Entre 15 et 30% de la population souffrent de troubles du sommeil, ce qui représente un enjeu majeur de santé publique et souligne la nécessité de mieux comprendre les mécanismes de régulation du sommeil. La régulation du sommeil est décrite comme un modèle à 2-processus comprenant un mécanisme circadien et homéostatique. La lumière exerce un effet sur le sommeil de deux manières distinctes: indirectement en resynchronisant l'horloge, et directement par des mécanismes qui restent mal compris. Cet effet direct est médié par des cellules spécialisées de la rétine intrinsèquement photosensibles et contenant un photopigment la mélanopsine (Opn4) mais aussi par les cônes et bâtonnets qui transfèrent l'information à ces cellules. Pour comprendre la façon dont ces effets directs influencent le sommeil et la veille, nous avons caractérisé des souris Opn4-/- et des souris sans horloge fonctionnelle (Syn10cre/creBmal1fl/-), ainsi qu’un rongeur diurne, Arvicanthis ansorgei. Les objectifs de cette étude étaient les suivants: (1) identifier les voies neuronales sous-tendant les effets directs de la lumière médiés par la mélanopsine ; (2) valider ces effets chez un rongeur diurne; (3) établir une relation entre lumière, Opn4 et homéostasie du sommeil. Ce travail a permis (1) de mettre en évidence que les effets directs de la lumière représente un troisième mécanisme majeur de régulation du sommeil permettant même de maintenir un rythme veille sommeil en l’absence d’horloge centrale (2) de démontrer que ces effets sont inversés entre espèces diurnes et nocturnes; (3) de démontrer que la mélanopsine et la lumière sont fortement liées à la modulation de l’homéostasie du sommeil. / Between 15-30% of the general population is affected by sleep disorders, representing a major public health challenge, and as such a need to better understand the regulatory mechanisms of sleep and waking. This has been previously described as a 2-process model; both a circadian and homeostatic process. Light exerts an effect on sleep and wake in two distinct ways: indirectly, through the resynchronization of the clock, and directly via mechanisms that remain poorly understood. This direct effect is primarily a result of interaction with specialized cells in the retina which are intrinsically photosensitive containing the photopigment melanopsin (Opn4) in addition to rods and cones, which to a lesser extent pass information through these cells. To understand the way in which these direct effects influence sleep and waking we characterized mice lacking Opn4, and a second group possessing a functionally disabled clock (Syn10cre/creBmal1fl/-), as well as a diurnal rodent, arvicanthis ansorgei. The aims of this study were to: (1) identify the possible neural pathways to the hypothalamus transmitting the Opn4-mediated direct effects of light; (2) validate these effects in a diurnal rodent; (3) demonstrate a biological link between light, Opn4, and sleep homeostasis. This work has provided (1) strong evidence for a third regulatory mechanism of sleep and waking (direct effects of light) that is able to maintain a sleep wake rhythm in the absence of central clock (2) an inversion of this mechanism between nocturnal and diurnal species; (3) demonstration that Opn4 and light are strongly related to the modulation of homeostatic sleep process.

Melanopsin (Opn4) is the opsin photopigment of intrinsically photosensitive retinal ganglion cells (ipRGCs). It has a conserved opsin structure and activation mechanism, yet demonstrates unusual functional properties that suggest it will possess unique structure-function relationships. The aim of this thesis was to characterise key OPN4 residues by examining the impact of non-synonymous mutations on melanopsin function. A genotype-driven screen of a chemically-mutagenized mouse archive led to the identification of a novel Opn4 mutant, S310A, located at a known opsin spectral tuning site. Action spectra from ipRGC and pupil light responses (PLR) of Opn4^S310A mice revealed no change in wavelength of peak sensitivity. However, Opn4^S310A PLR was significantly less sensitive at longer wavelengths, consistent with a short-wavelength shift in spectral sensitivity. This suggests S310A acts as a spectral tuning site in melanopsin. Next, the impact of naturally-occurring missense variants in human melanopsin (hOPN4) was examined in vitro. Fluorescent calcium imaging of 16 hOPN4 variants expressed in HEK293 cells revealed four hOPN4 variants abolished or attenuated responses to light (Y146C, R168C, G208S and S308F). These variants were located in conserved opsin motifs for chromophore binding or hydrogen-bond networks, functional roles apparently shared by melanopsin. Finally, two hOPN4 single nucleotide polymorphisms (SNPs) P10L and T394I, associated with abnormal non-image forming behaviour in humans, were explored in vivo. Using targeted viral-delivery of hOPN4 SNPs to mouse ipRGCs, a range of OPN4-driven behaviours, such as circadian photoentrainment and pupil light responses, were found to be comparable with hOPN4 WT control. Multi-electrode array recordings of ipRGCs transduced with hOPN4 T394I virus had significantly attenuated sensitivity and faster response offset, indicating this site may be functionally important for melanopsin activity but compensatory rod and cone input limits changes to non-image forming behaviour.

In addition to a central circadian clock in the suprachiasmatic nucleus (SCN), zebrafish (Danio rerio) have local clock systems in their peripheral tissues. These peripheral tissues express a complement of clock genes that can be synchronized with the 24 h light/dark cycle and thus may be entrained by light. To date, teleost multiple tissue (tmt) opsin identified from Fugu rubripes and Danio rerio is the only opsin that has been proposed as a candidate to mediate this cellular photoentrainment (Moutsaki et al., 2003). Here we report the discovery of a multigene family of tmt opsins found not only in the teleost fishes, but in vertebrates,including amphibians, birds, reptiles, and some mammals. Phylogenetic analysis demonstrated that this gene family consists of three main classes, tmtI, tmtII and tmtIII, with each duplicating further to give two paralogues in the zebrafish genome. Their predicted amino acid sequences contain most of the characteristic features for the function of a photopigment opsin, as well as seven transmembrane segments indicative of a G protein coupled receptor (GPCR) superfamily. Significantly, reverse transcription polymerase chain reaction (RT-PCR) reveals that the tmt opsin genes in zebrafish are both temporally and spatially regulated. To investigate if these tmt photopigments mediate light-activated currents in cells, each opsin was expressed in vitro and the responses characterised by calcium imaging, whole-cell patch clamp electrophysiology, UV-Vis spectrophotometric analysis, and bioluminescence reporter assay. Collectively, these data suggest that some of the opsin photoproteins signal via Gi-type G protein pathway. Interestingly, the spectral analysis obtained shows that most tmt opsins tested are UV-sensitive when reconstituted in vitro with 11-cis and all-trans retinal, indicating an intrinsic bistable dynamics. Using site directed mutagenesis on one of the tmt opsins, tmt10, the potential spectral tuning sites involved in UV detection were tested. As part of this study, tmt opsin cDNAs were isolated from three populations of Mexican tetra (Astyanax mexicanus): surface, Pachon and Steinhardt. This allowed for a direct comparison between the tmt opsins present in the dark adapted species (cavefish) versus those of the light adapted species (zebrafish). It is hoped that the findings from this project will contribute to our understanding of non-visual light detection in fish and the evolution of their non-image forming photoreception.