mistimed light exposure, and altered eating schedules in modern society. Deeper insights into clocks, sleep, and metabolism are necessary to understand these outcomes. In this work, extensive metabolic profiles of circadian systems were obtained from the development of new liquid chromatography mass spectrometry (LC-MS) metabolomics methods. These methods were applied to Drosophila melanogaster to discern relative influences of environmental and genetic drivers of metabolic cycles. Unique sets of metabolites oscillated with 24-hour circadian periods under light:dark (LD) and constant darkness (DD) conditions, and ultradian rhythms were noted for clock mutant flies under LD, suggesting clock-independent metabolic cycles driven by environmental inputs. However, this metabolomic analysis does not fully capture the inherently dynamic nature of circadian metabolism. These LC-MS methods were adapted to analyze isotope enrichments from a novel 13C6 glucose injection platform in Drosophila. Metabolic flux cycles were noted from glucose carbons into serine, glutamine and reduced glutathione biosynthesis, and altered under sleep deprivation, demonstrating unique energy and redox demands in perturbed sleep/wake cycles. Global isotopolome shifts were most notable in WT flies after lights-on, suggesting a catabolic rush from glucose oxidation early in the active phase. As the scope of these isotope tracer-based metabolomic analyses expand, attributing labeling patterns to specific reactions requires consideration of genome-scale metabolic networks. A new computational approach was developed, called the IsoPathFinder, which uncovered biosynthetic paths from glucose to serine, and extends to glycine and glutathione production. Carbon flux into glutamine was predicted to occur through the TCA cycle, supported by enzyme thermodynamics and circadian expression datasets. This tool is presented as a new mechanism to simulate additional isotope tracer experiments, with broad applicability beyond circadian research. Collectively, a new set of analytical and computational tools are developed to both produce dynamic metabolomic data and improve data interpretability, with applications to uncover new chronometabolic connections

