Circadian rhythms are the overt manifestation of an innate timekeeping system, i.e., a """"""""circadian clock,"""""""" that is synchronized (entrained) to the 24-hr day-night cycle by environmental light. The clinical and practical significance of the human clock is now well recognized. The overall objective of this research program is to understand the endogenous oscillatory mechanism of the circadian pacemaker in the suprachiasmatic nuclei (SCN) of mammals. During the current grant period, we began to study whether an alteration of gene expression might be involved in the entraining effect of light. We found that light regulates the expression of c-Fos and Fos-related transcriptional regulatory proteins in the ventrolateral SCN and in the intergeniculate leaflet (IGL) of rats. In this competing continuation application, experimental results are presented that lead to the hypothesis that events at the transcriptional level are part of the mechanism for photic entrainment of the circadian pacemaker to the light-dark cycle, and, more specifically, that Fos expression in the SCN may be a link in the entrainment mechanism. Protocols are proposed to study four questions directed to this hypothesis. (1) What are the effects of light and circadian phase on the expression of Fos, on its interaction with Jun, and on their binding to DNA? Patterns of Fos/Jun expression will be evaluated by immunohistochemistry and in situ hybridization, identification of the immunoreactive and mRNA species by Western and Northern blot analysis, and detection of sequence-specific DNA binding by mobility shift (gel retardation) assay. (2) What are the implications of the localization of Fos to the retino-recipient ventrolateral SCN and IGL? The possible colocalization of Fos to neurons that synthesize known neuropeptides and the dependence of light-induced Fos activation in the SCN on visual input from the IGL will be assessed. (3) Do stimuli that affect pacemaker entrainment modify Fos expression in the SCN? Changes of Fos expression will be investigated after injection of the N-methyl-D-aspartate antagonist MK-801, after daily injections of melatonin, and in the fetal SCN. (4) Will experimental reduction of intracellular Fos levels in the SCN inhibit the entraining action of light? An antisense oligonucleotide complementary to the 5' end of rat c-fos mRNA will be introduced by stereotaxic infusion in to the SCN via mini-osmotic pump, and its effects on light-induced phase shifts of overt behavioral rhythmicity will be measured.
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