To my father, who read to me when I was young of the wonders of the world. Who showed me science was a gift for everyone. Who encouraged every step I took, even if he thought it was the wrong one. Thank you for never telling me I couldn't do something and for always making me ask questions with answers that needed to be found.

To my mother, who instilled in me such desire for knowledge and provided me with all I ever needed. For pushing me always in the right direction, even if I didn't know what direction I was going. For always believing I could achieve anything. And for understanding that though my path was winding I found a

First and foremost I would like to thank my thesis advisor, my mentor, and above all, my friend, Professor Patrice Bourgin for giving me this amazing opportunity to be part of such fascinating research. There is no end to how much I can thank him for his time, patience, and constant reassurances as I navigated my way through these four years at the university.

He has far and away exceeded any expectations I would have had, and provided me with unwavering support.

To the members of my jury, Pr. Dr. Christian Cajochen, Dr. Pierre-Hervé Luppi, Pr. Pierre Philip, Pr. Vincent Lèlievre, and Dr. Marie-Paule Felder-Schmittbuhl, thank you for taking time out of your extremely busy schedules to take part in my thesis defense.

I would like to acknowledge Dr. Carmen Schroder who encouraged me to pursue a doctoral degree in Strasbourg, and who I thank for her enormous help and support.

In learning the analytical tools of sleep I would like to thank Dr. Paul Franken at the University of Lausanne who allowed me on multiple occasions to visit his lab and discussed with me the minutiae of the rodent EEG, and the joys of programming in Pascal.

To the other members of our research team at INCI, specifically Laurent Calvel, Ludivine Choteau, Claire-Marie Gropp. Your assistance with research protocols was invaluable and a majority of this work would not have been possible without you. And most importantly thank you for always providing such a friendly atmosphere to work in. I would also like to thank Caroline Allemann and Mio Frisk for their help with different projects which form parts of this thesis.

I wish to extend special gratitude to the clinical team at the Sleep service at the University hospital and all the researchers, engineers, technicians, and other students, at INCI for always treating me kindly, and always providing answers, no matter how ridiculous my questions.

To Dr. Elisabeth Ruppert without whom I would have been a lost little lamb in the bright foreign lights of Strasbourg. You helped me adjust in so many ways I feel I might never repay you. And to Dr. Nathalie Philippi who encouraged me to leave the lab sometimes to

Acknowledgments -Introductory Materials

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go rock climbing and have a drink, and for always being attentive in listening to my ridiculous complaints about experiments not working.

To Dr. Michael Reber, fourth floor compatriot and band mate. Thank you for everything both personally and professionally and for being a friend I could always count on, whether I needed a tube of polymerase or a cup of coffee.

To Dr. Domitille Boudard and Dr. Jorge Mendoza, who guided me through life in a biology laboratory, teaching me many things, and above all giving me lasting friendships.

To my PhD compatriots, Marc-Antoine Muller and Paul Chu Sin Chung, who always gave me great excuses to escape my data analysis and have a few laughs.

And of course to all the others who helped me find my place here in Strasbourg, please know that you have my unending gratitude.

I would also like to give a special thanks to Pr. Ruth O'Hara at Stanford University who took an enormous chance on me and set me upon the track of which I now find myself completing the first step. Please know that from the bottom of my heart that nothing I have done or will do in the future would be possible without your belief in me and your mentorship from the beginning.

To all my friends back in California thanks for being there during the good times and bad, or even the boring ones where we just watched the same movie for the 20 th time.

To Catherine Saunders. Thank for all the hilarious gifts you have sent me here in France.

There were many times when I really needed a good laugh and a picture of an unhappy cat in a cowboy hat was the perfect medicine.

To my parents-in-ODZ .U\VW\QD DQG 3LRWU ']LĊNXMĊ Eardzo. For everything, for your welcoming hearts and for making me feel so quickly like I was a part of your family. I know we have many more years to spend together and now that this thesis is done I won't have to analyze data on your couch instead of eating dinner.

And to my brother, who is so much my opposite and my mirror simultaneously. Thank you for never taking me too seriously and thank you for always engaging me with lively discussions so many times over the years, whether intellectually or not. You have no idea how much it prepared me for a career in academia.

generated by an endogenous clock whose period is approximately 24 hours and is located in the suprachiasmatic nuclei (SCN, master clock). The homeostatic process (S) is characterized by an increase in the level of sleep pressure as a function of time spent awake, and its subsequent decrease with sleep. In addition light is known to play a fundamental role in the regulation of sleep and behavior via two distinct types of nonvisual effects; indirect through the resynchronization of the clock (well-characterized mechanisms), and direct, acting on the expression of sleep and wakefulness via mechanisms independent of the circadian system that remain poorly understood. These non-visual effects are mediated by melanopsin (Opn4), a photopigment expressed in a subpopulation of intrinsically photosensitive ganglion cells of the retina, but also by both rods and cones. In recent years, research into the role of melanopsin has led several research teams to revisit the non-visual effects of light. Previously our group showed that by using through a transgenic mouse model with invalidation of the melanopsin gene (Opn4 -/-), the direct non-circadian effects of light interact with process C to influence sleep and wakefulness. Analysis of EEG delta power in Opn4 -/-mice also suggested that melanopsin may be a homeostatic sleep factor [START_REF] Tsai | Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice[END_REF]), yet needed to be further established.

The focus of this doctoral thesis was to demonstrate that the non-circadian effects of light on sleep represent a major mechanism capable of regulating vigilance states, and that it can contribute significantly to the daily distribution of sleep and waking. Specifically, we addressed the following three areas:

1) The direct effects of light (mediated principally by Opn4-based phototransduction) represent a regulatory mechanism capable of maintaining an organization of sleep and waking synchronized with the light/dark cycle in the absence of a circadian pacemaker.

Abstract in English -Introductory Materials

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2) The characterization of Arvicanthis ansorgei as a novel diurnal rodent model for the study of sleep and validation of the non-circadian direct effects of light.

3) A demonstration that light and melanopsin influences NREM sleep homeostasis in both diurnal and nocturnal rodents

Results from these three projects yielded the following conclusions. (I) Whereas the primary function of the SCN was thought to be almost exclusively to be a central circadian pacemaker, our findings allow for its reclassification as a relay system for approximately 50% of the melanopsin-dependent non-circadian direct effects of light. (II) The sleep-wake cycle was considered mostly determined by the circadian clock, entrained by light. Our results demonstrate that the melanopsin-dependent direct photic input to sleep across a normal 24-hour day (12hL:12hD light-dark cycle) determines approximately 1/3 of the daily sleep and wake distribution and allows mice to maintain a functional sleep-wake cycle even in the absence of a circadian drive. (III) The diurnal rodent, arvicanthis ansorgei, is a valid model for studying the regulatory mechanisms of sleep, including the direct effects of light, and will help to create a viable translational model between laboratory rodents and basic human research for the study of sleep. (IV) The homeostatic response characterized by the increase in EEG delta as a function of time spent awake, was dramatically altered in the absence of melanopsin, and independent of genetic background, as well as present in a diurnal rodent model. This finding establishes the proof of concept that a photopigment, melanopsin, can affect sleep homeostasis. Furthermore these results indicate the presence of the first biological link between light and sleep homeostasis and completion of the experimental analysis will allow us to further understand the lightmelanopsin-sleep homeostasis link.

The work in this doctoral thesis examines from several directions, the accepted 2process model for the regulation of sleep and wake, proposing a third mechanism both independent yet interrelated. These data, suggest a more complex role for light which may affect sleep and waking from multiple aspects: indirectly on the circadian drive, and directly on the expression of sleep and waking and through modulation of the sleep homeostat. In essence these results encourage a reevaluation on the role of light in mammalian physiology. Additionally, with further validation of this model in humans, this could provide a new conceptual framework for understanding the disturbances associated with sleep disorders, as well as the way we in our individual daily lives artificially expose ourselves to light and darkness.

Résumé en français -Introductory Materials

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Résumé en français

Les troubles du sommeil affectent 15% à 30% de la population générale représentant un enjeu majeur de santé publique. En l'absence de thérapeutiques suffisamment efficaces, il est nécessaire de mieux comprendre leur physiopathologie ce qui repose sur une meilleure connaissance des régulations du sommeil. Au cours des trois dernières décennies et jusqu'à aujourd'hui, ces mécanismes de régulation du sommeil ont été conceptualisés sous la forme d'un modèle à deux processus ; circadien et homéostasique (Borbély 1982). Le processus circadien (C) est généré par une horloge endogène dont la période est d´environ 24 heures et qui est localisée dans les noyaux suprachiasmatiques (NSC, horloge principale). Le processus homéostasique (S) est caractérisé par l'augmentation du niveau de pression de sommeil en fonction du temps passé éveillé et sa diminution avec le sommeil. Ce processus est évalué en réalisant des privations de sommeil, la quantité de sommeil perdu étant récupérée lors du rebond de sommeil, ce phénomène étant au mieux caractérisé par la mesure de la puissance spectrale delta de l'EEG.

La lumière joue un rôle fondamental dans la régulation du sommeil et des comportements en exerçant deux types d'effets non-visuels; indirect en resynchronisant l'horloge (mécanismes bien caractérisés), et directs agissant sur l'expression du sommeil et de la veille via des mécanismes qui restent mal compris et indépendants du système circadien. Ces effets non-visuels sont médiés par la mélanopsine (Opn4), un photopigment exprimé dans une sous population de cellules ganglionnaires de la rétine, leur procurant une photosensibilité intrinsèque, mais aussi par les cônes et bâtonnets. La caractérisation des processus de phototransduction liés à la mélanopsine au cours de ces dernières années a permis de revisiter les effets non-visuels de la lumière. Notre équipe a ainsi montré grâce à un modèle de souris transgénique avec invalidation du gène de la mélanopsine (Opn4 -/-) que les effets directs non-circadiens de la lumière interagissent avec les processus C et S pour influencer la veille et le sommeil. L'analyse de la puissance EEG delta de souris Opn4 -/-suggérait également que la mélanopsine puisse constituer un facteur homéostasique de sommeil [START_REF] Tsai | Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice[END_REF]. Ces résultats ouvrent des perspectives importantes pour améliorer notre compréhension des régulations du sommeil et ont été discutés dans un travail de synthèse (Hubbard et al., soumis, sleep medicine reviews, 2012).

Résumé en français -Introductory Materials

| 12 L'objectif de ce travail de thèse a été de démontrer que les effets non-circadiens de la lumière sur le sommeil constituent un mécanisme majeur de régulation des états de vigilance pouvant contribuer significativement à la distribution journalière veille sommeil.

Plus spécifiquement, nous avons abordé les trois axes suivants 1) Les effets directs de la lumière constituent-ils un mécanisme capable de maintenir une organisation temporelle veille sommeil synchronisée avec le cycle de lumière obscurité en l'absence de pacemaker circadien. En répondant à cette question nous proposons aussi de démontrer si les NSCs au-delà de leur fonction d'horloge, constituent une structure de relai de ces effets. En effet, l'analyse de modèles de souris sans horloge centrale permet de répondre à ces deux questions en parallèle. 2) caractériser un nouveau modèle de rongeur diurne, arvicanthis ansorgei pour l'étude du sommeil et déterminer si les effets non-circadiens directs de la lumière sont conservés et inversés chez les espèces diurnes. 3) démontrer que la lumière influence l'homéostat de sommeil via la mélanopsine chez le rongeur diurne et nocturne.

Pour réaliser ce travail, nous avons utilisé les modèles suivants : souris sans mélanopsine (Opn4 -/-) ; souris sans pacemaker circadien, obtenues soit par lésion électrolytique des NSCs, soit par invalidation de l'horloge par transgénèse (Synaptotagmin10 Cre/Cre , Bmal1 fl/-) ; Arvicanthis Ansorgei, un rongeur diurne provenant Les résultats montrent que chez la souris sauvage la lumière induit le sommeil et l'obscurité l'éveil, ces effets étant étudiés en réponse à des pulses d'une heure de lumière et d'obscurité respectivement. Cette réactivité est atténuée de moitié chez les animaux sans mélanopsine ou sans pacemaker circadien, et complètement abolie en l'absence des deux.

L'analyse d'un rythme ultradien lumière/obscurité de 1hL:1hD appliqué pendant 24 heures permettent d'étudier cette réactivité en fonction du moment de la journée. En l'absence de mélanopsine, ces modifications sont essentiellement observées pendant la période nocturne (ZT12-ZT24) (Figure 0.1). La qualité de l'éveil induit par l'obscurité a été évaluée en mesurant les rythmes EEG thêta et gamma, qui représentent des corrélats électrophysiologiques de vigilance et du fonctionnement cognitif. La stimulation de ces rythmes par l'application de pulses d'obscurité nécessitait la conservation des NSCs. groupes de souris montrent que les souris Opn4 -/-perdent environ une heure de NREM pendant la période de lumière. Les valeurs moyennes sont exprimées en comparaison au groupe contrôle (Sham-Opn4 +/+ ). Encadré : En présence ou en l'absence de lumière (obscurité pendant 24 hr), les souris Opn4 -/-présentent pendant la période subjective de lumière les mêmes taux de sommeil que les animaux contrôles mis en obscurité : ceci

Résumé en français -Introductory Materials

Résumé en français -Introductory Materials

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Perspectives

Ce travail a permis de démontrer que la mélanopsine et les effets directs noncircadiens de la lumière représentent un mécanisme majeur de régulation du sommeil chez les espèces diurnes et nocturnes. Ces données vont encourager de nouvelles voies de recherche pour réévaluer le rôle de la lumière sur la physiologie des mammifères et en particulier chez l'homme. Elles remettent en question le modèle de régulation du sommeil à 2-processus, nous incitant à proposer un nouveau modèle incluant une troisième composante. Nous pourrons ainsi proposer un nouveau cadre conceptuel de régulation du sommeil indispensable à une meilleure compréhension de la physiopathologie des troubles du sommeil afin de pouvoir développer à long terme de nouvelles stratégies thérapeutiques.

Glossary of Terms -Introductory Materials

| 25 Sleep is an ever present process in the existence of most organisms, occupying a significant portion of their lives. In humans, sleep accounts for a large amount of the 24hour period; the average adult will spend approximately one third of their time sleeping.

Glossary of Terms

From the smallest organisms, such as the Caenorhabditis Elegans, a nematode smaller than a centimeter [START_REF] Dabbish | GABAergic synaptic plasticity during a developmentally regulated sleep-like state in C. elegans[END_REF][START_REF] Schwarz | Reduced activity of a sensory neuron during a sleep-like state in Caenorhabditis elegans[END_REF], that shows clear periods of metabolic resting, to the blue whale, the largest animal on the planet, who can rest only one brain hemisphere at a time while keeping the other half conscious, and every organism in between; all of them sleep or rest. Some animals have functional adaptations, such as the horse which sleeps standing up, or certain insects which retract their antennae in order to reduce the amount of external stimuli they experience. In essence, sleep and resting, though incredibly varied amongst many animal species, always serves the same purpose, a restoration and regulation of the animal's internal processes so that they may better interpret and adapt to their surrounding environment when they are awake.

A brief history of sleep research

Since the advent of the human species it's likely that people were aware of the phenomenon of sleep, the idea that at some period during the day fatigue would set in and the body would become unconscious. As early as the Classical period in Greece, philosophers were engaged in rhetoric and discussion about the topic. Around 350 BCE, Aristotle proselytized that sleep was "an inhibition of sense perception", removing the person from their interaction with the physical world. However, it was not until the 20 th century that the idea of researching and observing sleep phenomena became a subject for scientific inquiry. electroencephalographic (EEG) recordings were crude by today's standards, differences between waveforms were easily identifiable (Fig. 1.1) [START_REF] Berger | Uber das Elektrenkephalogramm des Menschen[END_REF]. This was followed by Loomis and colleagues who in 1937 noticed significant differences between waking and sleeping brains [START_REF] Davis | Changes in Human Brain Potentials during the Onset of Sleep[END_REF], and soon after the development of new tools for measurement. By the 1930s and 40s, scientists had begun experimenting in cats, with not only EEG recording but artificial induction of sleep through electrical stimulation of the reticular formation [START_REF] Moruzzi | Brain stem reticular formation and activation of the EEG[END_REF]. During this time Nathaniel Kleitman, a physiologist at the University of Chicago, began investigating biological rhythms and sleep deprivation, giving birth to an entirely new research field. With the establishment of sleep specific brain activity across multiple mammalian species, the next step was to understand how the sleep-wake cycle was generated and regulated. Soon after, rapid-eye movement (REM) sleep was discovered when a student of Kleitman, Eugene Aserinksy, passed hours observing subjects and began noticing oscillating eye movements constrained to certain times during their sleeping period [START_REF] Aserinsky | Regularly occurring periods of eye motility, and concomitant phenomena, during sleep[END_REF]. Along with another student, William Dement, REM sleep was defined. Several years later Jouvet, a researcher in Lyon, began studying sleep in cats using EEG and noticed these "REM periods" characterized by a desynchronized EEG, more similar to waking in contrast to normal sleep EEG, when the animal was behaviorally sleeping and termed them "paradoxical" (Jouvet and Jouvet 1963;Jouvet, Jouvet et al.

Introduction -Chapter 1 -Sleep | 35
1963; [START_REF] Mouret | Electrical activity of the visual system during the paradoxical phase of sleep in the cat[END_REF]. Subsequently, Dement and colleagues identified the relationship between REM sleep and dreaming, giving rise to a greater understanding of both. In 1962, more extreme research into biological rhythms began, starting with Siffre who lived underground without light to study its effects [START_REF] Siffre | Hors du temps; l'expérience du 16 juillet 1962 au fond du guffre de Scarasson par celui qui l'a vécue[END_REF], and continued with Aschoff and Wever, who observed research subjects placed in sealed chambers [START_REF] Aschoff | Desynchronization of human circadian rhythms[END_REF]). In the 1970s the discovery of the suprachiasmatic nucleus, a central biological clock located in the hypothalamus led to the study of internal biological rhythms [START_REF] Moore | Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat[END_REF].

In subsequent years, a variety of sleep problems would be discovered and examined, starting with Gastaut, who first identified sleep apneas [START_REF] Gastaut | Polygraphic study of diurnal and nocturnal (hypnic and respiratory) episodal manifestations of Pickwick syndrome[END_REF]. Numerous disorders were identified from the extremely rare, such as Fatal Familial Insomnia in 1986, a disease linked with genetic factors [START_REF] Lugaresi | Fatal familial insomnia and dysautonomia with selective degeneration of thalamic nuclei[END_REF][START_REF] Medori | Fatal familial insomnia, a prion disease with a mutation at codon 178 of the prion protein gene[END_REF] to an amplifier which will allow for signal analysis and facilitate its interpretation.

