title: Rat retina shows robust circadian expression of clock and clock output genes in explant culture.
abstract: OBJECTIVE
  Circadian rhythms are central to vision and retinal physiology. A circadian clock located within the retina controls various rhythmic processes including melatonin synthesis in photoreceptors. In the present study, we evaluated the rhythmic expression of clock genes and clock output genes in retinal explants maintained for several days in darkness.
  
  
  METHODS
  Retinas were dissected from Wistar rats, either wild-type or from the Per1-luciferase transgenic line housed under a daily 12 h:12 h light-dark cycle (LD12/12), and put in culture at zeitgeber time (ZT) 12 on semipermeable membranes. Explants from wild-type rats were collected every 4 h over 3 days, and total RNA was extracted, quantified, and reverse transcribed. Gene expression was assessed with quantitative PCR, and the periodicity of the relative mRNA amounts was assessed with nonlinear least squares fitting to sine wave functions. Bioluminescence in explants from Per1-luciferase rats was monitored for several days under three different culture protocols.
  
  
  RESULTS
  Rhythmic expression was found for all studied clock genes and for clock downstream targets such as c-fos and arylalkylamine N-acetyltransferase (Aanat) genes. Clock and output genes cycled with relatively similar periods and acrophases (peaks of expression during subjective night, except c-fos, which peaked around the end of the subjective day). Data for Per1 were confirmed with bioluminescence monitoring, which also permitted culture conditions to be optimized to study the retina clock.
  
  
  CONCLUSIONS
  Our work shows the free-running expression profile of multiple clock genes and potential clock targets in mammalian retinal explants. This research further strengthens the notion that the retina contains a self-sustained oscillator that can be functionally characterized in organotypic culture.
authors.affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Daniella C
authors.initials: DC
authors.lastname: Buonfiglio
authors.affiliation: Département Neurobiologie des Rythmes, Institut des Neurosciences Cellulaires et Intégratives, CNRS, Université de Strasbourg, France.
authors.forename: André
authors.initials: A
authors.lastname: Malan
authors.affiliation: Département Neurobiologie des Rythmes, Institut des Neurosciences Cellulaires et Intégratives, CNRS, Université de Strasbourg, France.
authors.forename: Cristina
authors.initials: C
authors.lastname: Sandu
authors.affiliation: Département Neurobiologie des Rythmes, Institut des Neurosciences Cellulaires et Intégratives, CNRS, Université de Strasbourg, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Jaeger
authors.affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: José
authors.initials: J
authors.lastname: Cipolla-Neto
authors.affiliation: Département Neurobiologie des Rythmes, Institut des Neurosciences Cellulaires et Intégratives, CNRS, Université de Strasbourg, France.
authors.forename: David
authors.initials: D
authors.lastname: Hicks
authors.affiliation: Département Neurobiologie des Rythmes, Institut des Neurosciences Cellulaires et Intégratives, CNRS, Université de Strasbourg, France.
authors.forename: Marie-Paule
authors.initials: MP
authors.lastname: Felder-Schmittbuhl
mesh_terms: D000818:Animals; D001683:Biological Clocks / Q000235:genetics; D056926:CLOCK Proteins / Q000235:genetics* / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002940:Circadian Rhythm / Q000187:drug effects / Q000235:genetics*; D003470:Culture Media / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D056950:Period Circadian Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D046509:Tissue Culture Techniques
chemical_list: D003470:Culture Media; D056950:Period Circadian Proteins; D012333:RNA, Messenger; D008156:Luciferases; D056926:CLOCK Proteins