Circadian rhythms regulate a number of diverse physiological functions in the vertebrate eye. Inspite of the significant body of information clearly documenting important physiological roles for circadian rhythms in the retina, very little is known about the molecular mechanisms that generate circadian oscillations in the eye. Evidence from a diverse set of experiments suggests that visual sensitivity, photoreceptor metabolism and turnover, retinomotor movements, indoleamine biosynthesis, and gene expression of photoreceptor transduction proteins all vary with a circadian rhythm. What accounts for these 24-hour rhythms that persist in the absence of environmental timing cues? Research in circadian biology has established that these rhythms are the manifestation of a physiological timing mechanism with the properties of a self-sustained oscillator. Physiological experiments have shown that circadian oscillators are located in three diencephalic structures: the suprachiasmatic nucleus, the pineal gland and the retina. Our long-term objectives are to understand the cellular and biochemical events that underlie the temporal regulation of rhythmic photoreceptor metabolism. Primary cultures of embryonic chick retina and human retinoblastoma cell lines will be studied to determine if either of these preparations exhibit properties of a circadian oscillator in vitro. The phase-shifting effects of light on melatonin release will be studied because identifying the photic entrainment pathway should ultimately aid in identifying the cellular components of the oscillator. The potential entraining effects of dopamine will also be analyzed to determine whether an additional input pathway to the oscillator exists. Using purified photoreceptor cultures, we will determine whether photoreceptors are the melatonin-synthesizing cells and whether these cells are also the locus of the retinal oscillator. These experiments will determine if circadian oscillations are a cellular property of retinal photoreceptors. In recent work we have discovered that retinoblastoma cell synthesize melatonin. We propose to define the biochemical events regulating melatonin in these cells. In addition we will explore the intriguing possibility that retinoblastoma cells express circadian rhythms. The identification of a human retinoblastoma cell line that oscillates would provide a novel system in which to study the cell biology of ocular clocks using biochemical, molecular and genetic probes. Ultimately, an understanding of the biological basis of ocular circadian rhythms may lead to procedures useful in the diagnosis and treatment of pathophysiologic conditions in the retina.
