The present studies were undertaken to investigate the effect of 5-bromo- 2'-deoxyuridine (BrdU; 50 μM) or forskolin/3-isobutyl-1-methylxanthine (F/I; 10/500 μM) on TRH gene expression in cultured fetal diencephalic cells. BrdU as well as drugs such as F/I that raise intracellular cAMP levels had been previously termed differentiating agents because they cause morphological and functional differentiation of IMR-32 neuroblastoma cells. We postulated that neurons of fetal diencephalons may remain relatively undifferentiated in vitro and that this might be the reason for low or undetectable TRH production. We hypothesized that treatment with differentiating agents might increase neuropeptide expression. Both BrdU and F/I dramatically (P < 0.01) raised intracellular TRH and pro-TRH messenger RNA concentrations in cultured diencephalic neurons. Although a short BrdU exposure during the first 4 days of culture was sufficient to irreversibly change TRH neurons and to cause maintenance of high TRH levels after withdrawal of the drug, F/I had to be present continuously throughout the observation period of 16 days to significantly elevate TRH expression. This suggests that BrdU and F/I act at different intracellular sites to activate TRH expression in cultured diencephalic neurons. The reduction of glial cells that occurs concurrent with the BrdU treatment was not observed after F/I exposure, and therefore, this effect does not appear to be a key factor for the induction of TRH expression. As the intracellular accumulation of somatostatin and arginine vasopressin, which were determined in parallel, was similarly enhanced after treatment with BrdU or F/I, our culture system might provide a valuable tool for the study of these and possibly other neuropeptides in vitro.,
Department of Reproduction and Development

Bruhn, T.O, Rondeel, J.M.M, Bolduc, T.G, Luo, L.-G, & Jackson, I.M.D. (1996). Activation of thyrotropin-releasing hormone gene expression in cultured fetal diencephalic neurons by differentiating agents. Endocrinology, 137(2), 572–579. doi:10.1210/en.137.2.572