Regulated production and secretion of immunoreactive neuropeptide Y by aggregating fetal brain cells in culture

The aim of this study was to establish a culture system of fetal brain cells that could serve as a model for the study of the developmental regulation of the neuropeptide Y(NPY) neuron. Single cell suspensions were prepared from the hypothalamic-olfactory tubercle region of 18-day-old rat fetuses, and aggregates were formed by incubation in serum-free medium under constant rotation. Aggregate formation was complete within 24-48 h, and cultures were maintained for up to 23 days. The content of immunoreactive (IR) NPY in the medium and in the aggregates increased progressively with time in culture and at each time point, the medium contained 5- to 10-fold more NPY-IR. A 48-hour exposure to forskolin resulted in a 2-fold increase in the accumulation of NPY-IR in the aggregates and in the medium, indicating that both production and secretion of NPY are regulated by the cAMP intracellular pathway. Sephadex gel filtration revealed the presence of proNPY- and NPY-size substances. The ratio of NPY- to proNPY-size substances increased progressively with age of the aggregates as well as in tissues obtained from perinatal rats of comparable age. Thus, production and secretion of NPY-IR in the cultured aggregates are regulated processes and hence, this culture system can serve as a model to study regulatory processes in the developing NPY neuron.