Oscillatory neural activity exists between thalamus and cortex, wherein the thalamus gates information to the cortex as well as providing reciprocal feedback. This transfer of information between thalamic neurons and pyramidal cells in the cortex creates the thalamocortical loop and is the basis for the propagating electrical activity recorded by the EEG. This will be altered depending on the wake or sleep state of the brain. A schema of this interaction can be seen in Fig. 1.2. The signal which is seen by the observer is normally a subtraction between the surface electrode and a reference area of the body (in humans the contralateral mastoid process, for example). The EEG is an incredibly important device for evaluating a variety of neurological disorders. EEGs are vital to the field of epileptology where they are used to identify seizures, in conjunction with neuroimaging, and neuropsychological assessment, to determine the best course of action for a patient. However, in regards to the subject of this dissertation it is the use of EEG to examine sleep and wake that is most pertinent.

EEG as a tool for sleep

As previously stated, a single EEG is a sum of the depolarization of each cell, measured in voltage. However, these signals can be radically different, and have therefore been categorized into different subgroups to represent diverse stages of sleep and alertness.

Categories are determined by the amplitude, or height of the signal, and the frequency at which it repeats. Each waking or sleep stage has specific features in the EEG which under optimal circumstances make identification relatively simple.

Sleep stages in human beings, as defined by the American Academy of Sleep Medicine, consist of wake, NREM stage 1-3, and REM (paradoxical) sleep. Though NREM stage 1-3 are all generally described as non-rapid eye movement sleep, changes within these stages on the EEG can be quite striking. An EEG of a person who is awake will show desynchronized waveforms. However, this stage is still dominated by specific frequencies, principally alpha (8-12 Hz) and beta (13-30 Hz). As a person becomes drowsy they enter into the first stage of NREM sleep, which can also be thought of as a transitional stage between conscious and unconscious states. This stage is classified based upon the continued presence of alpha waves and inclusion of theta (4-7 Hz). This is followed by stage 2 sleep, defined by the appearance of unique waveforms, known as spindles and kcomplexes. Their function is still unknown yet possible functions have been suggested [START_REF] Tononi | Sleep function and synaptic homeostasis[END_REF][START_REF] Cash | The human K-complex represents an isolated cortical down-state[END_REF]), and it is still unclear as to whether they exist in other animals which display similar sleep patterns to human beings. In addition, the functional significance of sleep spindles also remains unclear though some have suggested a relationship with cognition. K-complexes, which are large negative The regulation of the sleep and wake cycle is classically thought to be under the control of 2 independent mechanisms: homeostatic (process S) and circadian (process C).

Process S can be described as an increase in the pressure to sleep as a function of time spent awake. The circadian process describes a regulatory mechanism under the control of a central clock, which lasts approximately 24-hours. The interaction of these two processes determines the timing and quality of sleep and alertness.

The circadian process

The term "circadian", is derived from two Latin words: circa, meaning "approximately", and dies, or "day". The adaptation to this 24-clock is consistent among nearly all plant and animal life. In mammals this is under the direct regulation of the suprachiasmatic nucleus, controlled principally by light; synchronizing the expression of the clock, based on a feed-back loop controlling the 24-hour expression of clock genes.

These markers were shown to exist in a variety of species including plants, drosophila, and mammals, leading to extensive characterization of the system in laboratory animals [START_REF] Vitaterna | Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior[END_REF]. The timing of the clock is a result of the regulation of specific proteins: CRY1-2, CLOCK, BMAL1, PER1-2, and others.

There is strong evidence supporting the claim that the SCN is the central biological clock, regulating the sleep and wake cycle, at least in terms of 24-hour distribution under light and darkness, coming from studies where the effect of removing these nuclei through electrolytic lesion, was examined. These results indicated that complete removal of this structure caused the animal to completely desynchronize its circadian rhythm, with sleep and waking more evenly distributed across the 24-hour period regardless of the amount of light [START_REF] Moore | Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat[END_REF][START_REF] Ibuka | Loss of circadian rhythm in sleep-wakefulness cycle in the rat by suprachiasmatic nucleus lesions[END_REF].

Additionally, light via the SCN can coordinate the release of melatonin, a hormone produced by the pineal gland, critical for the timing of sleep. Melatonin exerts a strong hypnotic effect and synchronizes the clock through the SCN. When the SCN is activated by light, neurons send information to the preautonomic neurons of the paraventricular nucleus (PVN), the sympathetic preganglionic neurons in the spinal cord, and noradrenergic neurons in the superior cervical ganglia (SCG), located in the lateral horn of the spinal cord, in turn projecting to the pineal gland (Fig. 1.4). A decrease in the amount of light promotes the production of the hormone, whereas exposure will suppress it. of starting a sleep deprivation [START_REF] Everson | Sleep deprivation in the rat: III. Total sleep deprivation[END_REF].

EEG observation of the homeostatic process can be seen in the increased propensity of delta wave activity (0.5-4 Hz) during NREM sleep. Recent evidence has indicated a genetic component may contribute in some part to the changes observed (Franken, Chollet et al. 2001).

The

Sleep distribution

Sleep stages do not always progress directly from stage 1-3 though, normally in order to reach the deepest levels of sleep, the person will pass from wake, to stage 1, 2, etc.

In most cases deeper sleep will be consolidated to the beginning of the night, with bouts of

Recording and scoring sleep in rodents

Quantitative sleep studies in rodents operate more or less on the same principles as human beings. However, the electrode implantation is less extensive and more invasive.

Unlike in humans, EEGs in mice are implanted underneath the skull and directly on to the cortex and thus these signals are referred to as electrocorticograms (ECoG). This creates a different signal as the electrical output of the brain is not diffused by any bone matter.

Standard implantation to record sleep in mice is a 5-channel chip attached to the head using dental cement with 3 ECoGs, and two EMGs placed in the muscles of the neck to record body movement, and in some experiments the recording of ocular movements (EOG). Throughout this thesis ECoG will be referred to as EEG.

Scoring sleep in rodents is based on specific changes in the EEG signal, in terms of dominant frequency, as well as differences in the EMG, or EOG. The scoring window is generally lower than in human beings, though a smaller window increases the accuracy of the power spectrum calculation (Franken, Malafosse et al. 1998).

Wake or vigilance in mice is dominated by theta (6-10 Hz) and gamma (40-70 Hz)

ECoG activities, ECoG correlates of cognition and alertness, respectively, which originate in the CA1-CA3 cell layers of the hippocampus (Chrobak and Buzsaki 1998;[START_REF] Montgomery | Theta and gamma coordination of hippocampal networks during waking and rapid eye movement sleep[END_REF]. SWS (NREM) represents the majority of the rodent resting period and is dominated by delta frequencies with long rhythmic oscillations that are high in amplitude. REM, is similar to humans and can be characterized by rhythmic rapid eye movements, and muscle atonia, as evidenced on the EMG. Furthermore, REM sleep is dominated by desynchronized theta activity.

The neuronal networks responsible for sleep and waking

Different structures within the brain are responsible for the induction of wake and sleep as well as the balance and regulation therein. To change from a sleeping state to awaking state, certain neurons must be activated in conjunction with the synthesis and release of specific neurotransmitters. The various brain structures involved in the regulation of sleep and wake are complex and require comprehensive explanations; however given the focus of this dissertation is not on neuroanatomical systems, it is only briefly discussed.

Sleep in rodents -Chapter 1 -Sleep

| 48

Key to understanding this process was the discovery of the reticular activating system, a region of the brainstem tegmentum, which arouses the animal if stimulated, and specifically a subset of cholinergic nuclei which project to thalamocortical neurons involved in sleep/wake regulation. In addition, the main wake promoting systems are represented by the noradrenalinergic (NA; locus coeruleus), serotoninergic (5-HT; dorsal and median raphe), and histaminergic (His; tubero-mammillary nucleus, TMN), neurons.

The hypocretinergic system, as mentioned before, is also involved in promoting wakefulness.

The majority of the control of NREM sleep rests within the hypothalamus including the VLPO. It is known that lesions in this region can severely disrupt sleep in both humans and laboratory animals, for example, causing insomnia (Von [START_REF] Economo | Encephalitis lethargica[END_REF]. The transition from waking to NREM sleep, and vice-versa, is based on a reciprocal inhibitory interaction between "sleep-promoting neurons" of the ventrolateral preoptic area (VLPO) that contain GABAergic cells and co-express galanine, and the wake-promoting systems (NA, His, 5-HT, Hcrt). NREM sleep depends on thalamocortical interactions or feedback loops that will control cortical synchronicity as measured at the level of the EEG.

For several decades the structures involved in REM sleep regulation were thought to be mainly located within the brainstem, dependent on an interaction between "REM on" the pons is relayed directly to the thalamus, which is then sent to the occipital cortex, referred to as ponto-geniculo-occipital (PGO) waves. Currently, the function of these eye movements is unknown as they require no visual stimulus as in wake.

Conservation of the 2-process model in rodents

The circadian function in rodents has been studied with a variety of methods.

Principle among them is observation based on actimetric recordings, measurements of locomotor activity during a given period. Since it is non-invasive, measurements have been taken for months or years at a time, identifying seasonal activity patterns and other similar behaviors. In addition, recording internal body temperature gives a clear pattern of variability depending on time of day. From the perspective of sleep, simple distribution of the cycle as seen previously (Fig. 1.9) is confirmation of a time-of-day organization. In rodents this cycle is approximately 24 hours, though there are slight changes depending on the species.

The homeostatic process can be challenged in rodents through sleep deprivation.

Though initially this involved forcing the animal to run on a moving wheel, a less aggressive method known as "gentle handling", is now used. However automated systems such as the multiple platforms technique are also implemented at certain labs. By forcing the animal to stay awake, researchers can increase the amount of homeostatic sleep pressure and then observe the response in the delta power. Delta (0.75-4 Hz) power is the most reliable marker for sleep need [START_REF] Franken | Sleep deprivation in rats: effects on EEG power spectra, vigilance states, and cortical temperature[END_REF] and increases are noted in the absence of sleep, with decreases following sleep bouts, thus the level of delta power is considered to reflect overall sleep need at a given time point. Human studies have confirmed this is a valid method, for testing the limits of the homeostatic sleep system. In a clinical lab, sleep deprivation may continue for 20-30 hours however, with mice, a sleep deprivation of just 6-hours or less at resting phase onset (light) has proven effective at eliciting a delta power response. Through comparison of the delta power from 24-hour period before the sleep deprivation, a strong drive from the homeostat can be seen, followed by a short recovery period lowering the delta power to its previous levels (Fig. 2.1 -Introduction

1.10).

Sleep in rodents -

Background

Once every twenty-four hours the planet Earth completes a single rotation along its axis, crucial for the survival of all life. The most essential feature of this rotation is the exposure to light during certain times, and darkness at others. Given the latitudinal position and time of year, the amount time spent in light or dark can shift drastically. Yet so immense is the impact that the evolution of thousands of species and their adaptation to certain behaviors is a result of this process. Light is responsible for vision, certain wavelengths for heating, and entrainment of the central biological clock. Animals need light to determine their feeding and reproductive cycles, through activation and stimulation of certain areas of the brain. In human beings, we use light to modify our natural environment to adapt to changing conditions. Light will affect cardiac rhythm, core body temperature, cognitive processing and alertness. Light changes sleep by promoting wake through the suppression of melatonin, a hormone secreted by the pineal gland, or change mood states, such as in people suffering from Seasonal Affective Disorder [START_REF] Vandewalle | Wavelength-Dependent Modulation of Brain Responses to a Working Memory Task by Daytime Light Exposure[END_REF]).

Light and the brain

Light enters the brain via the eye, which is composed of several key structures: the pupil, the iris, and the retina. The pupil controls the amount of the light able to pass into the eye. The iris, a circular structure surrounding the pupil will expand or contract depending on the requirements, meaning the further the pupil expands, the higher the quantity of light can enter, and inversely as it contracts, the lower. After light has entered the pupil it passes through the interior chamber of the eye to the retina, a thin membrane of cells on the outer wall, sensitive to changes in light (see Fig.

-The Retina and Visual Light

Retina

The existence of the retina and its structure has been known for many years and was clearly illustrated in 1900 by famed anatomist Santiago Ramon y Cajal. The mammalian retina is composed of distinct types of cells which serve a variety of functions, principally the interpretation of visual and non-visual light produced by the environment.

Visual light, composed of wavelengths within the observable electromagnetic spectrum, activates specialized cells in the outer layer called rods and cones (Fig.

2.2).

Rods, named for their elongated cylindrical shape, are extremely light sensitive cells important for vision, also known as scotopic vision, in environments with extremely low luminosity. The cell itself is composed of an inner segment, consisting of the organelles and nucleus, an outer segment, which functions as the light absorption area, and a synaptic terminal to transmit information to bipolar neurons or horizontal cells.

Color vision is constructed based on the reactivity of cone cells sensitive to specific wavelengths, and functions most efficiently under high luminosity. Though the basic cellular structure is nearly identical to rods, their physical shape is drastically different due to the requirements of photosensitivity. Several subtypes of cones are present: (i) the shortwavelength (S-cone), which is sensitive to blue, (ii) a medium-wavelength (M-cone), sensitive to green, and (iii) a long-wavelength (L-cone), sensitive to red, which makes up the majority of light entering the eye.

Thus it is a combination of photosensitive information from these cells which produces the interpretation of color. However, the distribution and density of rods and cones can vary widely depending on the animal, from over one hundred million in human beings, to far less in species of birds and reptiles. Following synaptic transmission to the bipolar and amacrine cells, which help to organize information from rods and cones, visual light, activates the retinal ganglion cells, neurons containing photo-opsins, which in turn communicate to the brain via the retinohypothalamic tract. | 57

-Non-visual Light and Melanopsin

Non-visual light

For many years, non-visual light was not considered substantial in its influence upon key regulatory systems in the brain, and the assumption was that previously known photoreceptors involved in vision were responsible for processes such as circadian regulation. Thus these subsets of intrinsically photosensitive retinal ganglion cells (ipRGCs), which are now known to specifically transmit this information, were largely ignored upon their initial discovery. Though first identified in 1923 by Clyde Keeler, it was not until the end of the 20 th century that their significance was truly understood. The principle finding in 1991 by Foster et al., was that this highly specialized cell was directly responsible for sending information to a variety of brain areas responsible for different functions [START_REF] Foster | Circadian photoreception in the retinally degenerate mouse (rd/rd)[END_REF]. It was shown that non-visual light mediated the circadian cycle through activation of the SCN, and additionally, controlled the pupillary light reflex, a function of the olivary pretectum nucleus (OPT). Furthermore, the function of melatonin, critical for inducing sleep, was altered depending on changes to environmental non-visual light. However, it was the discovery of melanopsin (Opn4), a new photopigment within these cells, in 2000 by Provencio and colleagues, which showed the most promise for scientific advancement [START_REF] Provencio | A novel human opsin in the inner retina[END_REF].

Melanopsin

Opn4, is a photopigment whose existence was initially suggested by the group of Foster, who in 1999 identified a continued response to light in mice which possessed neither rods nor cones [START_REF] Lucas | Regulation of the mammalian pineal by non-rod, non-cone, ocular photoreceptors[END_REF]). These mice when placed under different light and dark conditions were still able to entrain their circadian cycle despite a total lack of visual input. Following this research axis, Berson underlined that these photosensitive cells were completely separate and were able to depolarize in response to light even when synaptic transmissions from rods and cones were blocked [START_REF] Berson | Phototransduction in ganglion-cell photoreceptors[END_REF].

Later this same team would identify the sensitivity of Opn4 by using various levels of light and darkness to describe its responsiveness. Critical to understanding the contribution of this non-visual melanopsin-mediated information was to identify the areas of the brain mediated by Opn4 [START_REF] Altimus | Rods-cones and melanopsin detect light and dark to modulate sleep independent of image formation[END_REF]Lupi, Oster et al. 2008;[START_REF] Tsai | Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice[END_REF]). These findings will be more rigorously discussed in a review presented in chapter 3. These ipRGCs though extremely small in number, accounting for less than 3% of mammalian ganglion cells, have now been highly implicated in a variety of regulatory processes. As previously discussed, melanopsin is able to directly control circadian rhythms through modulation of photic phases [START_REF] Panda | Melanopsin (Opn4) Requirement for Normal Light-Induced Circadian Phase Shifting[END_REF]Ruby, Brennan et al. 2002).

Non-visual light and melanopsin-Chapter 2 -Light

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Innervations from the retina to the brain

Signals coming from the retina are transduced down the RHT, specifically innervating several cerebral structures (summarized in Fig. 2.5). However not all areas are innervated evenly. The areas of highest innervation are the SCN in the hypothalamus, the intergeniculate leaflet, and the olivary pretectum, responsible for circadian synchronization, interpretation of photic information, and the pupillary reflex, respectively.

To a lesser extent is the VLPO in the hypothalamus, an area with a high concentration of sleep-promoting (galanin-containing GABAergic) neurons. An additional area of high innervation is the superior colliculus, which has a variety of functions including control of eye movements, and sleep regulation. The weakest projections are seen to the dorsal lateral geniculate nucleus, which functions mainly as a visual relay, and the subparaventricular zone (SPVZ), involved in regulating body temperature and food-energy intake [START_REF] Hattar | Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity[END_REF]Hannibal and Fahrenkrug 2004). Opn4 and with an electrolytic lesion of the central circadian clock, were compared for differences with their controls under a variety of light-dark conditions. In addition, a mutant mouse possessing a disrupted central clock (Syt10 Cre/Cre Bmal1 fl/-), yet with intact cellular structures, was used as a complementary approach to the SCN lesion. Indeed, inhibition of the clock in two ways, through transgenes and lesion of the SCN will allow controlling for the limitations of each approach. Finally, the overarching goal was to not only identify the pathways responsible but to determine the proportional contribution of the direct effects of light on the sleep wake distribution under habitual environmental settings.

As the University of Strasbourg is one of the only laboratories to possess a diurnal rodent model for studying chronobiology, Arvicanthis ansorgei, a second project was designed to demonstrate that these direct effects of light exist across multiple species, even if the interpretation of light and dark is inverted. Primarily this required modification of the EEG system for use with a larger more active rodent, as well as verification of the vigilance states present. This approach used a similar protocol as with the mice, though slightly modified due to the diurnality of the species. The objective of this project was not

Introduction to research projects

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only to validate the existence of the direct effects of light in a separate species, but indeed on a long-term perspective, to understand more clearly the inherent differences which determine nocturnality vs. diurnality. Furthermore, it was in search of a stronger translational model to human research, available for manipulation within controlled laboratory settings.

