This is an application for a new project that seeks to clarify the mechanisms and significance of the diurnal variation in the polyadenylated (poly (A)) tail length of the messenger RNA encoding the peptide hormone vasopressin (VP) in the suprachiasmatic nucleus of the hypothalamus (SCN). Four sets of experiments are proposed. First, to determine whether the diurnal rhythm in SCN VP poly (A) tail length is a characteristic endogenous to the SCN, the rhythms of VP mRNA poly (A) tail length and VP peptide release will be followed in the SCN in organ culture. The manner and extent to which poly (A) tail rhythms may be affected by perturbation of second messenger systems known to influence other SCN rhythms, in vitro will be assessed. This will include assessment of the effects of treatment with cyclic nucleotides, low calcium and tetrodotoxin. Second, to determine whether the rhythm in SCN VP poly (A) tail length is dependent upon synthesis of the VP peptide, diurnal fluctuations in VP tail length will be followed in the Brattleboro rat, a mutant strain bearing a point mutation in the VP gene. Third, to determine whether the rhythm in SCN VP poly (A) tail length is dependent upon the presence of specific cis-acting sequences within the VP gene, transgenic mice bearing that intact rat VP gene will be studied. The mouse SCNs will be assayed for the presence of a poly (A) tail rhythm in the rat VP transgene. In addition, mice bearing mutant rat VP transgenes, already identified in heterologous cell transfection assays to have abnormal regulation of poly (A) tails, will be analyzed for any abnormality in the rhythm of rat VP mRNA poly (A) tail length. Finally, to determine whether the circadian rhythm in VP secretion by the SCN depends on the rhythm in VP poly (A) tail length, transgenic Brattleboro rats will be prepared with the normal rat VP transgene, and the rhythms of poly (A) tail length and VP secretion into CSF will be compared to the normal situation. Transgenic Brattleboro rats bearing mutant rat VP genes will also be prepared to evaluate the impact of dysfunctional VP genes on the mRNA tail and secretory rhythms.
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