Non-visual photoreceptors and oscillators in the inner retina; the role of serotonin-derivatives in the functioning of retinal circadian clocks
Mario E. Guido.
CIQUIBIC-Dpto. de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, 5000 Córdoba, Argentina
Purpose: Retinal oscillators may act like clocks to induce changes in the visual system by predicting ambient illumination variations. Retinal ganglion cells (RGCs) send photic information to the brain regarding the light:dark (LD) cycles that adjust central pacemakers. We investigated the capacity of chicken RGCs to function as autonomous circadian oscillators and non-visual photoreceptors. To test this, we examined if RGCs in culture: a) synthesize the serotonin-derivatives N-acetylserotonin and melatonin in a rhythmic manner, and b) detect light intrinsically that regulates gene expression and the synthesis of serotonin-derivatives.
Methods: Primary cultures of chicken RGCs were obtained at embryonic day 8 (E8), cultured for several days, synchronized at time 0 by medium exchange or exposure to a h LD cycle, and sampled at different times.
Results: Embryonic retinas expressed master specification genes for RGCs and ancestral rhabdomeric photoreceptor markers such as Pax6, Brn3, two melanopsin isoforms and the G-protein q at very early stages (E4-5) before the expression of markers for classical photoreceptors (Crx, c-opsins and α-transducin) were observed. Moreover, primary RGC cultures at E8 expressed Pax6, Brn3, two melanopsin isoforms, the G-protein q and serotonin N-acetyltransferase (NAT), the key enzyme in melatonin synthesis. RGC cultures synchronized by medium exchange or exposure to LD cycles, synthesized 3H-melatonin in the dark with levels peaking 8 h after synchronization and declining a few hours later. These cultures also showed self-sustained daily rhythms in NAT mRNA expression at least during three cycles with a 24 h period that persisted after synchronization by 100 µM glutamate. The daily variation in 3H-melatonin levels of dark-maintained RGC cultures was significantly inhibited by light. This effect was further increased by the chromophore all-trans retinal, and suppressed by specific inhibitors of the invertebrate photo-cascade involving phosphoinositide hydrolysis and Ca2+ mobilization.
Conclusions: The results demonstrate that chick RGCs are intrinsically photosensitive cells acting via an invertebrate-like phototransduction cascade and contain autonomous circadian oscillators synthesizing N-acetylserotonin and melatonin rhythmically. RGCs may be responsible for early detection of light before vision occurs and temporal regulation of development.
Supported by: Fundación Florencio Fiorini, FONCyT, CONICET, SeCyT-UNC.