Building upon a surprising result found by our group [START_REF] Tsai | Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice[END_REF] a third project sought to examine a biological link, the first ever seen, between sleep homeostasis and light via melanopsin. A deprivation protocol was designed around a dose-response curve to elicit a clear model of the homeostatic process. Analysis of the delta (0.75-4 Hz) power spectrum gave a clear impression of the changes between these experiments.

Genetically ablated Opn4 mice were used in conjunction with their controls to quantify this difference, in addition to an inverted, yet identical procedure for arvicanthis ansorgei.

Isolation of this association is vital to revealing the fundamental information gating of noncircadian light on the accumulation of and release of sleep pressure.

Chapter 4 -Laboratory techniques and materials

Animals -Chapter 4-Laboratory techniques and materials

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4.1 -Laboratory Animals

Opn4 Gene-ablated mice

Experimental studies on the melanopsin-mediated direct effects of light and sleep homeostasis in mice were realized through the use of Opn4 genetically ablated mice. The mutant mice were initially generated by Jackson laboratory. Gene disruption was made using a 38-bp fragment corresponding to a segment of the protein-coding region that was replaced by an IRES-lacZ reporter and neomycin resistance cassette (IRES-lacZ-neo). This mutation was designed to produce a loss-of-function mutation by deletion of amino acids 116 to 128, as well as insertion of the 6.9-kb reporter/resistance cassette. After electroporation into mouse embryonic stem cells of strain 129P2/OlaHsd, selecting those with G418 resistance, the cells were injected into blastocysts from a C57BL/6 strain. These chimeric mice were then crossbred with wild-type C57BL/6 producing the first generation heterozygotes. The mice were initially transferred from Stanford University, and the colony was extended at our facility in Strasbourg. Opn4 -/-mice from two different genetic backgrounds (C57/Bl6&129P2 and C57/Bl6 backcross) were studied during the course the doctoral program. The majority of the experiments were performed on Opn4 -/-C57/Bl6&129P2 strain due to the availability of mixed genetic background mice at the start of the PhD. To control for genetic heterogeneity (C57/Bl6 and 129P2) the experiments were performed on male littermate homozygous offspring (Opn4 +/+ & Opn4 -/-) obtained from cross-breeding heterozygous (Opn4 +/-x Opn4 +/-) males and females. In addition, a second colony of melanopsin KO mice with a homogeneous C57/Bl6 genetic background was developed. The backcross began at Stanford University, and subsequently continued in Strasbourg, arriving at 10 backcross generations by the end of the PhD. At all times prior to experimentation mice were kept under environmentally stable conditions (12hL:12hD; 25 ± 0.5° C, with food and water ad libitum. Cre knock-in cassette, and after cloning of the Syt10 Cre vector, the targeted clones were then injected into blastocysts of C57/Bl6 mice. These chimeric mice were then bred using wildtype C57/Bl6 to produce F1, followed by continued breeding to produce a colony by backcrossing to C57/Bl6. Syt10 Cre were then crossed with Bmal1 fl/fl to disable Bmal1 expression, solely in the SCN. Depending on the dosage of the Cre recombinase, mice with phenotypes ranging from minimal circadian perturbation to complete arrythmicity were obtained, confirming the usefulness of the Syt10 Cre driver line. These Syt10 Cr/Cree Bmal1 fl/-mice retained arrythmicity (locomotor activity, time course quantification of clock genes expression in the SCN) consistent with a disabled SCN. The controls used in this study were Syt10 Cre/Cre Bmal1 +/-mice that are rhythmic and allow us to control for Bmal1 and Syt10 gene expression (from our collaborators, data not shown). The genetic background of these mice was C57/Bl6 & C57/Bl6n, thus C57/Bl6 mice were used as an additional control for this study.

Arvicanthis Ansorgei

The Sudanian Grass Rat (arvicanthis ansorgei) is a diurnal rodent from the northern African grasslands. Though not extensively used in a laboratory setting, breeding facilities and previous research studies performed by the institute made this animal model available [START_REF] Challet | Circadian organization in a diurnal rodent, Arvicanthis ansorgei Thomas 1910: chronotypes, responses to constant lighting conditions, and photoperiodic changes[END_REF]. The laboratory colony began in 1998 using trapped animals from southern Mali, initially 10 males and 15 females. To confirm the species was correctly identified, karyotipic analysis was performed on colony animals. Arvicanthis are closer in size to laboratory rats and as such are at least 5-10 fold higher in weight as compared to mice (150-250g vs. 25-35g). All animals were maintained in 12:12 light-dark (LD) cycle under constant temperature of 22 ± 1 °C, with food and water ad libitum.

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-Methods

PCR Genotyping

Genotyping for Opn4 mice was performed using a standard PCR protocol. Before PCR DNA concentration was determined using a standard spectrometer (BioRad). Primers For WT, band was located at 426 bp, and for mutants, 538 bp. Bands were separated and run on a 1.5% agarose gel (Husse, Zhou et al. 2011).

Protein quantification through western blot

Western blot procedures were used to determine amount of the melanopsin protein in (1:1000 Provencio), or D-18 (1:1000, Santa Cruz Biotechnologies), primary antibodies.

Following this, the membranes were incubated with secondary antibodies, and then after exposition of the membranes to photoreactive film, the Opn4 protein was expected at ~53 and ~85 kDa (glycosylated form of the protein) via chemiluminescence (General Electric).

Unfortunately after performing several experiments attempting to optimize the protocol conditions, the results were not convincing enough to pursue due to specificity/sensibility of the different antibodies and low level of expression of the protein in the tissue. Additionally, other teams have failed to obtain relevant and reproducible results from melanopsin western blot.

Surgery:

All surgical procedures were performed under deep anesthesia delivered intraperitoneally with Nembutal (68 mg/kg; University IRB-approved).

Lesion of the suprachiasmatic nuclei

Under pentobarbital anesthesia removal of the two SCNs was achieved using radio frequency lesions, according to published protocols (Easton, Meerlo et al. 2004). Lesions handling [START_REF] Bourgin | Cortistatin promotes and negatively correlates with slowwave sleep[END_REF].

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Quantitative analysis of vigilance states

The behavioral states wakefulness (W), rapid eye movement sleep (REMS), and non-REM sleep (NREMS) were visually assigned for consecutive 4s epochs, either as Wake, NREM, or REM. In arvicanthis, EOG and a video capture system was used to verify the accuracy of the scoring with physiological behavior. All recordings were scored every 4-sec based on visual inspection of the EEG and EMG, as described previously [START_REF] Bourgin | Cortistatin promotes and negatively correlates with slowwave sleep[END_REF])and without knowledge of genetic background. If epochs contained signal artifacts they were included in the analysis for state amounts, yet excluded for power spectrum (see next). Amounts spent in each vigilance state were calculated in 5 and 30 min, and 1-, 12-, and 24h intervals.

Power spectrum analysis of the EEG

Once scoring was completed an average spectral profile was constructed using the entire experimental period, excluding epochs marked as artifacts. All EEG signals were converted using a Discrete-Fourier Transformation (DFT) giving a power spectra between 0 and 128 Hz (0.25Hz resolution), and using a rectangular calculation with an overlapping window of 4-sec. The frequency range 49-51 Hz was omitted from the analysis due to powerline artifacts in some of the recordings. For each vigilance state and animal, an individual EEG spectral profile was constructed, using a mean average of all 4s epochs.

For all NREM sleep epochs, any time-dependent changes in EEG power for the delta (0.75-4Hz) band were examined. For epochs scored as wake, both theta (6-10 Hz) and gamma (40-70 Hz) bands were measured as EEG markers of alertness. To normalize for delta power during NREM sleep the last 4h of the (subjective) light period, the lowest period of homeostatic sleep pressure, was used and all values measured against it in mice. Spectral profiles of theta and gamma were calculated using an overlapping 10 min. window of 5-min.

increments, yielding 13/hour, or for hourly values, were normalized against the lowest period of gamma and theta during the day (i.e. the least alert period). Custom programs were written for analysis in Pascal and then transformed into Visual C++.

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Actimetric recordings

Locomotor activity recording was performed using either single-wheel cages or infrared motion capture, simultaneous or not to EEG recording. When the animals were in individual cages equipped with wheels (diameter of 22cm), locomotor activity was measured based on the number of wheel turns, recorded via a standard actimetry system (DataportDP24, Minimitter). Animals were recorded for at least 10 days for each of the light/dark exposure to establish clear patterns of activity. Infrared motion detectors were used for EEG sleep recording periods, as a wheel would be prohibitive due to the data cable attached to the animal. Following acquisition, data was organized into 10 5-minute bouts of locomotor activity using software (VitalView, Minimitter). Actograms and chi-squared analysis were performed using Clocklab (Actimetrics) following data transformation via Matlab.

Anatomic Analysis -Chapter 4-Laboratory techniques and materials

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-Anatomy

Once experimental protocols were completed, animals were deeply anesthetized with pentobarbital and perfused with heparin/NaCl followed by transcardial fixation for 15 minutes with 4% paraformaldehyde in PBS, pH 7.4 for in situ hybridization and immunohistochemistry. After dehydration in 30 % sucrose for 48 hours, the brains were frozen and cut in a cryostat.

To prepare tissues for immunohistochemistry or in-situ hybridization, samples were still possess the physical structure of the SCN, yet this lack of difference, suggests an anatomically dysfunctional system not only for the circadian, but also the direct effects of light. Based upon results from our previous studies and others we subjected Syn10 Cre/Cre Bmal fl/-mice and their controls (Syn10 Cre/Cre Bmal +/-and C57/B6 WT) to a one-hour light pulse administered during the dark period (ZT15-16) 4,16,17 . Interestingly c-Fos immunoreactivity was severely diminished in Syn10 Cre/Cre Bmal fl/-following the pulse and was conserved to the retino-recipient level when compared to their controls (Fig 3a-d, h).

Furthermore, no AVP activation was seen in the SCN in the mutants (Fig 3e-g). This suggests that the interpretation of the direct effects of light is possible without a functioning SCN though is severely reduced. Moreover, disruption of clock gene expression, in this case Bmal1, seems to disable the functionality of certain light information cells in the SCN, or impede transmission to more posterior areas which project to areas of the brain involved in sleep and wake regulation.

Discussion -Chapter 5-Opn4 photic regulation in arrhythmic mice

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-Discussion

One can argue that animal studies, such as the present one, do not control for sleep homeostasis. However, animal research controls for environmental and circadian zeitgeibers, genetic background, and most major advancements in chronobiology come from research that did not control specifically for process S. When deciphering the acute photic effects under a 1hL:1hD schedule, we can assume that within the time frame of a 1hr pulse, there is not

-Methods Summary

Sleep and the EEG were analyzed under various lighting schedules in mice possessing a targeted disruption of the gene responsible for melanopsin (Opn4 -/-; see Ruby et al. 17 mediate the direct photic input to sleep and waking (for detailed calculations see supplementary methods). Values represent mean ± SEM (for both days Sham Opn4 +/+ n=9; Sham Opn4 -/-n=7; SCNx Opn4 +/+ n=10; SCNx Opn4 -/-n=8; C57/Bl6 n=7; Syn10 Cre/Cre Bmal1 +/-n=7; Syn10 Cre/Cre Bmal1 fl/-n=10). Light induced c-Fos in the retino-recipient zone of the SCN in WT (data not shown), in Syn10 Cre/Cre Bma1l +/-(a) and to a lesser extend in the Syn10 Cre/Cre Bmal1 fl/ mice (c).

Figures -Chapter 5-Opn4 photic regulation in arrhythmic mice

Figures -Chapter 5-Opn4 photic regulation in arrhythmic mice

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Syn10 Cre/Cre Bmal1 fl/ mice show a dysfunctional SCN (a-g). AVP -staining of the SCN is preserved in Syn10 Cre/Cre Bma1l +/-mice (a,b,g) and comparable of that found in WT animals (data not shown). Mutant mice do not show AVP expression in the SCN, whereas it remains unaltered in adjacent regions (c-e). The SCN is structurally intact as shown by DAPI-staining (grey) (f). Scale bar in a-d indicate 100µm, and in e-g 500µm.Histogram represents the number of c-Fos immunoreactive neurons per SCN section in control WT, Syn10 Cre/Cre Bma1l +/-, and Syn10 Cre/Cre Bmal fl/ mice after a 1h LP (mean ±sem) (h). In mice lacking a functional SCN an induction was observed though was significantly reduced (63%) (Two-way ANOVA, light pulse effect: p<0.001; an asterisk (*) indicates post hoc Fisher PLSD: p<0.05. No significant differences were observed between the two control genotypes.

A two-way ANOVA, the star indicates light pulse (genotype interaction: p<0.001). of 8.7-kb was then cloned. Targeted clones were then injected into blastocysts of C57/Bl6 mice and resulting offspring were bred using wild-type C57/Bl6 to produce F1, followed by continued breeding to produce a colony. Syt10Cre were then crossed with Bmal1 fl/fl to disable Bmal1 expression, solely in the SCN.

Figures -Chapter

Lesion of the SCN

A subset of mice (Opn4 -/-, n=8) and their littermate controls (Opn4 +/+ , n=10) were radio frequency lesioned around the area of the suprachiasmatic nucleus, before electrode implantation as previously described 19 . Lesions were performed under anesthesia by heating the (250µm) tip of a Radionics (Burlington, MA) TCZ electrode to 55°C for 20 sec by passing RF current from a RFG-4 lesion generator (Radionics). The mice were then placed in a stereotaxic instrument (Kopf Instrument), and a standard electric probe was lowered into four points of the SCN region (from zero ear bar, nose at +5°: lateral: +/-0.2 mm; antero-posterior:

+3.4 and +3.6 mm; dorso-ventral: +0.95 mm; 21 ). A lesion generator (Radionics Lesion

Generator System) was then used to control both the temperature and voltage of the probe, for 30 seconds at each lesion site. All other mice underwent Sham lesions for control, where the probe was used but the generator was not activated. All animals then underwent identical ECoG/EMG electrode implantation to the others (described below).

EEG implantation

Before undergoing implantation mice were anesthetized with an intraperitoneal injection of pentobarbital (68 mg/kg; protocol-approved). Male Opn4 -/-(n=16) , Opn4 +/+ (n=19), Syn10 Cre/Cre Bmal1 fl/-(n=10), Syn10 Cre/Cre Bmal1 +/-(n=7), and C57/Bl6 (n=7) mice were implanted with two EEG, one reference, and two EMG electrodes in order to record vigilance states, aged 10-12 weeks. Mice were given a minimum of 14 days to recover from surgery and habituate to the baseline control conditions before any further experiments were carried another and a minimum of 14 days under 12hL:12hD was used to habituate the animals to the control condition. The 24 hours of recording which preceded each experiment was used to confirm that the mice had returned to baseline, in terms of sleep-wake amounts and architecture had. All mice were recorded simultaneously.

Scoring of sleep and wake state

EEG and EMG signals were amplified, filtered, and analog-to-digital converted to 256Hz. The EEG signal was then modified using a Discrete-Fourier Transform (DFT) to yield a power spectra between 0 and 90 Hz (0.25Hz resolution) using a 4-s window. Any epochs containing EEG artifacts identified and excluded during further analyses. Differences between genotypes in sleep amounts were calculated by averaging time spent in each state over 5-min, and 1-, 12-, and 24-h intervals.

The amplitude of the light-dark dependent changes in wakefulness under the 1h:1h LD schedule were determined using sine-waves, fitted to the individual 5-min values for wakefulness obtained in the 1-h before, during, and after the 1-h dark periods. For each mouse, the 5-min values for the 6 dark-pulses given during the 12-h subjective light period and the 6 dark-pulses given during the 12-h subjective dark period were averaged. Sinewaves were fitted to the average time courses using SigmaPlot (Systat v. 12) with amplitude, phase, y-offset as free parameters and period set to 2 hours. These individual amplitudes for the subjective light and dark period were then further analyzed using a factorial 2-way ANOVA with genotype and SCN condition and the repeated measures for factor light or dark period.

For each vigilance state of the ECoG, an average spectral profile was constructed using all 4-s epochs scored with the same state. The frequency range 49-51 Hz was omitted due to power-line artifacts in some of the recordings. In NREM sleep, time-dependent changes in ECoG power for specific frequency bands, was performed for delta (0.75-4Hz).

During wakefulness, theta (6-10Hz) and gamma (40-70Hz) were measured instead. ECoG delta power during NREM sleep was normalized by expressing all values relative to the mean value obtained in the last 4-h of the (subjective) light period, the lowest period of homeostatic

Materials and Methods -Chapter 5-Opn4 photic regulation in arrhythmic mice

| 113 sleep pressure. Profiles were calculated using overlapping 10 min windows of waking at 5min increments (13/hour).

Detailed Calculations for Schema Figure 2f

Under a standard 12hL:12hD cycle, the intact circadian system is responsible for the majority of regulation of sleep and alertness and is abolished with the removal of the SCN, demonstrated in fig 1A. Melanopsinergic cell transmission under this condition passes both directly through and around the SCN, though at a smaller magnitude than under a noncircadian light/dark regimen.

During the 12hL:12hD cycle a combination of direct and indirect effects of light with or without Opn4 on sleep and alertness is observable. In order to determine the relative contribution of melanopsin and rods/cones through and external to the SCN, the groups must be parsed apart. The total contribution of effects mediated or relayed by the SCN is represented as the normalized difference between Sham Opn4 +/+ and SCNx Opn4 +/+ (72%), which consists of the circadian drive with light entrainment via melanopsin and rod and cones non-circadian and circadian effects of Opn4 via the SCN and well as the contribution of rods/cones and intrinsic circadian processes. The melanopsin direct non-circadian effect of light passing through the SCN during this condition is represented as the total amount of melanopsin contribution (the difference between the two Sham Opn4 +/+ and Opn4 -/-) subtracted from the difference of melanopsin contribution when the SCN is removed (SCNx Opn4 +/+ and Opn4 -/-) and normalized (13%). The contribution via external pathways from Opn4 would simply be the difference between the two SCNx genotypes (20%). The SCNx Opn4 -/-group would thus represent rod/cones contributions to the non-circadian extra-SCN response to light. Since further reduction is difficult under these experimental conditions the remainder is assumed to consist of all circadian processes contributions to the regulation of sleep and alertness via the SCN, which is calculated as the difference between the groups lacking Opn4 (Sham and SCNx). However, a certain percentage of this remaining 59%

represents the direct effects of light on rods/cones via the SCN, which involve the OPN4 ipRGCs as well. Thus by using a similar ratio as evidenced between melanopsin information via and external to the SCN, we can postulate that the percentage of rods and cones passing information via the SCN is approximately 5% (a ratio of the 8% previously stated above).

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Statistical methodology

All statistics were calculated using standard methods with Statistica (Statsoft v. 8) and graphics were generated either in SigmaPlot (Systat, v. 11) or Microsoft Excel (v. 2010).

