RHYTHMIC OUTPUT PATHWAYS OF THE AVIAN PINEAL BODY

$0P01FY2002NSNIH

Texas A&M University System, College Station TX

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Abstract

The avian pineal gland is an important model system for the study of circadian organization. Second, the avian pineal gland contains a population of autonomous circadian oscillators capable of generating rhythm of generating rhythms of the hormone-melatonin in vivo and in vitro. The cellular site of this oscillatory mechanism has been localized to one of just three cell-types in the gland, the pinealocyte. Third, photoreceptive elements within the gland enable to entrain to environmental light cycles in vitro, and the identify of several photoreceptive pigments has been discovered, although it is not known which of these photopigments is/are critical for entrainment. Fourth, the molecular mechanisms of a circadian output pathway, the biosynthesis of melatonin, have been thoroughly worked out. However, the mechanism of rhythm generation or of the coupling of this rhythm to the melatonin generating system is completely unknown. The present proposal will employ state of the art molecular screening techniques to identify putative clock components within the chick pineal gland. This will include high- density transcriptional profiling, high throughput sequencing of cDNA's and mRNA and protein levels will be tested for importance to the system by employing anti-sense oligonucleotides directed at these sequences to a cell culture system of chick pinealocytes. Melatonin has broad effects on avian physiology and behavior. The molecular biology of melatonin sites of action is beginning to provide us clues to the mechanisms of melatonin action. We known there are at least three melatonin receptor sub-type is expressed preferentially on astrocytes and other glial elements of the chick nervous system, where it affects cellular metabolic activity and ionic currents. We will determine the cellular mechanisms by which melatonin influence glial ionic homeostasis by employing whole-cell patch-clamping techniques on isolated chick Muller cells. We will also determine the metabolic effects of the hormone in cultured Muller cells. The molecular mechanisms of action of melatonin in cultured Muller cells will be determined antisense characterization using ASOs of genes unveiled in Specific Aim #1 that are expressed in brain using both patch clamping and metabolic tracing techniques.

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