Assessment of Corticotropin-Releasing Factor, Vasopressin and Somatostatin Secretion by Fetal Hypothalamic Neurons in Culture

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Abstract
The concomitant release of corticotropin-releasing factor (CRF), vasopressin (AVP) and somastatin (SRIF) has been followed from pituitary cultures of rat hypothalamic neurons. 18-day-old fetal rat hypothalami were dissociated enzymatically and mechanically, then plated and maintained in a serum-containing medium at a density of 2.5 x 106 cells per dish (equivalent to 3 hypothalami). Cultured neurons remained viable for up to 6 weeks, and peptide release was followed by immuno-assay between days 14 and 39 in culture. The incubation media were concentrated on C4 and C8 silica columns to facilitate detection of CRF and AVP. Peptide release was measured at various times up to 4 h, at which point it was still increasing. To optimise measurements, taking into account peptide degradation, a 1-hour incubation period was chosen for further studies. Release of CRF, AVP and SRIF by 56 mM K+ or 10 .mu.M veratridine was statistically significantly greater than basal (p < 0.01) and was Ca2+-dependent. For CRF and AVP, stimulated release increased considerably with the age of culture, whereas SRIF release was stadier. Basal release for all 3 peptides did not fluctuate greatly over this period. Basal and stimulated release of the peptides continued over at least 5 successive 1-hour periods. At day 35 of culture, the peptide content was still increasing in a pattern which paralleled the increasing content in hypothalami freshly removed from age-matched rats. In conclusion, we have demonstrated a development of CRF, AVP and SRIF production by neurons over extended periods in culture as assessed by their peptide content and increasing responses to depolarizing stimuli. Such a system should prove useful in the study of the effects of environmental factors such as steroids, peptides and neurotransmitters on the maturation of peptidergic neurons and the synthesis and release of CRF, AVP and SRIF.

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