Differences in n-values between certain light/dark regimes were due to signal problems on day of recording. For EEG spectrum analysis some animals were excluded due to the increased number of signal artifacts which allowed for the quantification of sleep and wake distribution, but hampered Fourier signal transformation.

Main Figures

For Figure 1 A one-way ANOVA for showed significant differences between Syn10 Cre/Cre Bmal1 fl/-and their controls (P = 0.005) Red asterisks indicate significant differences compared to C57/Bl6 and Syn10 Cre/Cre Bmal1 +/-(post hoc t-tests; P < 0.05, P < 0.01, P < 0.001 respectively).

Comparisons between SCNx Opn4 +/+ and Syn10 Cre/Cre Bmal1 fl/-showed no significance, as did comparison between Sham Opn4 +/+ and C57/Bl6 and Syn10 Cre/Cre Bmal1 +/-controls.

For figure 2, (a) A three-way ANOVA (genotype, SCN condition, time-course),

showed significance for "SCN condition x genotype" (P = 0.004), "SCN condition x timecourse" (P < 0.0001), "genotype x time-course" (P = 0.01), "SCN condition" (P < 0.001), and "genotype" (P = 0.01); post-hoc t-tests showed significant (P < 0.05) genotype differences

Materials and Methods -Chapter 5-Opn4 photic regulation in arrhythmic mice

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(red psi), and SCN differences (red delta). For Syn10Cre/CreBmal1fl/-a two-way ANOVA (genotype, time-course), showed significance for, "genotype x time-course" (P = 0.01),

"time" (P < 0.001), and "genotype" (P = 0.007); post-hoc t-tests showed significant (P < 0.05) genotype differences (red phi). Dunnet's test was used for (b). (c) One-way ANOVA (genotype) -P < 0.05(d) One-way ANOVA (genotype) -P = 0.005, post-hoc P < 0.05. € . A two-way ANOVA (genotype, SCN condition), showed significance for "SCN condition" (P = 0.002), and "genotype" (P < 0.001) [post-hoc t-tests against control group Sham Opn4 +/+ and C57/Bl6 ; P < 0.05, P < 0.01, P < 0.001 respectively, Red asterisks in (c)]. SCNx Opn4 -/-L-D difference in NREM was not significantly different from zero as determined using One-Sample Signed Rank Test [(P = 0.48) insert)].

EEG Immunohistochemistry (IHC)

AVP and CTB immunostaining was carried out as described previously 4 . Free-floating slices were heated to ambient temperature before being rinsed with a solution containing PBS and 0,25% of Triton-X-100. Then slices were blocked with a solution containing 0,25% of Arvicanthis ansorgei has been extensively analyzed for the study of circadian rhythms, Indeed the first article using this species clearly identified it's circadian organization as being significantly different from similar sized nocturnal species [START_REF] Challet | Circadian organization in a diurnal rodent, Arvicanthis ansorgei Thomas 1910: chronotypes, responses to constant lighting conditions, and photoperiodic changes[END_REF].

Challet et al, confirmed that of the 108 animals screened, 84% were clearly diurnal, with activity onset slightly before and after (06:20 AM and 07:40 PM, respectively) light-dark transitions. Subsequent studies examined the circadian expression of neuropeptides in the SCN [START_REF] Dardente | Daily and circadian expression of neuropeptides in the suprachiasmatic nuclei of nocturnal and diurnal rodents[END_REF], melatonin output of the pineal gland (Garidou-Boof, [START_REF] Garidou-Boof | Environmental control and adrenergic regulation of pineal activity in the diurnal tropical rodent, Arvicanthis ansorgei[END_REF], and resetting of the circadian clock through use of hypocaloric feeding [START_REF] Mendoza | Setting the main circadian clock of a diurnal mammal by hypocaloric feeding[END_REF]. These studies taken together confirm a strong, consistent, diurnal circadian rhythm, yet this was never established using EEG recording. To date only two sleep EEG studies exist which specifically examined a diurnal rodent, yet were limited into their characterization of sleep regulatory mechanisms. In 1989, Dijk and Daan, implanted a series of electrodes in a chipmunk Eutamias sibiricus, with the purpose of identifying sleep and wake distribution across the 24-hour day [START_REF] Dijk | Sleep EEG spectral analysis in a diurnal rodent: Eutamias sibiricus[END_REF]. Additionally, a significant increase in the level of delta power was seen following a 24-hour sleep deprivation, as measured through power spectrum. This was further explored in a follow-up study in 1991 by the same group, though with the purposes of examining drug interaction [START_REF] Dijk | Effect of clomipramine on sleep and EEG power spectra in the diurnal rodent Eutamias sibiricus[END_REF]. Though the initial results were promising, an animal which slept 75% during the dark period, further experimentation was stopped due to the isolated research conducted on a biologically poorly understood model, as well as the lack of laboratory colonies and difficulties to realize additional studies. .

It is currently unknown as to the evolutionary and physiological change necessary for an animal to be diurnal or nocturnal. Crucial to this understanding is comprehension of the sleep and alertness inducing areas of the brain and why they would operate in the opposite fashion when exposed to external light and darkness. Induction of sleep or waking is thought to depend on a reciprocal inhibitory interaction between structures within the brain, such as the galanin-containing GABAergic neurons of the ventrolateral preoptic nucleus (VLPO) for sleep, and the tuberomammillary nucleus, locus coeruleus, and raphe nuclei, containing of histaminergic, noradrenergic, and serotoninergic neurons, for arousal [START_REF] Saper | Homeostatic, circadian, and emotional regulation of sleep[END_REF]. Given our observation of c-Fos activation of "sleep promoting galaninergic neurons" neurons of the VLPO in response to light application [START_REF] Tsai | Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice[END_REF]), our hypothesis is thought that this "flip-flop" switch should therefore be inverted in a diurnal species, with light stimulating arousal, and darkness promoting sleep, as is the case in human beings [START_REF] Münch | Wavelength-dependent effects of evening light exposure on sleep architecture and sleep EEG power density in men[END_REF]).

In the current study we sought to address whether arvicanthis is a valid model for studying the regulatory mechanisms of sleep. In addition we aimed to determine whether it is indeed a diurnal rodent based on EEG activity, and whether the direct effects of light are inverted to those we and others recently characterized in nocturnal mice. Finally, our goal is to determine whether these effects inversely alter the balance of the flip-flop switch, as a first step towards understanding the change between nocturnality and diurnality.

Results -Chapter 6-Studying sleep in a diurnal rodent model

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-Results

EEG characterization of sleep and waking in arvicanthis ansorgei

Before examining the regulatory mechanisms of sleep, in arvicanthis ansorgei the first step was to characterize waking, NREM and REM sleep. During waking arvicanthis showed highly desynchronized EEG and high muscle activity on the EMG, as well non-rhythmic eye movements on the EOG. The EEG power spectrum analysis revealed, in comparison to sleep, a higher composition of high frequencies rhythms such as theta and gamma activities, also observed in nocturnal rodents during wake (Fig. 6.1a, b). In NREM sleep the animal showed highly synchronized EEG activity, associated with low muscle tone, consistent with a resting state. Additionally no eye movements were observed (Fig. 6.1c). The EEG Power spectrum was dominated by delta frequencies (0.75-4 Hz), characteristic EEG rhythm of NREM sleep (Fig. 6.1d). REM or rapid-eye-movement sleep, characterized by highly desynchronized EEG activity, coupled with complete muscle atonia, and rapid eye movements every several seconds, circled in red on EOG with highlighted corresponding portions of the EEG (Fig.

6.1e).

Note the EEG theta-waves characteristic of this state (Fig. 6.1f).

Circadian, homeostatic and crepuscular components regulate sleep and waking in arvicanthis

Daily and circadian distribution of sleep and waking in arvicanthis ansorgei

Through previous observation of wheel running activity showing that arvicanthis is mostly active during daytime, the diurnality of the species needed to be verified with daily sleep recordings under a standard 12hL:12hD cycle. During baseline, wake accounted for 62%

during the light period compared to 34% in the dark (Fig. 6.2a). Conversely, there is also a significant difference of time spent in NREM or REM between the light and dark periods.

These values are summarized below in Fig. 6.3e. NREM sleep is relatively mirrored to the wake pattern across the 24-hour period. However, REM sleep is almost twice as high during the dark period as compared to the light period, though this may be a function of the REM homeostasis mechanisms, especially from an ultradian driven mechanism dependent on previous time spent in NREM and not on circadian influence [START_REF] Benington | REM-sleep timing is controlled homeostatically by accumulation of REM-sleep propensity in non-REM sleep[END_REF].

Observation under standard 12hL:12hD cycle suggests a circadian distribution of sleep and waking across the day, yet confirmation in the absence of light remained to be determined, thus the animals were placed under 24-hour constant darkness. Under this condition there was an overall decrease in the amount of waking, a slight increase in NREM, and a significant increase in REM sleep. These differences between 12hL:12hD and DD are possibly due several factors: i.) a decrease in the amplitude of the clock signal; ii.) a sustained direct effect of light; iii.) the absence of light influence on melatonin suppression. Under the constant darkness protocol, data suggest that arvicanthis has a period closer to 12-hours due to crepuscular activity (see below), though displaying an overall daily distribution of sleep and waking relatively similar to baseline conditions (Fig.

Circadian regulation of locomotor activity and sleep and waking

Though arvicanthis ansorgei was previously shown to be a diurnal animal based on wheel running measures of locomotor activity, given the variability sometimes seen in this species confirmation was needed of their activity rhythm prior to EEG implantation. After several weeks under a standard 12hL:12hD cycle a clear pattern of daytime activity (wheel running), with an almost complete cessation during the night period, was observed (Fig.

6.3a).

Examination of the total number of wheel counts between the light and dark periods (Fig. 6.3b) further confirmed this rhythmicity in all animals included in the study. There was a highly significant difference (p<0.001), indicating use of the wheel preferentially during the first 12-hours of the day.

However, the circadian organization of sleep and wake as measured with the EEG suggests, as mentioned before, a weak sleep-wake rhythm in contrast to the high amplitude locomotor activity rhythm (Fig. 6.3c). Note the dramatic difference of distribution of locomotor activity compared to sleep and wake with light and dark. The main experimental difference between both conditions results from the wheel suggesting that the reward properties of locomotor activity might synchronize the clock. The direct effects of light are inverted to nocturnal rodents

Results -

The direct effects of light are known to highly effect sleep in nocturnal rodents, as discussed in chapter 3 and 4 [START_REF] Altimus | Rods-cones and melanopsin detect light and dark to modulate sleep independent of image formation[END_REF]Lupi, Oster et al. 2008;[START_REF] Tsai | Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice[END_REF], thus the next experiment was to confirm the significance of this regulatory mechanism in a diurnal species. Assuming that the neuroanatomical mechanisms required are inverted as compared to mice, exposure to light should awaken the animal, whereas exposure to darkness should induce sleep. A 1-hour dark-pulse was given 3 hours after light onset (ZT3). To remove any possible confounding factors animals were kept under a 12hL:12hD cycle. A significant induction of NREM sleep was seen when compared to the same ZT from the previous day under a baseline condition and consequently a decrease in wake occurred (Fig. 6.5a). REM increased slightly, but not significantly as compared to baseline. However, this is in line with what was seen under the 12hL:12hD condition, in that there is less REM during the light period. This confirms that at least around this time during the 24-hour period the animal is susceptible to an induction of sleep by darkness.

To specifically examine whether light could alert the animal in the same way as darkness in mice, a 1-hour light pulse was given 3 hours after dark onset at ZT15. Based on EEG analysis under the baseline condition, the animals are normally asleep at this time. As can be seen in Fig. 6.6b, reactivity to the light pulse was almost immediate, with the animal fully awake after 15 minutes. Waking was relatively maintained across the pulse, though the animals did not immediately return to sleep with its cessation. When the EEG spectra was analyzed across the light pulse a strong theta (6-10 Hz) and gamma (40-70 Hz) induction was observed correlating with the maximum period of wake (Fig. 6.6c,d). This confirms that the direct effect of light is indeed present and likely modulated by melanopsin, and that it remains inverted to the results seen in mice. repeated measures ANOVA across the dark pulse indicated significant differences for both theta and gamma (p<0.05; n=4).

Results

The alerting effect of light is dependent on time of day

Following a test of the direct effects via single 1-hour light and dark pulses, animals were placed under an ultradian 1hL:1hD cycle which examined at the effect of light pulses throughout the 24-hour day. This lighting condition was originally designed to study the effects of light under a circadian cycle [START_REF] Borbely | Control of sleep states in the rat by short lightdark cycles[END_REF]. Consisting of alternating light and dark pulses over a period of 24-hours, this protocol has shown efficacy in the past as a way of illustrating the time-dependent direct effects of light [START_REF] Altimus | Rods-cones and melanopsin detect light and dark to modulate sleep independent of image formation[END_REF][START_REF] Tsai | Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice[END_REF]) (Fig. 6.6a). When the reactivity is examined during each of the light pulses, wake promotion is almost only observed during the subjective dark period.

Another unanticipated finding was that the dark-induced wake promotion was delayed in arvicanthis contrary to what we previously noticed in mice [START_REF] Tsai | Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice[END_REF])(Fig. may be a consequence of the diurnality of the animal being weaker in the absence of the physical reward system generated by the wheel. However, the amplitudes of the locomotor activity and the sleep-wake rhythm may simply be dissociative with one another. To remove possible bias created by the wheel, locomotor activity should be recorded using infrared motion detectors which we propose to address in future experiments. Regardless of the reason for these differences, the circadian and diurnal components are well-conserved, being clearly present under a variety of light/dark conditions: DD, 1hL:1hD, and 12hL:12hD baseline.

Overall when assessed against nocturnal rodents, the sleep and wake distribution is relatively inverted providing the scientific community with a clear diurnal rodent model for sleep neurobiology.

Homeostatic sleep pressure builds more rapidly

The arvicanthis has been shown in the current study and those previous to be an extremely active animal. Increased activity is known to augment homeostatic pressure in animals [START_REF] Tobler | Sleep EEG in the rat as a function of prior waking[END_REF][START_REF] Dijk | EEG power density during nap sleep: reflection of an hourglass measuring the duration of prior wakefulness[END_REF]. The time course of delta activity following the two periods of high activity suggests that sleep pressure builds up faster than in mice or rats, yet this remains to be further established. Given that there are two peaks of activity it may be pertinent to perform a sleep deprivation at light onset (ZT0) as well as dark onset.

The assumption was made that as a diurnal animal the most obvious point in time would be at dark onset (ZT12). However given the results which were obtained, we might find similar increases in delta power if we were to shift the sleep deprivation starting point. It

Crepuscular activity overrides the circadian drive

Under standard 12hL:12hD cycles the animals have clear peaks of crepuscular activity which are conserved, albeit altered, when the light condition changes. Several reasons might explain this particular behavior. Primarily one must consider the environment in which the arvicanthis comes from. In the sub-Saharan grasslands, average temperature can reach above 40° C as the day progresses, forcing the animal to rest during certain times, such as the "siesta-like" period of NREM activity seen between ZT5-9. Following this, activity slowly increases, maximizing around sunset and dusk, continuing after "lights-off" in a laboratory environment. It is likely at this time that the animal searches for food and shelter before sleeping during the night. Video recording also confirms that at certain times during both the light and dark period the animal is engaged in a waking state with restricted movement, confirmed with EEG and EMG. Further video comparison between animals showed that during the light and dark transitions most were engaged in foraging behavior, having buried their food in the preceding period. Furthermore, this bout of activity is repeated around light onset or sunrise/dawn, suggesting rather an ultradian cycle of 12-hours rather than a circadian period of 24-hours. Additionally, given the fact that this crepuscular behavior persists under constant darkness (Fig. 6.5a) and under an ultradian 1hL:1hD cycle (Fig. 6.8b), this is probably an evolutionary trait which is unable to be suppressed even in animals reared in a research setting with consistent lighting conditions. Moreover, these animals have only been bred for a few generations for experimental work in comparison to mice and rats which have long term adaptations to laboratory reproduction. Of note is the fact that animals nearer to the equator are exposed to relatively the same numbers of daylight across the year, making the 12hL:12hD cycle even more appropriate than for other rodents. If in fact this direct effect of light can be overridden in this animal at certain times of the day by a progressively suppressed mechanism, it asks the question as to whether in human beings a similar system exists underneath our forced daily activities. Finally, given the arvicanthis is as adapted to experimentation as it is, yet still retaining certain characteristics closer to what is likely observed in most rodents and other mammals in their natural environment, makes the model even more valuable. Finally, it would be of critical interest to study the circadian and crepuscular regulatory mechanisms under dawn and dusk experimental conditions as recent publications suggested in flies [START_REF] Vanin | Unexpected features of Drosophila circadian behavioural rhythms under natural conditions[END_REF]) and another species, that a comprehensive re-examination of circadian behavior and its molecular readouts under in alertness as measured by theta and gamma induction; however this induction was not sustained across the pulse as is normally seen in mice (see chapter 4). Mice also vary in their reactivity, though are more consistent across the day in general with melanopsin and rod/cones compensating for one another. The complexity of the arvicanthis retina may be responsible for waking up the animal, without actually being able to consistently activate the arousal system. Previously these animals were shown to react to very low luminosities, and the increased number of cones or ipRGCs in the retina may play a role [START_REF] Boudard | Retinas of the diurnal rodent Arvicanthis ansorgei are highly resistant to experimentally induced stress and degeneration[END_REF]. Physiological studies on the retina of the arvicanthis have identified a highly complex light interpretation system unlike many nocturnal rodents. For example, Boudard et al. found highly light resistant structures in the retina which prevent the type of phototoxicity seen in nocturnal species, likely an adaptation for an active lifestyle in full sunlight [START_REF] Boudard | Retinas of the diurnal rodent Arvicanthis ansorgei are highly resistant to experimentally induced stress and degeneration[END_REF]. Further research from the same group showed that this animal possesses ten times the number of cones as compared to other laboratory rodent species, sending visual as well as non-visual photic signals to the brain. This retinal structure difference suggests that these systems may not counterbalance as well, and as in mice during the subjective dark period, the effects might be melanopsin-dependent. However, the amplitude of wake reactivity to light pulses in arvicanthis is nearly identical to what is seen during a dark pulse in a mouse, leading us to conclude that this represents a significant regulatory mechanism. Furthermore, a sustained effect from light may also be present, yet without transgenic models it is difficult to address this question. An alternative to removal of melanopsin would be exposure to monochromatic light, preferentially stimulating the retinal substructures of the photodetection systems, allowing us to examine their isolated functionality. The fact that the pulse is given three hours after dark onset, during a time when sleep pressure is at its highest in these animals, may suppress the reactivity of the system to non-circadian light. The system seems to react more efficiently during the subjective dark period Future experiments need to address these results and try to challenge the system more effectively, such as by increasing the length of the pulse to 2 or 3 hours, as analysis of the 1hL:1hD condition showed a promoting effect of light, though mostly consolidated to the second half of the pulses. Furthermore shifting the pulses further into the dark period away from the crepuscular activity zone around ZT12 persisting after light has been extinguished.

