Thèse Contrôle des Rythmes Veille-Sommeil en Conditions Lumineuses Naturalistiques H/F - 75

Établissement : Université Paris-Saclay GS Life Sciences and Health
École doctorale : Signalisations et Réseaux Intégratifs en Biologie
Laboratoire de recherche : Institut des Neurosciences Paris-Saclay
Direction de la thèse : François ROUYER ORCID 000000015641397X
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-05-05T23:59:59

Les rythmes veille-sommeil sont contrôlés par la pression de sommeil et l'horloge circadienne. Le comportement issu de cette interaction doit également s'adapter aux conditions environnementales, en particulier changements quotidiens et saisonniers du spectre lumineux solaire. Cependant, on connaît peu de choses sur la contribution des différentes molécules et circuits photorécepteurs au profil veille-sommeil. Une première partie du projet de thèse visera à déchiffrer les mécanismes par lesquels la lumière façonne le profil veille-sommeil des drosophiles dans des conditions naturalistiques. Il utilisera un système récemment développé au laboratoire, reproduisant les cycles naturels de lumière et de température, ainsi que les outils neurogénétiques permettant de perturber les systèmes photorécepteurs aux échelles moléculaires, cellulaires, et des circuits neuronaux. Par ailleurs, des données récentes montrent la capacité de mouches dépourvues d'horloge de présenter dans ces conditions naturalistiques, mais pas en conditions de laboratoire standard, un profil veille-sommeil semblable aux mouches sauvages. La deuxième partie du projet aura pour but de comprendre quelles sont les voies photoréceptrices qui permettent cette adaptation et de mettre en évidence les circuits neuronaux par lesquels ce comportement indépendant de l'horloge circadienne peut-être généré. Elle comprendra une analyse transcriptomique pour identifier les changements moléculaires qui sont associés à la capacité des conditions naturelles à induire ce comportement rythmique indépendant du système circadien.

Organisms living on the earth are exposed to 24h day-night cycles and have evolved a circadian clock. A brain clock controls sleep-wake rhythms and integrates light and temperature cues to adapt the behavior to daily and seasonal environmental changes 1.
Fruit flies are crepuscular animals and display a bimodal activity pattern in light-dark conditions, with a morning activity bout, a siesta that is more pronounced in males, an evening activity bout and a night sleep. Drosophila melanogaster has been instrumental in understanding the largely conserved molecular mechanisms of circadian oscillators as well as neuronal circuitry principles that control the sleep-wake behavior 2,3. The brain clock relies on about 150 clock neurons that show 24h oscillations of clock proteins and are organized in half a dozen anatomically defined subsets with specific contributions to the building of the sleep-wake cycle, in particular morning and evening cells 4,5. Light synchronizes the clock network through either the internal photoreceptor Cryptochrome (CRY) or the different rhodopsins expressed by the photoreceptor cells 1. CRY is a blue-light sensitive photoreceptor protein that is expressed in most clock neurons and captures photons inside the brain 6,7. Rhodopsin-containing photoreceptor cells are located in three photoreceptive structures of the head: retina, eyelet and ocelli and cover a broad light spectrum (from UV to red light) through six characterized rhodopsins, and are connected via poorly characterized circuits to the clock neuronal network 8.
The adaptation of the sleep-wake profile to seasonal variations of light and temperature implies large changes in the temporal position of the activity peaks 913. These behavioral adaptations thus result from complex interactions between environment and both the clock and the sleep homeostat 3. As in mammals 14,15, the spectral quality of light also plays a role in the Drosophila locomotor activity behavior 16. However, Drosophila sleep-wake rhythms have been studied essentially in white light and how the daily changes of both light intensity and light spectral quality are integrated by the brain to shape the sleep-wake profile is virtually unknown. Interestingly, a study made with flies recorded outside showed strong differences with laboratory conditions, indicating that light and temperature natural conditions include specific cues that remain to be characterized 17,18. The cellular and molecular mechanisms involved in this behavior are completely unknown. We reasoned that a simulation of natural environment in the laboratory would provide an easier, reproducible, and flexible manner to study behavior when combined with the powerful genetic tools of Drosophila.

