Light-Dark and Circadian Melatonin Rhythms Are Established de novo in Re-Aggregates of the Embryonic Chicken Retina

Abstract

A central function of chicken photoreceptors is the rhythmic synthesis of the neurohormone melatonin; however, it is unclear how they can achieve – already before hatching (i.e. without light exposure) – a circadian pacemaker system. Here we studied melatonin synthesis and secretion in rosetted spheroids, which are de novo histotypic spheres, re-aggregated from dissociated retinal cells of chicken embryos. Spheroids were cultured during a 12-hour light/12-hour dark cycle. Shortly after the onset of photoreceptor differentiation in spheroids near day 5 in culture, melatonin secretion set in and, by day 8 in culture, showed distinct dark-light oscillations, with high values during the dark and lower ones during the light period. Concomitantly, expression of two key enzymes of melatonin synthesis, arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) was detected by northern blot analysis from day 6 onwards, increasing until day 8. Only HIOMT, but not AA-NAT levels were 2- to 3-fold higher at the end of the light compared with the dark phase. Even under permanent darkness, a slight melatonin oscillation was revealed after 6 days. In conclusion, photoreceptors in de novo regenerated retinal spheroids become light-responsive, establish appropriate pathways for melatonin synthesis and secrete more melatonin in the dark. The underlying circadian pacemaker seems to depend on photoreceptor differentiation, not on previous light-dark entrainment.