Criticism of the 1hL:1hD protocol usually concerns the fact that a light or dark pulse given during one hour will influence the reactivity of other periods during the cycle. During the 1hL:1hD cycle this is clearly underlined by the lack of reactivity under subjective light and a very clear and significant reactivity under subjective dark. One possible explanation is that the animal sleeps enough during the subjective light period due to the pulse activity, that it is able to awaken more easily at a time period where it is not expecting light. However, the reactivity also seems to show that dark pulses are promoting NREM sleep at this time as well.

The direct effects of light and dark pulses on sleep have been recently revisited in mice (i.e.

melatonin deficient animals which represent a main difference with arvicanthis). Melatonin, a hormone secreted by the pineal gland, is responsible for sleep promotion in most mammals, and can also powerfully synchronize the clock. According to previous studies on pineal secretion in arvicanthis, the peak of melatonin release is during the second half of the dark period starting at ZT2 [START_REF] Garidou-Boof | Environmental control and adrenergic regulation of pineal activity in the diurnal tropical rodent, Arvicanthis ansorgei[END_REF]. Melatonin production is suppressed by light, thus these pulses should be affecting its output, or at least tapering it to some extent.

However, given the magnitude of the effects of the pulse during the first part of the dark period, it is unlikely that the alerting effect of light in arvicanthis depends only on melatonin suppression. Further studies in pinealectomized animals should clarify this point. Moreover, the amplitude of reactivity is the same in mice lacking melatonin and would be significantly different if this were a factor, though this is not the case.

In conclusion, the current study validates Arvicanthis ansorgei as a model for the translational approach in connecting findings from transgenic mouse models to human beings.

The model represents a great compliment to others for improving our understanding on the neurobiology of sleep, as well as the switch between nocturnality/diurnality. Ultimately this will lead to a greater understanding of a variety of pathologies and sleep disorders seen in human beings.

Introduction

Introduction and rationale

Light exerts pervasive effects on mammalian physiology and behavior, including sleep and alertness, yet its putative influence on sleep homeostasis remains unclear and is still under discussion. In 2009, our group reported that the peak of EEG delta activity reached after a 6 hour sleep deprivation was half decreased in mice lacking melanopsin [START_REF] Tsai | Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice[END_REF]). This observation from a single sleep deprivation experiment and performed in a knockout mice from a mixed genetic background, implied the need for further investigation.

However, this original finding raised the question as to whether a photopigment can act as a sleep homeostatic factor and thus whether melanopsin could be a biological link between light and sleep homeostasis. Therefore, we chose to revisit this possible influence of light. To answer these questions we performed various sleep deprivation experiments in melanopsin deficient mice of different genetic backgrounds and under various light and dark conditions.

Additionally, because the effects of light in mice (i.e. nocturnal animals are inversed to humans), we also conducted the same protocol in a diurnal rodent model (a section of which is included in the previous chapter), arvicanthis ansorgei, that we previously validated as a model for sleep study. Ultimately, the aim is to examine whether light and/or sleep homeostasis might affect melanopsin expression, a part of the project which I participated in but is still underway by other members of the team. All mice used in the study were implanted specifically for this protocol. Due to time constraints arvicanthis were used which had previously underwent protocols as described in Chapter 6.

Methods -Chapter 7-Melanopsin as a homeostatic factor

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-Materials and Methods

Experimental light conditions

To determine the homeostatic response in both mice and arvicanthis, a comprehensive ATT CAT CGA C-3'from Integrated DNA Technologies, Inc.), previously described (Ruby, Brennan et al. 2002).

Each animal was implanted using a standard set of electrodes. EEG power spectrum was analyzed and quantified according to standard criteria (Franken, Malafosse et al. 1998).

EEG implantation

Prior to implantation, animals were anesthetized via intraperitoneal injection of pentobarbital (68 mg/kg; protocol-approved), implanted with two EEG, one reference, and two EMG electrodes. After two weeks of recovery from surgery and habituation to the recording conditions a baseline recording was performed during 48 under 12hL:12Dh (white fluorescent lights, 150 lux, measured at the bottom of the cage) using commercially available hardware and software (Micromed France, SystemPLUS Evolution version 1092). Animals were subsequently kept under standard LD conditions and subjected to sleep deprivations of varying lengths (see above), realized with gentle handling to prevent over-stressing. Each individual experiment occurred on differing days approximately one week apart. All animals were recorded simultaneously with their respective controls.

Delta power spectrum analysis

For the purposes of analysis, all EEG and EMG signals were amplified, filtered, and converted from analog-to-digital into 256Hz. Following this, a Discrete-Fourier Transform (DFT) was applied producing power spectra between 0 and 100 Hz (at 0.

-Results

All EEG recording conditions (i.e. the whole protocol described above) were completed in melanopsin KO mice of both genetic backgrounds and their controls, as well as in arvicanthis (Animals available for power spectrum analysis: C57/BL6 & 129P2 Opn4 +/+ n=6, Opn4 Mice were recorded for 48-hours consecutively under 12hL:12hD cycle and EEG delta power spectrum during NREM sleep bouts was analyzed. A pronounced and significant difference between Opn4 +/+ and Opn4 -/-was observed across this light/dark condition, yet was most evident during periods of darkness (Fig 7 .2), confirming previously published results [START_REF] Tsai | Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice[END_REF]. As expected in wild type mice, EEG delta power was higher during the dark period due to the buildup of sleep pressure in relation to higher levels of activity at this time of the day. Delta activity was significantly reduced in melanopsin deficient mice, suggesting an attenuation of the homeostatic process in the absence of melanopsin. Opn4 -/-mice (a) showed smaller increases in delta power following SD as compared to

Results

Opn4 +/+ mice (b). In both groups, delta power increased with the length of the SD, though at a higher rate for Opn4 +/+ . Following initial recovery there were not clear differences dependent on the length of deprivation for either group. Delta power is measured as a normalized value against the baseline period where sleep need is lowest, the end of the light phase (ZT8-12).

Values represent mean ± s.e.m. Note only a single preceding baseline day is shown to more clearly illustrate the differences. (Opn4 +/+ -1.5 hr: n=6, 3.0 hr: n=4, 6.0 hr: n=6, 9.0 hr: n=5;

Opn4 -/--1.5 hr: n=8, 3.0 hr: n=7, 6.0 hr: n=4, 9.0 hr: n=4) A Repeated measures ANOVA was performed between time x genotype (1.5 hr, time-p<0.001; 3.0 hr, time-p<0.001; 6.0 hr, time-p<0.001). One-way ANOVAs showed genotype significance (p<0.05) at certain time points (indicated by filled red circles)

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Interestingly, delta power during recovery sleep in mice lacking melanopsin reached a plateau beyond 3-hours of time spent wake with similar levels of delta activity after 6-hour or 9-hour SD as compared with the 3-hour protocol (Fig. 7.4). This suggests a saturation of the system, and thus a severe alteration of the sleep homeostat. These noticeable and significant differences between groups imply a role for melanopsin in the regulation of sleep homeostasis. Mean values for peak delta activity during the recovery period following SD identify significant differences, with Opn4 +/+ increasing substantially with the length of time spent awake. One-way ANOVA for genotype was performed for all experiments. Values represent mean ± s.e.m. Red-asterisk represents statistical significance (p<0.05) after post-hoc analysis, and red-xi symbol a statistical trend (p<0.1). (Opn4 +/+ -1.5 hr: n=6, 3.0 hr: n=4, 6.0 hr: n=6, 9.0 hr: n=5; Opn4 -/--1.5 hr: n=8, 3.0 hr: n=7, 6.0 hr: n=4, 9.0 hr: n=4)

Characterization of the buildup rate of sleep homeostatic pressure in a diurnal rodent, arvicanthis ansorgei

As a first step before examining the putative influence of light and dark on the sleep homeostatic response, we aimed to characterize the sleep homeostat in arvicanthis ansorgei, (i.e. to determine the kinetic of the buildup of sleep pressure as a function of time spent awake). As this animal is a diurnal rodent, the SD protocol was inverted as compared to mice,

Results -Chapter 7-Melanopsin as a homeostatic factor

| 158 with all conditions beginning at ZT12 or dark onset, the period of highest pressure to sleep in these animals. As expected, the delta power observed during recovery sleep increased with time spent awake under deprivation (Fig. 7.5a). Unfortunately due to a system malfunction, recordings during the 3-hour SD were unusable, though will be implemented in a future experiment. Clear differences between protocols were seen, with the highest levels of delta Mean values for peak delta activity during baseline and the recovery period following SD identify differences, with a steady state increase depending on the length of the deprivation.

Values represent mean ± s.e.m. One-way ANOVA for period was performed for all experiments. Red-asterisk represents statistical significance (p<0.05) after post-hoc analysis.

(BL: n=8; 1.5 hr: n=8, 6.0 hr @ ZT12: n=6, 9.0 hr: n=4; 6.0 hr @ ZT0: n=4) through melanopsin may influence the homeostatic process of sleep [START_REF] Tsai | Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice[END_REF]). Here, we aimed to further explore this hypothesis by subjecting mice lacking melanopsin, as well as a diurnal rodent, arvicanthis ansorgei, to a variety of sleep deprivation (SD) under various lighting conditions.

Discussion

At this stage of advancement of the analysis, we propose below to briefly discuss the preliminary results and perspectives.

The proof of concept that a photopigment, melanopsin, affects sleep homeostasis

Besides sleep deprivation, the reference condition to challenge the sleep homeostat, the analysis of the time course of EEG delta activity can give some indication of the homeostatic process. Under a 12hL:12hD cycle, melanopsin deficient mice display a one hour sleep loss. Despite this reduction in sleep time, delta power was dramatically decreased in Opn4 -/-mice, mostly during the dark period, at a time when sleep pressure builds up due to the higher activity of the animal. This observation, in line with our previous analysis suggests an alteration of the homeostatic process that would affect the daily buildup of homeostatic sleep pressure [START_REF] Tsai | Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice[END_REF]).

The realization of sleep deprivation of various lengths allows characterization of the time-course of the homeostatic process to definitively establish its relationship with Opn4.

The buildup rate of EEG delta power peak after sleep deprivation increases as a function of SD lengths and decreases exponentially during recovery sleep, a time-course that nicely describes the homeostatic process. In mice lacking melanopsin, the delta power reached a plateau beyond 3-hours of time spent wake showing a saturation of the system, in essence a Discussion -Chapter 7-Melanopsin as a homeostatic factor

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severe alteration of the sleep homeostat. This might imply that the sleep homeostat would be set up to a different level in absence of melanopsin.

Finally, because these experiments have been conducted in KO mice of a mixed genetic background C57/BL6 & 129P2, one could argue that the background strain used to create the genetic modification is inappropriate for phenotypic analysis of the mutation. In order to control for this, we backcrossed the animals on a pure C57/BL6 background.

Preliminary results (data not shown) suggest similar alterations of the sleep homeostat in OPN4 -/-of different genetic backgrounds, strongly reinforcing this finding, as it is observed in a homogenous, as well as an enriched and diverse genetic group.

Quantification of the sleep homeostatic process in a diurnal rodent, arvicanthis ansorgei

The effects of light, either indirect through the phase shifting of circadian rhythms, or direct on the expression of sleep and waking, are inversed in mice that are night-active animals as compared to humans. Thus the need to study these effects in a diurnal model, for which arvicanthis ansorgei was used. This model has been extensively studied in the chronobiology field, and we previously validated it for sleep study (Chapter 6). To examine the influence of light and darkness on sleep homeostasis in this species, one must first characterize the homeostatic response. The buildup rate of EEG delta power with time spent awake, as well as the exponential decrease during subsequent recovery sleep, describes a nice time-course of the homeostatic process. In preliminary experiments the buildup of sleep pressure seems to be significantly higher as compared to mice, perhaps due to the animal's increased activity during waking periods. The inclusion of a diurnal animal into this protocol further confirms that sleep homeostasis is well conserved across multiple species. Previously, Dijk and Daan exposed a diurnal rodent, eustamicus sibericus to a 24-hour sleep deprivation, finding a 50% increase in the amount of power during the first 3-hours of sleep recovery [START_REF] Dijk | Sleep EEG spectral analysis in a diurnal rodent: Eutamias sibiricus[END_REF][START_REF] Dijk | Effect of clomipramine on sleep and EEG power spectra in the diurnal rodent Eutamias sibiricus[END_REF], though spectrum analysis was broadened to include 1-10 Hz, thus not representative of "delta" power, and therefore not really describing the homeostatic response.

Discussion -Chapter 7-Melanopsin as a homeostatic factor

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Sleep homeostatic response is altered under various light and dark conditions

In addition to the analysis shown in this article, several additional experimental light/dark conditions were used. Animals were placed in constant darkness for 24 hours to observe whether the effect of melanopsin is suppressed in the absence of light. In addition animals were given a 6-hour SD under constant darkness, as well as after 3 continuous days of constant darkness. Finally, animals were placed under constant light for 3 days and given a 6hour SD, to observe the effects after an overexposure of light. Unfortunately logistic reasons inherent with a project this size inhibited completion of the analysis, though preliminary results are promising. Before publication this analysis will be realized to form a more complete story in addition to the data already presented.

Modulation of melanopsin

Constant exposure to light and dark modulates the melanopsin system

Though not yet fully analyzed, one important question still unknown is the homeostatic response to mice in the absence of melanopsin following a sleep deprivation after several continuous days under constant days of light or darkness. Previous studies in rats by [START_REF] Hannibal | Light and darkness regulate melanopsin in the retinal ganglion cells of the albino Wistar rat[END_REF] on melanopsin showed that after several days of Discussion -Chapter 7-Melanopsin as a homeostatic factor

| 163 darkness, overexpression of mRNA in the RGCs was evident, increasing with the number of days. Conversely prolonged exposure to light suppressed the melanopsin mRNA expression.

It is therefore possible that the homeostatic process if related to this expression of melanopsin will similarly be affected if sleep deprivations are performed after these light-dark conditions.

Perspectives

This study represents the first comprehensive experimental protocol to examine the link between light and sleep homeostasis, utilizing not only a transgenic model, but a comparable diurnal rodent as well. After continued exploration on the remaining experimental conditions, in addition to melanopsin RNA and protein analysis, comprehension of this elusive regulatory mechanism will be closer to fruition. Furthermore, with the results seen in arvicanthis, this may serve as a valid translational model to human beings, allowing similar protocols to be implemented and studied in clinical settings. Sleep deprivation caused by insomnia and other sleep disorders is known to drastically affect mood, behavior, memory, and many other abilities essential to daily life. Understanding the mechanisms underlying the relationship of light to sleep homeostasis is a major step towards finding ways to treat dysfunctions.

Conclusions and Perspectives

| 164

Conclusions and Perspectives

The data presented in this dissertation represents strong evidence to support the existence of a third regulatory mechanism for sleep and alertness. The two-process model has The first research project identified a secondary non-circadian function of the SCN, previously alluded to, as a relay system for the direct effects of light on sleep and alertness.

The prevailing consensus on the functionality for the SCN was that it serves as the master circadian pacemaker, responsible for timing peripheral clocks, and synchronizing around a 24-hour day. However, its physical location at the terminus of the retinohypothalamic tract makes it well-placed to receive non-visual direct light signals, transduced via rods and cones and melanopsin cells in the retinal ganglia. Indeed, removal of the clock removed half of the acute direct effects of light and darkness on sleep and waking, suggesting a relatively even functional distribution of retinal fibers extending both through and around the SCN.

Conclusions and Perspectives

| 165

More importantly, when the EEG was altered across the different mice groups under sustained exposure to light (normal 12hL:12hD conditions), leading to the conclusion that melanopsin-based direct photic input drives 1/3 of the daily distribution of sleep and waking.

This observation calls into question years of chronobiology research that paid little consideration to the direct non-circadian effects of light, also termed "masking effects" given as well as the way we in our individual daily lives artificially expose ourselves to light and darkness.

  d'Afrique Subsaharienne et maintenu au laboratoire depuis une quinzaine d'années. L'activité locomotrice (actimétrie), le sommeil et l'EEG (puissance spectrale) des animaux ont été analysés (après implantation d'un set classique d'électrodes sous anesthésie et après une période d'habituation aux conditions d'enregistrement) dans différentes conditions de lumière obscurité. Concernant les lésions des NSCs, l'intégrité des structures avoisinantes ainsi que des projections rétiniennes vers les structures cérébrales autres que les NSCs ont été vérifiées par anatomie (injection à la fin du protocole d'un marqueur rétrograde, la sous-unité B de la toxine cholérique, dans la chambre postérieure de l'oeil et immunmarquage post-mortem).Résumé en français -Introductory Materials| 13 1 er volet) Effets directs de la lumière médiés par la mélanopsine : un mécanisme suffisant pour maintenir une organisation journalière veille sommeil en fonction du cycle de lumière/obscurité en l'absence de pacemaker circadien (Hubbard et al, prêt pour soumission) Il est généralement admis que le processus circadien est principalement responsable de l'organisation journalière de la veille et du sommeil. En l'absence d'horloge centrale, le rythme d'activité locomotrice reste cependant partiellement synchronisé avec le cycle de lumière obscurité, un phénomène mal caractérisé et suggérant l'existence d'un mécanisme supplémentaire indépendant de l'horloge et qui reste à identifier. Les cellules ganglionnaires intrinsèquement photosensibles de la rétine (ipRGCs) exprimant la mélanopsine, relaient les « informations non-visuelles » à des nombreuses structures cérébrales. L'activation des NSCs par la lumière, mise en relation avec l'entrainement de l'horloge, ne permets pas d'exclure que les NSCs, au-delà de leur rôle d'horloge, influencent le sommeil en relayant les effets directs de la lumière. Ces deux questions ont été abordées en parallèle en étudiant d'une part des animaux rendus arythmiques après lésion des NSCs ; d'autre part des souris transgéniques également arythmiques et présentant une invalidation de l'horloge mais des NSCs intacts (invalidation d'un gène, la synaptotagmine 10, fortement exprimé dans les NSCs et invalidation restreinte aux NSCs d'un gène d'horloge Bmal1 ; Synaptotagmin10 Cre/Cre , Bmal1 fl/-).

| 14 Figure 0

 140 Figure 0.1-Effets directs d'un cycle ultradien de lumière obscurité (1h :1h) sur le sommeil et l'éveil chez des souris sans SCNs et/ou sans mélanopsine

Figure 0 . 2 -

 02 Figure 0.2-Distribution journalière du sommeil en l'absence d'horloge centrale et/ou de mélanopsine et modélisation des voies cellulaires médiant les différents effets de la lumière sur le cycle circadien veille-sommeil (a) Le sommeil lent est diminué pendant la période de lumière en l'absence de mélanopsine (souris Sham Opn4 -/-). La distribution journalière jour/nuit du sommeil est très nettement atténuée en l'absence de NSCs (SCNx) et complètement abolie en l'absence des deux (SCNx-Opn4 -/-). (b) Les différences accumulées (moyenne sur 2 jours) entre

  Figure 0.3-Quantification des effets directs de la lumière sur le sommeil en l'absence de mélanopsine, de noyaux suprachiasmatiques ou d'horloge circadienne.

