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2016

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Mammalian retinal photoreceptors and integration of photoperiod

Research unit

UPR 3212 - Institut des Neurosciences Cellulaires et Intégratives (INCI)
5, rue Blaise Pascal, 67084 STRASBOURG

Group

Name: Rythme, Vie et Mort de la rétine

Group leader: HICKS David - photoreceptor67@hotmail.com

Group leader's phone: 0388456723

Website: Visit website

Group organization:
- Chercheurs: 7
- ITA: 3
- Doctorants: 7
- Post-Docs: 1
- Autres: 3

Publications of the team linked to the topic (3 last years):
1) Gianesini C., Hiragaki S., Contreras-Alcantara S., Laurent V., Hicks D., Tosini G.; Cone viability is affected by interruption of melatonin signaling. Investigative Ophthalmology and Visual Science 2016, Jan 1; 57(1):94-104.
2) Gianesini, C., Clesse, D., Tosini, G., Hicks, D., & Laurent, V. (2015). Unique regulation of the melatonin synthetic pathway in the retina of diurnal female Arvicanthis ansorgei (Rodentia). Endocrinology, EN.2015–1267.
3) Bobu C., Sandu C., Laurent V., Felder-Schmittbuhl M-P., Hicks D. Prolonged light exposure induces widespread phase shifting in the circadian
clock and visual pigment gene expression of the Arvicanthis ansorgei retina. Molecular Vision, 2013; vol. 19.

About PhD

PhD Director: LAURENT-GYDé Virginie - gydelaurent@inci-cnrs.unistra.fr

Phone: 0388456675

Junior advisor: non

Co-tutely: non

Co-Director: non

About PhD topic :

Title: Mammalian retinal photoreceptors and integration of photoperiod

Project: Background: Photoperiodic anticipation and integration are essential for adaptation to the environment, including in humans (Rosenwasser and Turek, 2015). Melatonin constitutes the endogenous messenger of seasonal variations used by organisms to synchronize reproduction (Simonneaux et al, 2013; Revel et al, 2009). Melatonin is synthesized and secreted by the pineal gland at night, with duration of secretion proportional to night length. However, the cellular mechanisms by which seasonal changes in daylight are transmitted to the brain are still unknown. Intrinsically photosensitive retinal ganglion -melanopsin containing cells- (ipRGCs) project towards the hypothalamic suprachiasmatic nuclei (SCN) to modulate circadian photo-entrainment (Schmidt et al, 2011). In turn, SCN control melatonin secretion by the pineal gland (Larsen 1998). Rod- and cone-less mice still display acute light suppression of melatonin secretion (Tri Hoang Do and Yau, 2010), suggesting a role for ipRGCs in regulating photoperiod. Understanding the detailed mechanisms of photoperiodic encoding is thus crucial to address related physiological disorders in human.
Objectives: Our goal is to determine i) whether ipRGCs are the only cells transmitting photoperiodic information to the brain; and ii) how retina encodes photoperiod length.
Experimental Procedures: Different mouse models will be used in order to analyse the role of each photoreceptor-type. They will include: A-C3H rd1 mice whose rods and cones degenerate rapidly (99% and 90% respectively), leaving only ipRGCs to perform photoreception. B. Opn4 KO (melanopsin KO) backcrossed with CBA/CaJ (melatonin-proficient mouse without the rd1 mutation); C. Triple KO (C3H x Opn4 KO mouse) completely lacking photoreceptors; D- WT CBA/CaJ (intact retina). I)- In preliminary experiments, melanopsin-saporin immunotoxin will be injected intravitreally into sub-groups of each model in order to specifically eliminate ipRGCs. Then, the various models will be placed 4 weeks in short photoperiod (SP) (8 hours light, 16 hours dark) or long photoperiod (LP) conditions (16 hours light, 8 hours dark) and then circulating night-time melatonin levels will be assayed by ELISA. Then, in order to determine which retinal cells project to SCN, retrograde labeling by cholera toxin injection into the SCN will be conducted. RGC markers (melanopsin, Brn3a-c, Thy1, NF-200) will be used to identify back-labeled cells. Ii) In a second axis, we will analyze the mechanism involved in the photoperiod integration by the retina. Retina contains circadian clocks (Jaeger et al, 2015) controlling rhythmic processes essential to the physiology of the tissue (McMahon et al, 2014). Moroever, nocturnal melatonin synthesis is rhythmic and can act on clock (Hiragaki et al, 2014) We will establish retina clock gene expression profiles (by qPCR) from the described mouse models under the different SP and LP conditions. Melatonin is also rhythmically secreted in the retina (Tosini et al, 2012) and synchronizes clocks and modulates physiology of the tissue via MT1/MT2 receptors (Gianesini et al, 2015; McMahon et al, 2014). The indolamine can notably modify M4-type ipRGC activity (Pack et al, 2015). In order to monitor whether photoperiodic message integration depends on melatonin within the retina, we will study endogenous retinal melatonin synthesis under SP and LP conditions. To do this we will quantify the melatonin synthetic enzymes Aryl-Alkylamine N-Acetyl Transferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) by qPCR and immunohistochemistry in CBA/CaJ mice placed 4 weeks in SP or LP conditions beforehand. The data generated from these experiments will provide novel and solid information on the role of the retina in controlling photoperiod.

Wished skills: Neuroscience training and neuroendocrinology background.
Theoretical knowledge of animal physiology and genetics.
Willing to undertake in vitro and in vivo experiments as well as learning multielectrode array technique.
Basic knowledge of laboratory techniques in cell and molecular biology.

Expertises which will be acquired during the training: Immunocytochemistry; qPCR, Western blotting ; ELISA, electroretinograms; MEA, blood sampling by tail-snip technique,
intraocular injections. Collaboration : HPLC/MS