Consciousness unbound: social simulation theory of dreaming

Every night during sleep we experience an immersive world of dreams, woven together by our sleeping brain unbound by external stimulation. Despite considerable effort the question of why we dream has eluded a conclusive answer. Understanding dreams also arguably makes progress toward answering the broader question of consciousness: why do we experience anything at all? I attempt to illuminate these questions by concentrating on the quintessentially social nature of dreams. First, in Study I a novel theoretical account —the Social Simulation Theory of dreaming (SST)—is proposed, together with the first outlines of a research program for its empirical study. SST suggests the world simulation form of dreams provides clues for its function by preferentially simulating certain kinds of scenariosnamely social interactions. Second, in Studies II and III specific hypotheses derived from the SST in Study I are empirically evaluated. These provide evidence for dreams to contain more social content than corresponding waking life and to remain so even when social interactions are removed from waking life (Sociality Bias). Furthermore, the Strengthening Hypothesis that suggests dreams serve to maintain and/or increase social bonding with close others gains partial support. The Practise and Preparation Hypothesis gained support as dreams simulated positive interactions in one fifth of dream interactions and overall simulate complex social behaviours. The Compensation Hypothesis suggests dreams simulations to increase when waking social contacts are abolished, but this was not supported in the data as dream sociality remained stable despite social seclusion. When excluded from others our dreams reconfigure to decrease simulations of interactions with strangers. However, dreams during normal day-to-day life do not preferentially simulate bond-strengthening interactions with close others. In opposition to previous findings, Study II found no differences in social dream contents between either stage of sleep or time of night. In Study III a short social seclusion showed not only differences in dream content, but also in sleep structure, with an increase in REM sleep. Third, methodological development was undertaken by, both, developing a content analysis method for extracting social episodes in narrative reports (Social Content Scale, SCS; Study II), and by assessing the validity of a novel home sleep monitor device, the Beddit Sleep Tracker (BST). While the SCS proved useful for categorizing the social features in both studies II and III, BST failed to provide accurate sleep data as measured against a polysomnogram. Overall, the development of SST and the initial empirical evidence for some of its hypotheses brings us closer to understanding the twin problems of dreaming and consciousness.Kahlitsematon tajunta: unennäön sosiaalisen simulaation teoria Nukkuvat aivomme kehittävät joka yö ajankohtaisesta aistitiedosta riippumattoman monipuolisen ja todentuntuisen kokemuksen maailmasta—unen. Kysymykseen siitä miksi koemme unia ei ole yrityksistä huolimatta vielä saatu kattavaa vastausta. Unien luonteen ymmärtäminen toisi meitä todennäköisesti lähemmäs myös suuremman, tajunnan luonnetta koskevan kysymyksen ratkaisua: miksi ylipäänsä koemme mitään? Pyrin valottamaan näitä kysymyksiä keskittymällä erityisesti unien sosiaaliseen luonteeseen. Osatutkimuksessa I kehitämme uuden sosiaalisen simulaation teorian (SST) sekä esittelemme tutkimusohjelman sen väitteiden empiiriseen arviointiin. SST hyödyntää näkemystä unien maailma-simulaatio-muodosta ymmärtääkseen niiden funktiota, keskittyen erityisesti unien taipumukseen painottaa sosiaalisten tilanteiden simulointia. Osatutkimuksissa II ja III tutkimme SST:n hypoteeseja empiirisen unitutkimuksen keinoin. Sosiaalisuusvinouma unista poikkeuksellisen sosiaalisina kokemuksina saa vahvistusta löydöksestä, jossa unissa havaitaan olevan merkittävästi vastaavaa valvetta enemmän sosiaalisia tilanteita (II), ja vaikka sosiaalisia tilanteita ei esiintyisi arjessa, pysyy niiden määrä unissa ennallaan. Lisäksi vahvistushypoteesi, jonka mukaan unet vahvistavat erityisesti läheisiä ihmissuhteitamme, saa osittaista tukea. Osatutkimus III:ssa lyhyt sosiaalinen eristys johtaa muutoksiin unihahmojen luonteessa, unien alkaessa sisältää vähemmän tuntemattomien kanssa koettuja vuorovaikutustilanteita. Harjoitushypoteesi sai osin tukea unien simuloidessa monimutkaisia, ja viidenneksen positiivisia vuorovaikutustilanteita. Kompensaatio-hypoteesin mukaan vuorovaikutusunet lisääntyvät arjen sosiaalisten suhteiden poistuessa, mutta tämä ei saanut tukea unisosiaalisuuden pysyessä entisellään eristyksestä huolimatta. Normaalisti unissa emme kuitenkaan erityisesti simuloi vahvistavia vuorovaikutustilanteita läheisten kanssa, eivätkä unien sosiaaliset sisällöt eroa univaiheen tai nukkumisen keston mukaan (II). Sosiaalinen eristys kuitenkin lisäsi myös REM-unen osuutta. Lopuksi, väitöskirjassa menetelmäkehitystä edistettiin sekä luomalla uusi sisällönanalyysimenetelmä sosiaalisten tilanteiden luokitteluun (SCS) että tutkimalla unta mittaavan Beddit-unimittarin (BST) tarkkuutta mitata nukkumista ja univaiheita. Siinä missä SCS osoittautui käyttökelpoiseksi menetelmäksi sosiaalisten tilanteiden kategorisointiin, BST ei kyennyt esittämään luotettavaa tietoa unimuuttujista verrattuna unipolygrafiaan. Lopputulemana, SST ja sen ensimmäisten hypoteesien tutkimus tuo meidät lähemmäs unennäön ja tajunnan kaksoisongelmien ratkaisua

Exploration of neuronal ensembles responsible for sleep and body temperature regulation

Sleep is a behaviour we experience every day but the fundamental function(s) and the neuronal circuitry underlying it remain mystery. Previous study from our laboratory suggests the lateral preoptic (LPO) area of the hypothalamus plays an important role in recovery sleep (RS) after sleep deprivation (SD) as well as 2-adrenergic agonist (dexmedetomidine)-induced sedation and hypothermia. Preliminary data of whole-brain mapping of the neuronal activity of 5-hr SD mice and 2- hr RS (followed 5-hr SD) mice by cFos expression confirmed that the LPO shows higher neuronal activity in RS mice compared to SD mice. Cell-type specific ablation of galaninergic neurons in the LPO abolished sleep homeostasis in mice, in terms of the amount of RS as well as the increased slow wave activity (SWA) of RS after SD which is a hallmark of sleep homeostasis. In addition, mice with ablation of LPOGal neurons have a permanent elevation in their body temperature compared to control mice. LPOGal neurons are also involved in mediating dexmedetomidine (DEX)-induced sedation and hypothermia. Mice without LPOGal neurons have reduced effects: administration of DEX cannot induce high-power oscillations or sustained hypothermia. Together, LPOGal neurons unite sleep homeostasis and 2-adrenergic sedation. Preliminary whole-brain cFos mapping also revealed a few other potential brain regions that might be involved in sleep/wake regulation, including the ventral tegmental area (VTA). Chemogenetic activation and inactivation increase the neuronal activity of VTAVglut2 and VTAVgat neurons, respectively, and both increase wakefulness. VTAVglut2/Nos1 neurons promote wakefulness by sending excitatory projections to both the lateral hypothalamus (LH) and nucleus accumbens (NAc), whereas the wake-inhibiting effect of VTAVgat neurons is achieved by sending inhibitory projections to local VTAVglut2 and VTADA neurons as well as to the orexin neurons in the LH, implying the significance of the VTA in sleep/wake regulation.Open Acces