Figure 0

 0 Figure 0.5: Activité EEG delta en sommeil lent chez les souris Opn4 +/+ and Opn4 -/-en réponse à des privations sommeil de durée variable

Figure 30 Figure

 30 Figure 1.1: An early EEG recording from Hans Berger Figure 1.2: Schema of the thalamocortical loop Figure 1.3: The two-process model of sleep and wake regulation Figure 1.4: SCN control of the pineal gland and secretion of melatonin Figure 1.5: Proposed mathematical equations to quantify the 2-process model Figure 1.6: Hypnogram of healthy adult subject Figure 1.7: Continuous and periodic EEG signal calculation Figure 1.8: Example of the power spectrum of a clinical subject during N3 Figure 1.9: Hypnogram for 24-hours in a normal mouse Figure 1.10: Delta power following a 6-hour sleep deprivation

Figure 1 . 1 :

 11 Figure 1.1: An early EEG recording from Hans Berger (Berger 1929)

Figure

  Figure 1.2: Schema of the thalamocortical loop

  First described in 1982 by Borbély, this model has driven both the clinical and fundamental sleep research fields for the past three decades (Borbely 1982). Fig. 1.3 summarizes the two models.

Figure

  Figure 1.3: The two-process model of sleep and wake regulation

  Figure 1.4: SCN control of the pineal gland and secretion of melatonin in rodents

  In order to remove some of the abstraction of the theory, several attempts have been made to mathematically quantify the 2-process model. In 1999, a review by Borbély and Achermann, put forth several equations to describe these two phenomena, illustrated below in Fig.1.5.

Figure

  Figure 1.5: Proposed mathematical equations to quantify the 2-process model from (Borbely and Achermann 1999)

  Figure 1.6: Hypnogram of healthy adult subject (NREM stages are listed in varying shades of blue)

Figure 1 . 7 :

 17 Figure 1.7: Continuous and periodic EEG signal calculation

Figure

  Figure 1.9: Hypnogram for 24-hours in a normal mouse (personal data)

  and "REM off" structures, respectively activated and silent during REM sleep[START_REF] Pace-Schott | The neurobiology of sleep: genetics, cellular physiology and subcortical networks[END_REF]. According to this model of reciprocal interaction, the level of activity of PRF (pontine reticular formation) neurons, which are under control of these on/off components, is critical for REM sleep generation. Based on more recent data, other researchers, especially the group of Lyon, propose an updated integrated model. This hypothesizes that transitions from NREM to REM are due to the stimulation of the GABAergic neurons comprising melanin concentrating hormone (MCH) in REM-on contained in the ventrolateral periaqueductal gray, the dorsal paragigantocellular reticular nucleus, and the posterior hypothalamus. Conversely, REM episodes terminate with the inhibition of these neurons by using a REM-gating system composed of these GABAergic neurons of the ventrolateral periaqueductal gray, and other systems involved in waking such as those containing noradrenergic and hypocretinergic neurons. Serotonergic and monoaminergic neurotransmitter levels rapidly decrease during REM, yet cholinergic neurotransmission increases to levels seen in a waking brain. REM sleep is also characterized by rapid eye movements, generated at the pontine level, whose signal fromSleep in rodents -Chapter 1 -Sleep| 49

  Figure 1.10: Delta power during 24-hours of baseline followed by a 6-hour sleep deprivation and recovery in a mouse adapted from Franken et al., 2001 (Franken, Chollet et al. 2001)

  Figure 2.1: Model of the human eye

56 Figure 2

 562 Figure 2.2: Simplified schematic of the mammalian retina outlining location of cell types and interrelation -Lok, 2011 (Lok 2011)

  Non-visual light and melanopsin-Chapter 2 -Light| 58 being targeted by projections from the retinohypothalamic tract (RHT). Groups lead bySaper and Hannibal (Gooley, Lu et al. 2001; Hannibal and Fahrenkrug 2004) demonstrated that these melanopsin ganglion cells projected directly via the RHT to the SCN and other areas of the brain, thus confirming the initial experiments by Foster. Several years later researchers identified the absorption peak of the photopigment as centered to the blue light (460-480nm), close to the visual spectrum of the S-cones, suggesting wavelength specific adaptation of the system (Fig.2.4). Use of a rodless-coneless animal was extremely important in proving the existence of Opn4, however it was not until the use of melanopsin knock-out mice that the contributions were specifically elucidated. In 2008 and 2009, concurrent research studies using this model from Tsai et al. and Altimus et al, and Lupi et al., identified a direct non-circadian influence of non-visual light on sleep and alertness,

Figure 2 . 4 :

 24 Figure 2.4: Melanopsin photopigment absorption peak adapted from Hankins et al. (Hankins, Peirson et al. 2008)

Figure 2

 2 Figure 2.5: retinal targets to different brain areas, adapted from Hannibal and Fahrenkrug (Hannibal and Fahrenkrug 2004).

4. 1 . 2

 12 The synaptotagmin10-Cre Bmal1 conditional KO mice (conditional deletion of the clock gene Bmal1 using the Syt10Cre driver)The second transgenic mouse model included in the first experimental study was composed of Synaptotagmin10-Cre (Syt10 Cre ) Bmal1 conditional knock-out (Bmal fl/-) mice coming directly from the Max Planck Institute for Genetics in Göttingen, Germany. TheseAnimals -Chapter 4-Laboratory techniques and materials| 74 mice were used specifically for transgenic modifications which invalidated their circadian clock in the suprachiasmatic nucleus. Here, we briefly describe the generation of this model; for details see Husse et al(Husse, Zhou et al. 2011). Based on the Allen Brain Atlas(Lein et al., 2007) our collaborators from the Göttingen identified Synaptotagmin10 (Syt10) as a gene strongly expressed in the SCN with relatively few auxiliary expression sites in the CNS, even during development. The generation of a Syt10 Cre driver line enables SCN targeting without targeting of peripheral, non-neuronal clocks. The exon 1 of the Syt10 gene was replaced by a

  : 5'-TCA TCA ACC TCG CAG TCA GC -3'; Reverse-Mel2: 5'-CAA AGA CAG CCC CGC AGA AG -3'; Forward-TodoNeo1: 5'-CCG CTT TTC TGG ATT CAT CGA C-3'. PCR consisted of 30 cycles: 95°C for 30s, 60°C for 30s, 72°C for 1 min. WT band was seen at 290 bp, mutant band at 950 bp. Genotyping of Syt10 mice was performed using the following protocol: 38 cycles with DQ DQQHDOLQJ WHPSHUDWXUH RI Û& XVLQJ WKH SULPHUV-Forward-ƍ-AGA CCT GGC AGC AGC GTC CGT TGG-ƍ Reverse-ƍ-AAG ATA AGC TCC AGC CAG GAA GTC-ƍ and for the KI Reverse-ƍ-GGC GAG GCA GG CCA GAT CTC CTG TG-ƍ

  mice retinas at different time points across the day. For all western blots a standard extraction protocol was used consisting of lysis buffer: 100 µL (120 mg Tris Base 20mM, 435 mg NaCl 15mM, 500 µL Triton 1%, 18.6 mg EDTA 1mM) and16 µL protease inhibitor cocktail (Invitrogen). The lysates were then placed upon polyacrylamide gel for electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Boston, MA). Reaction was then blocked with 5% skim milk and membranes were incubated overnight in a cold URRP DW Û & with either: PA1-781 (1:1000, Affinity), ab65679 (1:4000, Abcam), UF006 Methods -Chapter 4-Laboratory techniques and materials | 76

  were achieved by heating the (250µm) tip of a Radionics (Burlington, MA) TCZ electrode to 55°C for 20 sec by passing RF current from a RFG-4 lesion generator (Radionics). Our group previously refined the various parameters to that we are able to create minimal lesions that spared surrounding brain structures. Briefly speaking, the lesions were performed stereotaxically (Kopf Instrument) with electrodes (0.3mm in diameter) introduced at the following coordinates (stereotaxic coordinates from zero ear bar, nose at +5°: lateral: +/-0.2 mm; antero-posterior: +3.4 and +3.6 mm; dorso-ventral: +0.95 mm;(Paxinos and Franklin 2001)) (Radionics Lesion Generator System) corresponding to the two SCN nuclei.Arrythmicity was confirmed by actimetry recordings under 12-12 LD cycle (10 days) and constant darkness (10 days), and effectiveness of lesions was assessed by periodogram analysis of locomotor activity (ClockLab, Actimetrics, Wilmette, IL, USA). Following the experiments lesions were verified histologically by performing Nissl staining on coronal brain sections.All animals underwent identical EEG implantation procedures, with the only exception being the size of the chip for arvicanthis. The chip used for these animals was more robust due to their increased size and activity as compared to mice.Adult male mice and arvicanthis were implanted with a classical set of electrodes including two EEG, one reference, and two EMG electrodes at an age of 10-12 weeks at the time of surgery. Two gold-plated wires were inserted into the neck muscle tissue to record electromyogram (EMG) and two EEG electrodes were implanted on the dura skull over the right frontal and parietal cortex, respectively. In mice the electrodes were positioned in the frontal: 1.7mm lateral to midline, 1.5mm anterior to bregma, and parietal: 1.7mm lateral to midline, 1.0mm anterior to lambda.In a subset of arvicanthis, EOG electrodes were implanted under the surface of the skin to record eye movements in order to characterize REM sleep. The five (or seven in case of EOG) electrodes were soldered to a connector and cemented to the skull. Each animal was housed in an individual cage with water and food provided ad libitum. After recovery from surgery (4-7 days) mice were connected to a swivel contact (6/12-Channel, Plastics One) through recording leads and allotted at least one week for cable adaptation. EEG, EMG and EOG signals were amplified, filtered, and their signals analog-to-digital converted and stored at 256 Hz (64-channel bipolar amplifier, Micromed France, SystemPLUS Evolution version1092).All animals were given a 48-hour baseline assessment under 12h:12h light-dark conditions (150-200 lux-white fluorescent lights measured at the bottom of the cage using a lux meter). Following baseline, continuous sleep recordings were taken under a variety of experimental conditions. A minimum of 14 days under 12h:12h LD was used to habituate animals to their baseline condition before proceeding to the next recording condition. Prior to each experiment, a 24-hour period was assessed to ensure that the sleep wake distribution of the animals had returned to baseline levels. Sleep deprivations were performed by gentle

  removed from -Û & VWRUDJH FRQWDLQHUV DQG SODFHG LQ D VWDQGDUG FU\RVWat at -Û & /HLFD Slices were then placed in a free-floating buffer solution for later use. Immunohistological and in-situ hybridizations were performed by other members of the research team, Elisabeth Ruppert and Ludivine Choteau. | 85Given the significance of the aforementioned acute effects of light, we assessed whether this would influence the organism for longer sustained periods, specifically if Opn4based direct photic information could significantly contribute to the temporal organization of sleep and waking within the light-dark cycle. Thus we quantified the proportions of sleep and waking and their distribution across 48-hours under a 12hL:12hD cycle. The dynamics in hourly amounts of NREM sleep (Fig.5.2a and Supplementary Table5.1) showed a clear genotype difference, with Sham Opn4 íí mice displaying an accumulated NREM sleep deficit of approximately 1-hour, lost exclusively during the 12h light period, as we previously suggested 4 (Supplementary Fig.5.5a). Syn10 Cre/Cre Bmal1 fl/-showed similar reactivity to SCNx animals, consistent with previous activity recordings in these animals (Supplementary Fig.5.5b)13 . Time course and total sleep amounts for Syn10 Cre/Cre Bmal1 +/-were nearly identical to both Sham Opn4 +/+ and C57/B6 mice. To confirm that this genotype difference was due solely to a loss of the direct component of the Opn4 photic input and not related to a weakened circadian drive, we performed a complimentary experiment placing both Sham Opn4 +/+ and Opn4 íí mice under constant darkness (DD) for 24-hours. During the first 12 hours the amounts of NREM, REM, and wake in both groups under DD were similar to those of Sham Opn4 íí mice under a 12hL:12hD cycle (i.e. decreased as compared with Sham Opn4 +/+ under the same condition; NREM-Fig.2band REM/wake-Supplementary Fig.5.6) confirming that the 1-hour sleep loss and subsequent attenuation of the daily sleep-wake cycle results is an Opn4-mediated direct photic regulation. The capacity of light to induce sleep was decreased but not abolished in mice lacking SCNs suggesting that the sustained sleeppromoting effect of light for longer periods is mediated partly by the SCN, yet also by other brain relays. The SCNx animal groups, due to missing circadian input, lacked the reduction in sleep observed in control animals during the dark period and their daily sleep-wake cycle was flattened (Fig.5.2a, Supplementary Fig.5.5a). Syn10 Cre/Cre Bmal fl/-mice displayed similar reactivity patterns to SCNx Opn4 +/+ . Arrythmicity of this animal was verified by placing animals under DD and scoring their vigilance states (Fig.5.2c), which showed no differences in NREM between subjective light and dark periods. In SCNx Opn4 +/+ a significant portion (28%) of the daily sleep/wake distribution was still entrained by light and dark phases (Fig. 5.2d), which indicates a direct sleep photic input relayed by brain areas external to the SCN. The role of the SCN remains predominant, supporting 72% of the sleep/wake cycle largely through its function as a clock, but also in relaying the direct effects of light. Nevertheless, in animals lacking both Opn4 and SCNs the sleep/wake cycle is flat with a non-significant difference in NREM between light and dark periods representing 8% of that observed in Results -Chapter 5-Opn4 photic regulation in arrhythmic mice | 86 control animals (Fig. 5.2d). Through measurement of these inter-group differences in NREM sleep in comparison to one another we arrive at a modelization of the relative contributions of each system for the transmission of the direct effects of light (Fig 5.2e). Thus, these direct Opn4-mediated effects of light are responsible for 1/3 of the distribution of wake and sleep across a normal 24-hour light-dark cycle, distributed between the SCN (13%) and other extra-SCN pathways such as the VLPO (20%).Interestingly, the light-dark difference in REM sleep amount was not affected by genotype in either Sham or SCNx mice, suggesting that regardless of its effects on NREM sleep, the direct Opn4-mediated light information poorly influences the 24-hour REM sleep cycle(Supplementary Fig. 5.7; Supplementary Fig. 5.8). EEG theta and gamma partition during wake was significantly attenuated during the dark period in the absence of melanopsin and was annulled in both SCNx groups (Supplementary Fig.5.9), a finding that suggests the alerting effect of darkness in nocturnal animals is mostly mediated by the SCN, a structure known to influence, albeit indirectly, hippocampal activity and possibly theta/gamma rhythms generators5,14 . This lack of induction in theta and gamma was also seen in Syn10 Cre/Cre Bmal fl/- mice, though to a lesser extent (Supplementary Fig.5.10). Currently, there is no proof of a direct link between the alerting effects of light and Opn4 in humans, although several studies comparing the effects of green (555nm) and blue (480nm) light suggest this relationship (for review see 15 ). Our findings provide compelling supportive evidence that humans, like mice, use Opn4-based pathways relayed by the SCN to provide a non-circadian acute, but also a sustained alerting effect of light.Post-mortem analysis of arginine vasopressin (AVP) and 4',6-diamidino-2phenylindole (DAPI) via immunostaining in lesioned mice confirmed the preservation of areas surrounding the SCN in comparison to their Sham controls (Supplementary Fig 5.11ad). Furthermore, detailed analysis using Cholera-Toxin B (CtB) retrograde fiber tracing, showed that retinal projections were conserved in similar proportions to those observed in Sham mice, both in highly innervated brain areas such as the ventral geniculate leaflet nucleus or the superior colliculus and in other areas less innervated but critically relevant for sleep regulation such as the VLPO (Supplementary Fig 5.11e-h). Additionally, the amplitude the direct effects of light observed in SCNx animals was not attenuated, implying the conservation of retinal projections innervating brains areas outside the SCN. The Syn10 Cre/Cre Bmal fl/-mice showed similar reactivity to light and dark pulses as SCNx Opn4 +/+ animals, confirming that effects must be a result of information passing external to it. These clock-disabled mutantsResults -Chapter 5-Opn4 photic regulation in arrhythmic mice| 87

  enough time for the animal to build up sleep homeostatic pressure importantly. This can be compared to multiple nap protocol designed to study sleep regulation in human subjects with low sleep pressure15 . The time-course for building up sleep pressure in mice requires several hours, leading the rodent to display recurrent naps over the course of a 24-hour day 18 . Thus, the dynamics of the homeostatic process are long enough that any effects of the 1-hour light/dark pulses are minute so as to not significantly hinder the sleep balance between the 12hour light and dark phases.The SCN promotes alertness and allows for the maintenance of a daily sleep-wake organization in case of weakened circadian rhythms. However, the direct effects of light now seem critical for adaptation to the light/dark cycle, characteristic of life on earth. This may explain why the direct photic input to sleep and waking is based on two phototransduction systems, melanopsin and the rods/cones, which compensate for one another in case of failure in either of the two signaling pathways. The SCN was treated for years as having one function to serve as the master circadian pacemaker in the body; however these data indicate it has an important role in the transmission of the direct effects of light. Furthermore, until now the role of the direct influence of light on sleep and waking was thought to be negligible, remaining confined to acute transitory effects. The current study demonstrates that direct photic influence corresponds to this unidentified mechanism, capable of compensating for the circadian drive to preserve a synchronization of sleep and waking with the external light/dark cycle in the absence of a functioning clock. These findings challenge the classical two-process model of sleep regulation and the way in which animals interpret light in their environment.Additionally, this calls for a reassessment for the role of light on not only mammalian, but human physiology and paves the way for the development of new concepts for better usage of societal lighting and to tackle sleep disorders and other neuropsychiatric illnesses.Methods Summary -Chapter 5-Opn4 photic regulation in arrhythmic mice| 89

