392 Brain Research, 456 (1988) 392-396 Elsevier BRE 23011 The vasopressin gene is expressed prior to regulation in the supraoptic nuclei of fetal rats Steven M. Reppert and George R. Uhl Laboratory of Developmental Chronobiology and Howard Hughes Neuroscience Group, Children's and Neurology Services, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114 (U.S.A.) (Accepted 19 April 1988) Key words: Supraoptic nucleus; Vasopressin; Gene regulation; Fetus The development of the regulation of vasopressin (prepropressophysin) mRNA in the supraoptic nuclei (SON) of rats was studied using quantitative in situ hybridization. On day 21 of gestation, vasopressin mRNA levels in the SON were increased by osmotic stimu- lation, with significant positive correlation between vasopressin mRNA levels and plasma osmolality. On day 19 of gestation, vaso- pressin mRNA levels in the SON were not significantly increased by osmotic stimulation, and no correlation between vasopressin mRNA levels and plasma osmolality was noted. The results suggest that the mechanisms for osmotic regulation of vasopressin mRNA levels develop between days 19 and 21 of gestation, a time course consistent with the initiation of afferent innervation of the SON. In adult rats, plasma osmolality is a potent regula- tor of arginine vasopressin gene expression in the neurons of the supraoptic (SON) and paraventricular (PVN) nuclei 4'8'14'17,19,20 and of vasopressin release from terminals of these neurons in the posterior pitu- itary gland 23. These regulatory activities are crucial for maintaining normal fluid and electrolyte balance. Our recent studies of the development of vasopres- sin gene expression in rats show that levels of vaso- pressin mRNA are consistently detectable in the SON on day 16 of gestation, when the nuclei are first discernible by light microscopy 12. Although this ini- tial expression is necessary for gene regulation, it may not be sufficient. Circuit elements that bring os- motic information to the SON, cell-surface receptors for this information, intracellular cytoplasmic media- tors and nuclear factors that interact with the vaso- pressin gene should all be present for osmotic regula- tion to occur. Study of the developmental emergence of osmotically regulated gene expression could pro- vide insight into the cellular components underlying gene regulation. With these issues in mind, we exam- ined the prenatal regulation of vasopressin mRNA levels in the SON by osmotically challenging preg- nant rats and their fetuses. Timed pregnant Sprague-Dawley rats were pur- chased from Zivic-Miller Laboratories (Allison Park, PA). Each animal was singly housed in a clear plastic cage; the cages were contained within well-venti- lated, light-proof environmental compartments that were located in a temperature-controlled (22 ___ 1 °C) animal room. The animals were housed in an auto- mated diurnal light-dark cycle with lights on from 06.00 to 18.00 h daily. Pregnant dams were killed by decapitation on either day 19 or day 21 of gestation (day 0 of gesta- tion = day of sperm positivity). The abdomen was then opened, and the fetuses were removed and de- capitated. Trunk blood from dams and fetuses was collected at decapitation and plasma osmolalities were measured by freezing point depression. Trunk blood from the fetuses of each litter were pooled so that enough plasma was available for an osmolality determination for each litter. Maternal and fetal brains were rapidly dissected out of the skull and im- mersion-fixed in cooled (4 °C) 0.5% depolymerized paraformaldehyde, 75 mM lysine, 37.5 mM Na2H- PO 4 (pH 7.4), 10 mM sodium periodate, and 1% glu- taraldehyde for 30 rain, soaked in buffered 7% su- crose for 45 min, and frozen in cooled 2-methylbu- Correspondence: S.M. Reppert, Warren 10, Massachusetts General Hospital, Boston, MA 02114, U.S.A. 0006-8993/88/$03.50(~) 1988 Elsevier Science Publishers B.V. (Biomedical Division)