Expression of the photopigment melanopsin in cultures of chicken retinal ganglion cells
M. E. Guido, P. S. Nieto, M. A. Contin, A. R. Carpentieri and E. Garbarino-Pico
CIQUIBIC-Dpto. de Química Biológica, Facultad de Cs. Químicas, Universidad Nacional de Córdoba, 5000 Córdoba, Argentina
mguido@dqb.fcq.unc.edu.ar
Purpose: We recently reported that chicken retinal ganglion cells (RGCs) synthesize melatonin with higher levels during the day. Cultured RGCs at embryonic day 8 (E8) synchronized by medium exchange convert radioactive tryptophan into a 3H-melatonin-like indole in a rhythmic manner and display circadian variations in the mRNA expression of the key melatonin-synthesizing enzyme, serotonin N-acetyltransferase (NAT) for at least three cycles (Garbarino-Pico et al., 2004). Retinal oscillators may act like clocks to induce changes in the visual system according to the phase of the day by predicting environmental changes. The oscillatory and photoreceptive capacities are likely to converge all together on selected retinal cell populations. In the present study we investigated the presence and the temporal regulation of the mRNA for melanopsin, a novel photopigment involved in the circadian phototransduction in mammals, in RGC cultures, free of other cellular types and systemic factors.
Methods: RGCs obtained from E8 by Thy-1 immunopurification (Brocco and Panzetta, 1997) were cultured in B27 supplemented DMEM (GIBCO) for several hours, synchronized the following day by a medium exchange (time 0) and fixed at different phases across 32 h. Melanopsin RNA levels were assessed by Northern blot and in situ hybridization with a specific synthetic radiolabeled oligonucleotide.
Results: We found detectable levels of melanopsin mRNA in cultures of RGCs, which displayed a circadian variation (p<0.001 by ANOVA) with higher levels during the first hours after synchronization and 24 h later, and the lowest mRNA levels at 15-18 h. Post hoc comparisons revealed that levels at 15 h differ from those at 0, 24 and 32 h whereas levels at 0 h were significantly greater than those at 7, 10, 15 and 18 h.
Conclusions: The results demonstrate that immunopurified chicken RGCs contain the photopigment melanopsin and this expression is controlled by a circadian clock located in the cells themselves.
Supported by: Fundación Antorchas, FONCyT, CONICET, SeCyT-UNC, CAEN-ISN Beca Carrillo-Oñativia, and Agencia Córdoba Ciencia.