FiguresFigure 5 Figures

 5 Figures -Chapter 5-Opn4 photic regulation in arrhythmic mice

| 94 Figure 5 . 3 :

 9453 Figure 5.3: Effects of a 1-h light pulse on c-Fos immunoreactivity in the SCN in Syn10 Cre/Cre Bmal1 +/-and Syn10 Cre/Cre Bmal1 fl/-mice

Figures

  Figures -Chapter 5-Opn4 photic regulation in arrhythmic mice

  out. 48 hours was recorded under 12hL:12hD light-dark conditions (white fluorescent lights, 150lux, measured at the bottom of the cage) for use as a baseline measurement and made using commercially available hardware and software (Micromed France, SystemPLUS Evolution version 1092). Following this, other continuous sleep recordings were taken under several experimental conditions: (1) a 1hL:1hD cycle for 24 hours, (2) 24-hour cycle of Materials and Methods -Chapter 5-Opn4 photic regulation in arrhythmic mice | 112 constant darkness under red light. All experiments occurred on different days from one

  , (b) Sine waves were calculated to show best fits for the data points using SigmaPlot(Systat, v. 11). The lack of reactivity in SCNx Opn4-/-mice is represented with a dashed line (purple). A four-way ANOVA with factors ''genotype,'' "SCN condition", 'time of day'' (subjective light versus subjective dark period), and ''time course'' (5-min values) revealed that for wake (W) and NREM (N), time course was significantly affected by SCN condition (W: P=0.004; N: P<0.0001) and genotype (W: P=0.003; N: P=0.028) (interactions: genotype x SCN condition: W: P=0.04; genotype x time course: W: P=0.0005 and N: P=0.00001; SCN condition x time course: W: P<0.0001 and N: P=0.0001). Red tridents denote significant genotype differences, red triangles denote significant SCN differences as compared to Sham Opn4+/+ (p<0.05; post hoc t-tests). Psi symbol represents statistically significant genotype differences either between Sham or SCNx groups. Delta symbols represent SCN-condition differences between Sham Opn4+/+ and SCNx groups. (c)

  Photomicrographs

Introduction -Chapter 6 -

 6 Studying sleep in a diurnal rodent model | 125

Figure 6

 6 Figure 6.1: EEG characteristics associated with wake and sleep states

130 Figure 6 . 2 :

 13062 Figure 6.2: Sleep and waking under 24-hours of 12hL:12hD and constant darkness (DD) (a) Vigilance states represented as number of minutes per hour across a 24-hour 12hL:12hD cycle. All subsequent values are expressed as average ± s.e.m. (b) Theta and gamma EEG power spectrum analysis of waking epochs per hour. (n=11) (c) As in (a) though under 24hours constant darkness. Time-course of vigilance states under 24-hours of constant darkness expressed as minutes per hour. A one-way ANOVA between subjective light vs. dark was not found to be significant. A repeated measures ANOVA over the course of 24-hours between one day of 12hL:12hD and DD showed significance (W: p=0.006; N: p<0.001; R: p<0.001). Filled black circles represent significant differences after post-hoc analysis in vigilance states as compared to 12hL:12hD (n=8) (d) Example of NREM sleep per 5 minute bouts of an individual arvicanthis under 3 different light/dark conditions: 12hL:12hD, 1hL:1hD, and 24 hours of constant darkness.

  Figure 6.3: Comparison of daily wheel-running activity and vigilance state distribution a standard 12h:12h LD cycle

Figure 6 . 4 :

 64 Figure 6.4: EEG delta power under sleep deprivations @ ZT12 (a) EEG delta power expressed as a percentage of ZT16-ZT22 during baseline, determined to consistently be the period with the lowest sleep pressure. 6-hour sleep deprivation is displayed as well as the preceding baseline day. (b) Histograms representing delta power during the three first recovery hours after the sleep deprivation as compared to the same baseline ZT (n=8). (c) Sleep deprivation dose response experiment showing an increase in delta power during recovery with an increase in sleep deprivation length.

- 136 Figure 6 . 5 :

 13665 Figure 6.5: The direct effects of a single 1hr LP at ZT15 and 1hr DP at ZT3 (a) Difference in vigilance states during 1-hour dark (grey) and 1-hour light (yellow) pulses as compared to the same ZT and surrounding two hours during the previous day under 12hL:12hD. One-Sample Signed Rank Test confirmed significant changes for all states except REM under dark pulse (wake: DP-p<0.05, LP-p<0.01; NREM: DP-p<0.01, LP-p<0.01; REM: DP-n.s., LP-p<0.01) (n=11). (b) Time-course of wake during the 1-hour light pulse @ ZT15. Values represent amount of wake per 5 minutes bouts. (n=11) (c) and (d) Induction of EEG theta and gamma activities, EEG correlates of alertness, across a 1-hour light pulse (d) a

138 Figure 6 . 6 :

 13866 Figure 6.6: The direct effects of 1-hour light and dark pulses during a 1h:1h LD cycle (a) Time-course of vigilance states under a 1hL:1hD ultradian cycle, measured as number of minutes per hour. (b) Wake reactivity during the subjective dark period (second 12 hours of the 1hL:1hD cycle) of arvicanthis (centered on light pulse). Data represent average of the six 6 light/dark transitions during the subjective dark period expressed as minutes of wake per 5minute bout. Sine-waves were calculated and superimposed. (n=10) (c) Theta and Gamma progression across the light/dark pulses. Note the increase in alertness during the second half of several consecutive light pulses of the subjective dark phase (n=5). Note the maximum amount of waking around ZT0 and ZT12, as a reflection of the powerful influence of the crepuscular regulation.

  is possible that the "gentle handling" required for the sleep deprivation entrains higher activity patterns less research adapted animals in comparison to laboratory rodents, potentially increasing fatigue and thus the rate of sleep pressure buildup. Our complimentary experiment which examined delta power with different sleep deprivations showed a clear dose dependent relationship between the length of the deprivation and the peak of delta activity during sleep rebound. A more complete analysis of all increases in delta in relation to periods of waking needs to be examined to further comprehend the mechanism.Discussion-Chapter 6-Studying sleep in a diurnal rodent model | 141

Discussion -Chapter 6 -

 6 Studying sleep in a diurnal rodent model | 142 simulated natural conditions provide a more authentic interpretation of the regulatory mechanisms controlling the behavior. The alerting effect of light and sleep promoting effect of darkness depend on time of day When the animals are exposed to a single 1-hour light or dark pulse, reactivity occurs which is inverted to what is seen in a nocturnal rodent, however not consistently across all hours of the day. EEG analysis of the signal under the 1-hour light pulse showed an increase

Discussion -Chapter 6 -

 6 Studying sleep in a diurnal rodent model | 143

  in this chapter are given in the back of the manuscript)

  Figure 7.1: Sleep deprivation protocol for mice and arvicanthis

  25Hz resolution) with a 4-s window. Vigilance states during each 4-s epoch were scored either as waking, REM sleep, or NREM sleep, via visual inspection of the EEG and EMG signals, from standardized criteria [3], without prior knowledge of either the genotype or condition. Those epochs which contained severe signal artifacts in the EEG, were removed and did not count for part of the spectral analysis. Additionally, frequencies between 49-51 Hz were taken out, as power-line interference artificially could modify the signal. Power spectrum analysis was focused specifically on the delta area of the spectrum (0.75-4 Hz) during all epochs classified as NREM. Values were normalized in relation to mean values during the last 4-hours (ZT8-12) of the light period (mice) or the ZT20-23 of the dark period (arvicanthis). Results -Chapter 7-Melanopsin as a homeostatic factor | 154

- 155 Figure 7 (

 1557 Figure 7.2: Time course of delta power during NREM sleep across 48-hours under a standard 12hL:12hD cycle.

Figure 7 . 4 :

 74 Figure 7.4: Level of delta power reached during recovery after sleep deprivation of various lengths in melanopsin KO mice.

  power during recovery sleep seen after 9-hours of SD (Fig 7.5a). As arvicanthis ansorgei are known to be active at both light and dark onset, and show significant periods of NREM activity during the light period (see chapter 5), a 6-hour SD was performed starting at ZT0 (Fig. 7.5b). Arvicanthis responded to the increased hours of wake, yet during sleep rebound the delta power was slightly lower than the corresponding SD at ZT12.

Figure 7 . 5 :

 75 Figure 7.5: Arvicanthis ansorgei: Time course of delta power during NREM sleep following various lengths of sleep deprivation beginning at ZT12.

Figure 7 . 6 :

 76 Figure 7.6: Level of delta power reached during recovery after sleep deprivation of various lengths in arvicanthis ansorgei.

  a fundamental characteristic of almost all animals living on earth and especially humans. The kinetics of sleep homeostasis vary greatly among species, possibly as an evolutionary consequence to different environmental conditions, especially changes in the natural ambiance light and dark. The link between sleep homeostasis and light, if any, remains to be clarified. A surprising finding by Tsai et al., has suggested that light

  RNA and protein expression across the day, and it's alteration under sleep deprivationUnder a 1hL:1hD cycle as shown inTsai et al, and Hubbard et al (Chapter 4), the effects of melanopsin vary depending on the time of day. To more clearly understand why changes exist, regardless of a direct pulse of light or darkness, it is pertinent to examine the modulation of melanopsin across the day. Initial analysis of qPCR and RT-PCR experiments in both mice and arvicanthis (data not shown), ongoing from another member of the team (CM Gropp), has shown encouraging results, indicating a time-dependent change on expression which is altered when a sleep deprivation protocol is enforced. The final component will be to successfully adapt the western blot procedure to identify protein expression across the same periods, and determine the timeframe between RNA transcription and appearance of the melanopsin protein. Previously a western blot was attempted but with little success likely due to the minute quantity of melanopsin protein available for extraction from a murine retina, in conjunction with anti-bodies not specifically designed for this task.

  been rigorously tested for years and established many of the concepts we currently know in the fields of chronobiology and sleep research. However, since the discovery of melanopsin and non-visual direct light effects on physiology, the model required revisiting. Along this axis the preceding research projects have therefore established several concepts: (I) Reclassification of the suprachiasmatic nucleus as a relay system for approximately 50% of the melanopsin-and rod/cone-dependent non-circadian direct effects if light outside of its role as the central circadian pacemaker, to areas of the brain responsible for the regulation of sleep and waking. (II) Impact of the sustained melanopsin-dependent direct effects of light across an habitual 12h:12h light-dark cycle, accounting for approximately 1/3 of the daily sleep and wake distribution under normal conditions. (III) Efficacy of using a diurnal rodent, arvicanthis ansorgei to study the regulatory mechanisms of sleep, including the direct effects of light, and creating the first steps towards a translational model between classical laboratory rodents and basic human research for the study of sleep. (IV) Establish that melanopsin affects the sleep homeostat to propose a first biological link between melanopsin, light, and sleep homeostasis, in both nocturnal and diurnal species.

  their ability to mask circadian rhythms. The daily partition of sleep and waking with light and dark was thought to be controlled mainly by the circadian clock, entrained by light. In fact, our findings establish, at least in nocturnal animals, that the Opn4-mediated direct effects of light represents a regulatory mechanism that can maintain a sleep-wake cycle with light and dark to keep the animal adapted to life on earth in the absence of a circadian drive. Lesioning of specific brain areas implies the possibility of damage to structures surrounding the SCN even through this point has been checked by functional anatomy. Fortunately transgenic mice possessing intact yet functionally disabled SCN tissue were available for comparison. Nearly identical results as compared to lesioned wild-type animals further validated our findings, each model controlling for the bias related to the other approach. Future experiments need to examine these sustained effects under different lighting conditions, such as using a dawn/dusk protocol, or increasing and decreasing the luminosity to mimic differing latitudes. However, the data should encourage revisiting the role of non-circadian effects of light in mammalian physiology.The evolutionary switch between nocturnality and diurnality, in terms of the internal mechanisms which cause functionality to invert, remain misunderstood. Additionally the majority of basic rodent research is performed in animals which have inversed activity and sleep cycles to humans, presenting a fundamental issue for transfer applying these findings to human physiology. The second project sought to help elaborate on this problem through EEG recording in the Arvicanthis ansorgei, which allowed us to precisely examine the regulatory processes of sleep and wake in such an animal. Sleep EEG and the circadian and homeostatic regulation of sleep were characterized in arvicanthis ansorgei. The results confirmed the animal's diurnal preference for waking, even if this proved less effective than in some of their murine counterparts, validating arvicanthis ansorgei as one of the first diurnal rodent models for the study of sleep. Moreover, a strong direct alerting effect of light, similar to humans and inversed to mice, was noted under several light/dark protocols. Further experiments by the team are underway, specifically seeking to identify brain structures which are activated by light, work previously performed in mice. These data will allow us to better understand theConclusions and Perspectives| 166 neuronal switch responsible for the inversion of the direct effects of light in nocturnal and diurnal species. On a long-term perspective, arvicanthis ansorgei can be a useful model to further decipher the mysteries of nocturnality and diurnality.Though the influence of light on the circadian process is well-known, the connection between light and sleep homeostasis remains to be clarified. Initial observations by our group, found a significant difference in sleep homeostasis between mice with and without melanopsin, a proof of concept that a photopigment can affect the sleep homeostat, though interpretation of this finding in the context of light remains to be established. In order to identify this, the sleep homeostat was challenged in mice lacking melanopsin through various sleep deprivation experiments with determination of the time course of EEG slow wave activity, the most reliable marker of sleep homeostasis. The homeostatic response, characterized by the increase in EEG delta as a function of time spent awake, was dramatically altered in the absence of melanopsin, and was independent of the genetic background. To put this finding in the context of the effects of light, these experimental conditions were also conducted during exposure to constant light and darkness over several days, though time constraints and the complicated nature of power spectrum analysis has postponed completion to a later date. Additionally, the protocol was performed in diurnal rodents with the final goal of deciphering how light and melanopsin might affect sleep homeostasis differently in nocturnal vs. diurnal rodents. Since melanopsin affects the homeostatic process, one could hypothesize that sleep homeostasis is influenced by melanopsin expression, a result confirmed by our team (at least at the mRNA level as at the protein level our experiments failed given the lack of efficient antibodies and low expression of the protein). Completion of this analysis will establish the role of light and melanopsin on sleep homeostasis, leading to future experiments in humans with possible applications to the study of sleep disorders related to neuropsychiatric diseases.In essence this thesis work challenges, from multiple directions, the accepted 2process model for the regulation of sleep and wake, proposing a third process which is independent yet also interrelated. These findings, taken together, suggest a more complex role of light which could affect sleep and waking in different ways: indirectly on the circadian drive, and directly on the expression of sleep and waking and through modulation the sleep homeostat. These results encourage a reevaluation on the role of light in mammalian physiology, and necessitate further validation of this model in humans, perhaps providing a new conceptual framework for understanding the disturbances associated to sleep disorders,

Index of Figures/Tables -Introductory Materials

	Glossary of Terms -Introductory Materials Glossary of Terms -Introductory Materials	| 26 | 27
	GNAT1 PS	guanine nucleotide-binding protein subunit alpha-1 paradoxical sleep (REM)
	HIS rd/rd cl	histamine rodless/coneless
	3V Hz REC	third ventricle hertz recovery following sleep deprivation
	5HT IGL REM	serotonin intergeniculate leaflet rapid eye movement sleep
	ȝ9 2 IPRGC RHT	microvolt-squared intrinsically photosensitive retinal ganglion cells retinohypothalamic tract
	aDTA KO S-CONE	ablated diphtheria toxin A knock-out short-cone
	AVP L SAD	arginine vasopressin light seasonal affective disorder
	BMAL1 L-CONE SC	brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like long-cone superior colliculus
	BSL LC SCG	baseline locus coeruleus superior cervical ganglion
	CA1-CA3 LD SCN	cornu ammonis area 1 and 3 light-dark suprachiasmatic nucleus
	CLOCK LH SCNx	circadian locomotor output cycles kaput lateral hypothalamus suprachiasmatic nuclei lesion
	CNGA3 LP SD	cyclic nucleotide-gated channel alpha 3 light pulse sleep deprivation
	CRY1-2 M-CONE SPVZ	cryptochrome 1 and 2 medium-cone subparaventricular zone
	CTBCtB N3 SWS	cholera toxin subunit B NREM sleep stage 3 slow-wave sleep
	D NA SYN10	dark noradrenaline synaptotagmin-10
	DAPI NIF TMN	4',6-diamidino-2-phenylindole non-image forming tuberomammillary nucleus
	DD NREM VGL	constant darkness or Dark-Dark non-rapid eye movement sleep ventral geniculate leaflet
	DFT OC VLPO	discrete Fourier transformation optic chiasm ventrolateral preoptic area
	DGL OPN4 WT	dorsal geniculate leaflet opsin 4 or melanopsin wild-type
	DP OPT ZT	dark pulse olivary pretectal nucleus zeitgeiber-time
	ECoG PER1-2	electrocorticogram period 1 and 2
	EEG PET	electroencephalogram (used interchangeably when discussing ECoG positron emission tomography
	PGO	in animals) ponto-geniculo-occipital
	EMG PPT	electromyogram pedunculopontine tegmentum
	EOG PRF	electrooculogram pontine reticular formation
	FFT PROCESS C	fast Fourier transform circadian process of sleep regulation
	GABA PROCESS S	gamma-aminobutyric acid homeostatic process of sleep regulation

Introduction -Chapter 1 -Sleep | 34

	The first major contribution came in 1913 from French psychologist
	Henri Pieron who with his text, "Le Probleme Physiologique Du Sommeil" described the
	physiological aspects of sleep he had seen in his patients. In 1929 the most important
	scientific breakthrough for studying sleep was invented when German psychiatrist Hans
	Berger attached the first electrodes to a human scalp. Though these initial

and the field continues to grow. The EEG -Chapter 1 -Sleep | 36 1.2 -The EEG 1.2.1 The EEG

	, to less rare (narcolepsy), or highly frequent, such as restless leg syndrome or the sleep respiratory syndromes. For example, narcolepsy, first observed and studied in the 19 th century, was not fully explained until the discovery of hypocretin in 1998 (de Lecea, Kilduff et al. 1998), changing interest towards the examination of the underlying mechanisms of this disease. In the 21 st century, research in sleep continues to grow exponentially, achieving a higher place within the domain of neuroscience, and involving a variety of other scientific disciplines including but not limited to, genetics, molecular biology, internal medicine, pulmonology, neurology, and psychiatry. Currently there are thousands of scientists in hundreds of clinical treatment and research centers produced in the brain in human beings, which is achieved by placing electrodes on the surface of the scalp in specific configurations. The signal itself is a by-product of communication between pyramidal cells, producing electrical activity which can then be amplified and analyzed. As millions of consecutive cells are aligned in the same direction, a signal propagation wave is produced, eventually reaching the scalp where the metal contained within the electrodes, usually Au or AgCl, will push or pull the ions and devoted exclusively to the study of sleep, Electroencephalography or EEG is the method for assessing electrical activity generate a voltage signal. Electrical signals coming from the electrode are then transmitted

The EEG -Chapter 1 -Sleep

	| 38
	spikes in EEG activity, are topographically different, and normally follow a group of sleep
	spindles.
	The final stage of NREM sleep is called stage 3, or slow-wave-sleep, due to the
	presence of low frequency delta waves (0.5-4 Hz) which are highly synchronized. This
	type of sleep is the primary component of the restorative part of the sleep/wake cycle and
	will be discussed further in section 1.3.3.
	In contrast to NREM, REM sleep is characterized by a desynchronized EEG, more
	similar to waking, though with complete muscle atonia and rapid eye movements. During
	this stage of sleep, rapid low-voltage, desynchronized EEG activity is evident, as well as
	activation in region-specific brain areas such as the visual cortex and those important to
	memory such as the hippocampus, evidenced by the propensity of theta and gamma
	oscillations.

