Synthesis of Melatonin in Chick Retinal Ganglion Cells. Light Effect and Circadian Regulation

Abstract

Abstract: : Purpose: We have previously reported that retinal ganglion cells (RGCs) synthesize melatonin rhythmically with maximal levels during the day, both in constant darkness (DD) or under the light-dark (LD) cycle. This rhythm is preceded by the elevation of cAMP levels, the 2nd messenger involved in melatonin synthesis regulation. In this context, the aim of the present work was to study the interaction between circadian and photic regulation of the RGC melatonin as well as the autonomous capacity of these cells to synthesize the methoxyindole when they are maintained in culture. Methods: Chicks entrained to a 12:12 h LD cycle were released to DD, constant light (LL) or maintained under the previous LD cycle for 48 h. Then, chicks were killed at different times, RGC preparations were obtained from lyophilized retinas (Guido et al., 1999) and the activity of arylalkylamine N-acetyl transferase (AANAT) was assessed (Champney et al., 1984). RGCs obtained from 8 day-old chicken embryos by Thy-1 antibody immunopurification (Brocco and Panzetta, 1997) were cultured in B27 supplemented DMEM (GIBCO) for several hours and synchronized by a medium exchange at time 0. Results: AANAT activity in RGCs, was significantly higher during the subjective day than during the night either in DD, LL or LD. Moreover, an acute light pulse in animals maintained in DD did not affect the diurnal peak of the enzymatic activity in RGCs. RCG cultures were capable of synthesizing melatonin, and AANAT mRNA expression showed a circadian rhythm that persisted at least during three cycles of 24 h each. RGCs were also synchronized by the addition of glutamate, the neurotransmitter involved in the retinohypothalamic tract, and rhythms of AANAT mRNA expression were observed. Conclusions: These findings support the idea that RGCs contain an endogenous circadian clock that may constitute a primary retinal oscillator during development, and that light differentially regulates the synthesis of RGC melatonin in a different manner from that previously described for photoreceptors and pineal gland.

Keywords: ganglion cells • melatonin • circadian rhythms