1: Define the contribution of the different wavelengths and photoreceptors to the sleep-wake profile in naturalistic conditions. Wild type flies and photoreceptive mutants will be tested in the different seasonal conditions (full light spectrum or depleted for specific wavelengths, presence or absence of temperature cycles) to reveal the key components that define bring light information to the circadian clock and sleep circuits. How the circadian oscillator is regulated by these conditions will be investigated.

2: Identify the cellular and molecular pathways that generate sleep-wake rhythms in the absence of a clock in naturalistic conditions. Preliminary data show that clockless flies behave similarly to wild type flies in naturalistic conditions, even in the absence of temperature cycling). Photoreceptive mutants will be tested in a clockless background and neuronal circuits will be investigated to decipher the clock-independent pathways by which light can build a sleep-wake cycle, molecular analyses will be done to identify putative pathways.

1: Define the contribution of the different wavelengths and photoreceptors to the sleep-wake profile in naturalistic conditions.
A customized lighting box was developed with an industrial partner, with different LEDs covering wavelengths from 365 to 650 nm and being independently controlled. We will use the light and temperature data (average of 8 years) provided by J-C Dupont (Site Instrumental de Recherche par Télédétection Atmosphérique, Palaiseau) for June, August, and October, when Drosophila populations are abundant. We plan to improve the similarity of the light spectrum with the natural conditions with Alpheus (UV coverage). The naturalistic system will be further improved by providing moonlight and possibly mimicking artificial light pollution at night.
Different rhodopsins contribute to circadian synchronization with day-night cycles 19,20. The contribution of the different wavelengths will be studied by modifying the LED program, and the role of the different photoreceptors (rhodopsins, cryptochrome) and downstream circuits will be analyzed with genotypes affecting these pathways. Multiple rhodopsin mutants in a cry0 background generated by the laboratory show that cry0 flies with none of the six (RH1-6) rhodopsins are circadianly blind in standard light-dark conditions (Thejaswini et al., in prep.). We will study how flies missing a single rhodopsin or with a single active rhodopsin and no CRY behave. This will tell us what are the key photoreceptive molecules for generating the sleep-wake behavior in naturalistic conditions. Circuits downstream of the retinal photoreceptor cells will be analyzed by testing mutants of the histaminergic and cholinergic pathways 21,22. The contribution of light and temperature will be analyzed by comparing the behavior with naturalistic light with or without temperature cycling.

2: Identify the cellular and molecular pathways that generate sleep-wake rhythms in the absence of a clock in naturalistic conditions.
Outdoor experiments revealed that clockless mutants displayed a bimodal activity similar to wild-type flies 17, see also 23. This surprising result (see 18) suggested that rich environmental inputs can bypass the circadian system to control behavior. Preliminary experiments with our setup show that clockless flies (per0 mutant) generate anticipatory activities which do not occur standard light-dark conditions. Importantly, this occurs in the absence of temperature cycling, suggesting that naturalistic light is sufficient.
We will thus investigate, in the naturalistic conditions, the male and female behavior of clockless flies with different genetic backgrounds affecting the light input pathways (see above). This will tell us which photoreceptors are involved. We will then ask whether clock neurons play a role in the clock-independent behavior by either manipulating or ablating different subsets, and will investigate the neuronal circuits, neuropeptides, and neuromodulators known to be involved in sleep control 3,24. These experiments will to understand how naturalistic conditions generate rhythmic behavior in the absence of a circadian clock.
Finally, we will investigate the effects of naturalistic light on brain gene expression in the absence of a clock. The behavior of clockless flies suggests that clock-independent cycling gene expression occurs in these conditions. We will thus do a transcriptional analysis of the brain at different time points through the 24h of the naturalistic light-dark cycle, in comparison to standard LD conditions to identify gene expression that could be specifically regulated by the naturalistic light and drive clock-independent sleep-wake rhythms.