The two-process model of sleep regulation -Chapter 1 -Sleep | 39 1.3 -The Two-Process Model of Sleep Regulation 1.3.1 Two separate but connected processes

5-Opn4 photic regulation in arrhythmic mice

						| 96
	Supplementary Figures
	Schedule	Time Spent	Condition	Opn4 Genotype	Waking (h)	NREMS (h) REMS (min)
	LD 12:12	12-h light period	Sham	-/-	5.48±0.48	5.23±0.45	79.9±3.3
				+/+	4.14±0.10	6.58±0.14	76.8±3.7
			SCN Lesion	-/-	4.71±0.28	6.24±0.21	63.1±4.6
				+/+	4.50±0.19	6.43±0.16	64.4±2.6
		12-h dark period	Sham	-/-	7.80±0.29	3.44±0.27	40.0±3.7
				+/+	7.38±0.30	3.96±0.27	39.9±3.8
			SCN Lesion	-/-	4.90±0.40	6.06±0.30	62.3±6.5
				+/+	5.33±0.21	5.68±0.17	52.1±3.0
		LD difference	Sham	-/-	1.57±0.81	1.29±0.49	34.2±4.5
				+/+	3.35±0.20	2.75±0.12	36.0±0.1
			SCN Lesion	-/-	0.19±0.12	0.18±0.28	0.79±1.9
				+/+	0.83±0.03	0.75±0.02	12.3±0.4
		24 h	Sham	-/-	13.49±0.61	8.69±0.53	125.8±8.3
				+/+	11.62±0.43	10.44±0.43	116.6±8.3
			SCN Lesion	-/-	9.61±0.68	12.30±0.52	125.4±11.2
				+/+	9.82±0.40	12.11±0.33	116.48±5.7
	LD 1:1	Subjective 12-h light period Sham	-/-	6.25±0.44	4.79±0.33	56.72±9.0
				+/+	5.30±0.19	5.73±0.22	59.0±7.6
			SCN Lesion	-/-	5.10±0.18	5.87±0.17	52.4±2.2
				+/+	5.12±0.15	6.03±0.14	50.8±2.6
		Subjective 12-h dark period	Sham	-/-	6.31±0.31	5.06±0.43	37.7±8.6
				+/+	6.79±0.23	4.50±0.20	39.6±7.6
			SCN Lesion	-/-	5.13±0.22	6.03±0.23	50.1±2.6
				+/+	5.06±0.16	6.11±0.15	49.98±1.5
		24 h	Sham	-/-	12.56±0.65	9.86±0.49	94.4±17.4
				+/+	12.08±0.31	10.23±0.32	98.6±14.8
			SCN Lesion	-/-	10.23±0.31	11.9±0.25	102.6±3.3
				+/+	10.18±0.25	12.14±0.25	100.75±3.2
		12 1-h Light periods	Sham	-/-	5.49±0.38	5.55±0.38	57.4±12.2
				+/+	4.54±0.16	6.41±0.18	62.3±8.8
			SCN Lesion	-/-	4.83±0.18	6.29±0.16	53.0±4.7
				+/+	4.36±0.22	6.73±0.22	55.2±2.7
		12 1-h Dark periods	Sham	-/-	7.07±0.20	4.31±0.19	37.0±12.1
				+/+	7.54±0.24	3.82±0.22	38.6±5.6
			SCN Lesion	-/-	5.41±0.36	5.78±0.33	48.6±2.7
				+/+	5.82±0.26	5.42±0.24	45.6±2.7

The time course of EEG delta power under a 12hL:12hD cycle suggests an attenuation of the homeostatic response in the absence of melanopsin (C57/BL6 & 129P2 strain)

	-/-
	n=8; C57/BL6 backcross Opn4 +/+ n=5, Opn4 -/-n=7; Arvicanthis ansorgei n=8). Given the
	length of time of the PhD, data analysis has not yet been completed and we present here
	preliminary results from the few experimental conditions that have already been investigated.
	One experimental result (fig. 7.5a) from the previous chapter is included here as well due to
	its compatibility with the mice protocol.

Acknowledgments -Introductory Materials

| 6 Acknowledgements Acknowledgments -Introductory Materials

-Abstract

Regulation of sleep and waking is classically defined as a two-process model, consisting of circadian (C) and homeostatic (S) components 1 . Light entrains process C aligning sleep and waking with time of day, and additionally exerts a direct effect promoting alertness in diurnal and wake in nocturnal, respectively 2-5 . Melanopsin (Opn4)-containing intrinsically photosensitive retinal ganglion cells (ipRGCs) are essential to relaying nonimage-forming light (NIF) and convey this information to various areas of the brain including the master clock, the suprachiasmatic nucleus (SCN), and others 6-10 . However, what is currently unknown is whether Opn4-dependent direct photic regulation could also shape the sleep/wake pattern consistent with the external light-dark cycle. Here we establish that the Opn4-dependent direct non-circadian effects of light contribute to one third of the temporal organization of sleep and waking of mice under a normal light/dark cycle (12hL:12hD). We analyzed sleep and the EEG under various light/dark regimens in mice lacking Opn4, SCNs, or both, as well as mice lacking a functional clock through transgenesis (Syn10 Cre/Cre Bmal fl/-).

Acute light/dark effects and the temporal organization of sleep and waking under a standard 12hL:12hD schedule, were dramatically decreased in SCN-lesioned or Opn4 -/-mice, and completely flattened in those lacking both. Clock-disabled transgenic Syn10 Cre/Cre Bmal1 fl/- mice reacted similarly to SCN-lesioned (SCNx) wild-type animals. This study demonstrates for the first time, at least in nocturnal animals, that the Opn4-dependent direct effects of light partition sleep and waking within the daily light/dark cycle in the absence of circadian drive.

Additionally, the SCN, beyond its primary function as the central clock, is one of several structures relaying these effects. These findings challenge the accepted 2-process model of sleep/wake regulation and calls for a reassessment for the role of light on not only mammalian, but specifically human physiology.

Results -Chapter 5-Opn4 photic regulation in arrhythmic mice

| 84

-Results

To examine the acute effects of light and darkness on sleep and waking across the day we exposed seven groups of mice to an ultradian 1-hour alternating light-dark cycle (1hL:1hD) over 24-hours and analyzed via EEG, their waking and sleep states. This allowed us to determine the direct photic input to sleep and the respective contribution of different photic pathways as arrhythmic mice such as SCNx and Syn10 Cre/Cre Bmal1 fl/-do not interpret zeitgeiber time (ZT). Under this condition in control animals (Sham Opn4 +/+ ), 1-hour periods of light readily induced NREM sleep whereas dark pulses promoted wakefulness (Fig. 5.1a, Supplementary Table 5.1). Dark to light transitions also favored REM sleep (Supplementary Fig. 5.1a,b). This reactivity to light and dark was significantly higher (P < 0.001) during subjective dark (ZT13-ZT24) as compared to subjective light (ZT1-12). When all light/dark induced changes in wake and NREM sleep were averaged and parsed into 5-minute bouts, a clear pattern of responsiveness emerged (Fig 5.1b; for time course over 24-hours see Supplementary Fig. 5.2). Sham Opn4 íí showed similar reactivity to Sham Opn4 +/+ during the subjective light period but failed to respond as efficiently during the subjective dark period. In the absence of SCNs, the effects of the light and dark pulses were decreased by half, demonstrating that the SCN, independent of its clock function, exerts a strong acute influence on sleep and waking by relaying a large amount (approximately 50%), though not all, nonvisual, non-circadian light information. Removal of both melanopsin and the SCNs, almost completely abolishes these responses with, over 24-hours, [18% (respectively 14% during subjective dark and 25% during subjective light phase)] of this acute reactivity to light mediated by rods/cones through brain relays exterior to the SCN (Fig. 5.1c). Acute light pulses also promoted REM sleep, and similarly to NREM, this effect was also dependent on the presence of both Opn4 and the SCNs (Supplementary Fig. 5.1a,b). Darkness promoted waking, but this wake-enhancing effect was associated with a rapid and prolonged induction over the course of all 1h dark pulses in EEG theta (7-10 Hz) and gamma (40-70 Hz) activity (Supplementary Fig. 5.3a,b and Supplementary Fig. 5.4), (i.e. EEG correlates of alertness and exploratory behavior in rodents 4,11,12 ). Syn10 Cre/Cre Bmal1 fl/-mice showed a nearly identical lack of theta and gamma induction as compared to the SCNx animals. Syn10 Cre/Cre Bmal1 +/- were similar to both Sham Opn4 +/+ and C57/B6, reinforcing the reliability of this control group. This alerting effect of darkness differed between genotypes and suggests that the SCN has a critical role in mediating the acute alerting effect of darkness. Waking values expressed as minutes/5 minute bout. In Sham Opn4-/-, mice do not have as strong an induction of wake from the dark pulse, especially during the subjective dark period, as compared to Sham Opn4 +/+ . SCNx Opn4 +/+ still react to the dark pulse but at a much lower interval, and does not seem to be dependent on circadian time-of-day. SCNx Opn4 -/-mice are unable to anticipate or react to the dark pulses regardless of ZT. X-axis numbers denote the time before and after the dark pulse (centered). or SCNx animals, and delta represents significant SCN condition differences. EEG power was normalized against the period of lowest power for theta and gamma (ZT4-7 for each day). For day 1 (Sham Opn4 +/+ n=8; Sham Opn4 -/-n=6; SCNx Opn4 +/+ n=5; SCNx Opn4 -/-n=5), and day 2 (Sham Opn4 +/+ n=7; Sham Opn4 -/-n=4; SCNx Opn4 +/+ n=5; SCNx Opn4 -/-n=5) Significance is represented by beta symbols. Power spectrum is calculated as in Supp. Fig. 9.

For day 1 and 2 (C57/B6 n=6; Syn10 Cre/Cre Bmal1 +/-n=5; Syn10 Cre/Cre Bmal1 fl/-n=8).

-Materials and Methods

Animals

All experiments were performed on adult male Opn4 -/-mice and wild-type littermates (as controls), were approved by university animal care facilities and pursuant to local and international rules. To disable the circadian pacemaker we performed under deep anesthesia a state-of-the-art 4-position electrolytic lesion of the SCN as detailed in supplementary material. The same surgical and stereotaxic procedure without injected current was used for Sham control animals. Locomotor activity was recorded using a standard infrared motion detector. All mice were implanted with a classical set of electrodes. Sleep and EEG power spectrum were analyzed and quantified according to standard criteria 20 . After completion of the protocol, the retrograde tracer CTB was injected under anesthesia into the posterior chamber of the eye. After sacrifice and perfusion of the animals three days later, DAPI and immunostaining to AVP and CTB was performed and visualized using a confocal microscope.

Sleep experiments were performed using male Opn4 íí C57/Bl6&129P2 mice, age approximately 2 months at time of implantation. These mice were originally obtained from Deltagen Laboratory, and subsequently bred in the Institute for Cellular and Integrative

Neurosciences breeding facility at the University of Strasbourg. Animals were raised under environmentally stable conditions (12hL:12hD; 25 ± 0.5° C, food and water ad libitum) and handled properly in agreement with the ethical guidelines for laboratory animal experimentation in the European Union. All experimental protocols were approved by the appropriate committees at the university and supervised by a veterinarian. Genotyping to verify the genetic background was performed using a standard PCR (primers: Mel4: 5'-GCT CAC TAT ACC CTA GGC AC-3'; Mel2: 5'-GTC CAT GGC TAT GGC TGT CA-3'; TodoNeo1: 5'-CCG CTT TTC TGG ATT CAT CGA C-3'from Integrated DNA Technologies, Inc.), described previously in Ruby, et al. 17 A complimentary group of transgenic mice were used to verify the effects in absence of a working SCN without lesioning the tissue. Synaptotagmin10-Cre (Syt10Cre) Bmal1 knock-out (Bmal1 fl/-) mice from the Max Planck Institute for Genetics in Göttingen, Germany, were subject to identical photo periods as the Opn4. These mice, created with a Syt10Cre All experimental sleep protocols were supervised by a veterinarian and approved by the appropriate committees.

Locomotor activity monitoring

Prior to electrode implantation, 14 male arvicanthis ansorgei were placed in individual running-wheel cages under 12hL:12hD conditions (DataportDP24, (VitalView, Minimitter).

Analysis and production of the actograms was performed by Clocklab (Actimetrics) via Matlab.

Surgery

Surgery protocol for implantation consisted of anesthesia (intraperitoneal injection of pentobarbital-68 mg/kg; protocol-approved), followed by implantation of electrodes, which were then secured using dental cement and sutured. Animals were implanted with two EEG on the dura, one reference, two EMG, and in a subset of rodents, two EOG electrodes and video recording to verify behavioral state in conjunction with EEG. Given the limited laboratory use of arvicanthis as compared to mice, the animals continue to retain certain savage tendencies such as increased activity and stress reaction to this environment. For this reason we designed a specific cable in order to prevent decoupling of the electrode chip as well as reinforcing it with INOX metal to prevent destruction by the animal. The increased weight of this system was offset by the size of the arvicanthis which averaged 150g at time of implantation. All sleep and light experiments were performed using male arvicanthis ansorgei, aged approximately 2 months at time of implantation. A minimum of 14 days was given to recover from surgery and habituate to the baseline conditions before any protocol began. Animals were recorded under 48 hours 12hL:12hD) condition (white fluorescent lights, 150 lux, measured at the bottom of the cage) as a baseline measurement. Signals were recorded for analysis using commercially available hardware and software (Micromed France, SystemPLUS Evolution version1092). Other continuous sleep recordings were taken under several experimental conditions: (1) a 1hL:1hD) cycle for 24 hours, ( 2 Video recording in several 4 animals was used to confirm concordance between behavior and EEG scoring (active wake vs. quiet during wake, cessation EMG activity during NREM, cessation of EMG activity coupled with EOG measured rapid-eye movements during REM sleep). In order to determine the qualitative aspects of these states, signals were modified using a Fast-Fourier Transform (FFT) to determine power spectra between 0 and 128 Hz (0.25Hz resolution) using a moving 4-s window. Power line artifacts disrupting the EEG were omitted with the frequencies between 49-51 Hz. For each vigilance state of the EEG, an average spectral profile was constructed using all 4-s epochs scored with the same state.

Epochs which had visible signal artifacts were identified and excluded from power spectrum analysis. Epochs scored as wake showed high levels of desynchronized activity, which EEG power concentrated in the theta range (6-10 Hz), with reduced delta power (0.75-4 Hz), and active EMG indicating increased levels of movement. NREM sleep was identified according to highly synchronized slow oscillating delta waves, with a negligible presence of other frequency bands, as well as a lacking any significant waveforms on the EMG, indicating stillness. REM sleep was categorized according to the presence of high peaks of theta in the absence of delta.

Under the 1hL:1hD schedule light-dark dependent changes were determined using sine-waves. For each animal, the 5-min values for the 6 dark-pulses given during the 12-hour subjective light period and the 6 dark-pulses given during the 12-hour subjective dark period were averaged. Sine-waves were then fitted using SigmaPlot (Systat v. 12) with amplitude, phase, y-offset as free parameters and period set to 2 hours. Time-dependent changes in EEG power for specific frequency bands under NREM sleep, was performed for delta (0.75-4Hz), and during REM, theta (6-10Hz), and in wake, both theta (6-10Hz) and gamma (40-70Hz). EEG delta power during NREM sleep was normalized by expressing all values relative to the mean value of the period consistently showing the lowest period of homeostatic pressure, ZT20-23. The final hour was not included due to the crepuscularity of the animal. Changes in theta and gamma were normalized against ZT2-5, which represented the lowest power in these frequency ranges during both baseline and 1h:1h LD conditions. A heat map for the 1-hour light pulse was made by splitting the pulse into 13 time periods of overlapping 10-minute windows, normalized to identical times during the baseline period.

Anatomy (data not shown)

Prior to perfusion animals were sedated using high doses of pentobarbital and perfused with PBS pH 7.4 solution followed by paraformaldehyde transcardial fixation and dehydration for 48 hours in a 30% sucrose solution. Brains tissue was then cut at 18µm systematically from rostral to the caudal area. Immunostaining is underway in the laboratory using standard free-floating slice protocols routinely used in the lab [START_REF] Tsai | Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice[END_REF].

Photomicrographs are analyzed using a filtered microscope for detecting Cy5, Alexa

-Perspectives

Here we have demonstrated for the first time that arvicanthis ansorgei is a valid model for studying the regulatory mechanisms of sleep and waking in a diurnal rodent. In addition, quantification of vigilance states and power spectrum analysis concluded the diurnality of the animal, though with a difference, in comparison to laboratory mice and rats, of a powerful crepuscular process overriding the circadian drive, as well the light dark influence.

Furthermore, the acute photic regulation in this animal is similar to humans in terms of the alerting effect of light and sleep promotion of darkness. At this stage, we are in the process of determining whether the direct effects of light can alter the balance of the flip-flop neuronal switch responsible for the occurrence of sleep or waking with the final goal to propose the first neuronal mechanisms underlying the inversion of light direct effects between nocturnal and diurnal species. This would then represent a first step under the way of understanding the mystery of nocturnality and diurnality. The anatomy experiments are underway and should be completed within a few months. Thus, this article is in the stage of preparation for publication, yet we are still awaiting completion of anatomic analysis by another member of the research team, which will hopefully outline the brain regions involved in this switch. In the future, it remains to be seen as to whether pinealectomized animals, in conjunction with sleep recording, are necessary so as to evaluate the effect of melatonin suppression on reactivity to light and dark pulses at certain times of the day.