Extracting circadian clock information from a single time point assay

A working internal circadian clock allows a healthy organism to keep time in order to anticipate transitions between night and day, allowing the temporal optimisation and control of internal processes. The internal circadian clock is regulated by a set of core genes that form a tightly coupled oscillator system. These oscillators are autonomous and robust to noise, but can be slowly reset by external signals that are processed by the master clock in the brain. In this thesis we explore the robustness of a tightly coupled oscillator model of the circadian clock, and show that its deterministic and stochastic forms are both significantly robust to noise. Using a simple linear algebra approach to rhythmicity detection, we show that a small set of circadian clock genes are rhythmic and synchronised in mouse tissues, and rhythmic and synchronised in a group of human individuals. These sets of tightly regulated, robust oscillators, are genes that we use to de ne the expected behaviour of a healthy circadian clock. We use these “time fingerprints" to design a model, dubbed “Time-Teller", that can be used to tell the time from single time point samples of mouse or human transcriptome. The dysfunction of the molecular circadian clock is implicated in several major diseases and there is significant evidence that disrupted circadian rhythm is a hallmark of many cancers. Convincing results showing the dysfunction of the circadian clock in solid tumours is lacking due to the difficulties of studying circadian rhythms in tumours within living mammals. Instead of developing biological assays to study this, we take advantage of the design of Time-Teller, using its underlying features to build a metric, ϴ, that indicates dysfunction of the circadian clock. We use Time-Teller to explore the clock function of samples from existing, publicly available tumour transcriptome data. Although multiple algorithms have been published with the aims of “time-telling" using transcriptome data, none of them have been reported to be able to tell the times of single samples, or provide metrics of clock dysfunction in single samples. Time-Teller is presented in this thesis as an algorithm that both tells the time of a single time-point sample, and provides a measure of clock function for that sample. In a case study, we use the clock function metric, , as a retrospective prognostic marker for breast cancer using data from a completed clinical trial. ϴ is shown to correlate with many prognostic markers of breast cancer, and we show how could also be a predictive marker for treatment efficacy and patient survival

Entrainment dynamics organised by global manifolds in a circadian pacemaker model

This is the final version. Available on open access from Frontiers Media via the DOI in this recordCircadian rhythms are established by the entrainment of our intrinsic body clock to periodic forcing signals provided by the external environment, primarily variation in light intensity across the day/night cycle. Loss of entrainment can cause a multitude of physiological difficulties associated with misalignment of circadian rhythms, including insomnia, excessive daytime sleepiness, gastrointestinal disturbances, and general malaise. This can occur after travel to different time zones, known as jet lag; when changing shift work patterns; or if the period of an individual’s body clock is too far from the 24-hour period of environmental cycles. We consider the loss of entrainment and the dynamics of re-entrainment in a two-dimensional variant of the Forger-Jewett-Kronauer model of the human circadian pacemaker forced by a 24-hour light/dark cycle. We explore the loss of entrainment by continuing bifurcations of one-to-one entrained orbits under variation of forcing parameters and the intrinsic clock period. We show that the severity of the loss of entrainment is dependent on the type of bifurcation inducing the change of stability of the entrained orbit, which is in turn dependent on the environmental light intensity. We further show that for certain perturbations, the model pblackicts a counter-intuitive rapid re-entrainment if the light intensity is sufficiently high. We explain this phenomenon via computation of invariant manifolds of fixed points of a 24-hour stroboscopic map and show how the manifolds organise re-entrainment times following transitions between day and night shift work.Medical Research Council (MRC)US-UK Fulbright CommissionEngineering and Physical Sciences Research Council (EPSRC)National Science Foundation (NSF