Abstract
The retina is a highly complex tissue, it is involved in the cogniative perception of the visual range of the electromagnetic spectrum of an organism's environment. There is an increasing need for an accessible model of the human retina to advance the understanding of how the retinal clock and melatonin synthesis integrate to influence retina physiology/pathology. The study of the human retina is limited to the use of post-mortem specimens; the use of which has inherent draw backs. The Y79 retinoblastoma cell is the only available retinal cell line that has been shown to exhibit retina-like responses (cf refs Table 3) and possess a reliable marker (melatonin) of the retinal clock (Ding 1992, Pierce 1991). Here we propose to assess the expression of clock components or circadian production of melatonin in this cell line. The aims of this thesis were to investigate: (a) the temporal profile of melatonin synthesis and clock gene expression, (b) the effect(s) of alternating nutrient composition (e.g serum factors) on melatonin output, (c) the glutamatergic control of melatonin production in this cell line. Y79 cells were maintained on an entraining 12:12 LD cycle from seeding of experimental cultures. Measurement of melatonin synthesis (RIA) and clock gene expression (RT-PCR analysis) was assessed under DD at 4 h intervals, in two separate studies, over at least 4 days. Melatonin production and the expression of clock genes was detected throughout, however they were not found to oscillate. Melatonin synthesis was not cumulative indicating presence of an endogenous feedback mechanism and/or an active metabolising process. The nutrient composition of the experimental media had a significant differential effect on total melatonin output. Melatonin production of cells in media containing glutamate or without FCS was 10 fold less compared to cells in basic media. The presence of all known clock genes suggested that this cell line is capable of generating circadian rhythms. The glutamate content of media used in previous studies is known to activate iGluR and mGluR responses in vivo targeting per1 expression catabolic turnover of BMAL1 (Tamurau 2000; Oishi 1998, Akiyama 1999). A dose-response effect was observed between mGluR3 agonist (DCG-IV) and melatonin output when cells were seeded/maintained on glutamate free media, under conditions described above. RT-PGR profiling of mGluRs in this cell line was shown to strongly express mGluR 3,5 and 7. Future studies should include: (a), antagonist study to determine if the dose response effect is reversable and real, (b). effects of glutamate analogues on clock genes (and proteins) and cAMP levels in Y79 cells, (c) repeat temporal analysis of clock gene expression and melatonin output in glutamate-free media. The data from these studies would provide a mechanistic link between stimulation of mGluR and the suppression of oscillatory melatonin synthesis and clock gene expression. These data suggest that the clock mechanism (lack of rhythmicity in melatonin synthesis) in this cell line is suppressed via the tonic effect of glutamate on 6MAL1 via mGluR signaling pathway. These studies further confirm the potential utility of this cell line in human circadian biology.