Primary cultures of rat hypothalamic neurones were maintained either in a serum-supplemented medium or in a serum-free chemically defined medium for up to 6 weeks. The release of the 41 amino acid-containing peptide, corticotrophin-releasing factor (CRF-41), vasopressin (AVP) and somatostatin (SRIF) were followed using immunoassays. In response to K+ (56 mmol/l) depolarization both the quantities of peptides released and the magnitude of responses were significantly greater from cultures maintained in the fully supplemented defined medium. As a consequence, release of CRF-41 and AVP could be measured directly, without requiring the concentration step necessary for cultures grown in serum. The response to K+ depolarization increased with the age of the culture, suggesting neuronal maturation.
Responses to K+ depolarization were Ca2+-dependent, and the addition of corticosterone (100 nmol/l) to the defined medium caused a significant reduction in the response of neurones secreting CRF-41 and AVP, but not those secreting SRIF, to depolarization. This suggests the retention in vitro of the responsiveness of stress-associated neuropeptides to the negative feedback effects of corticosterone. Neurones producing CRF-41 and AVP responded significantly in a dose-dependent manner to acetylcholine stimulation, whereas those producing SRIF did not. As cultures matured, the CRF-41- and AVP-producing neurones became more sensitive to acetylcholine with the maximal response at 1 nmol acetylcholine/1.
In conclusion, the culture of rat hypothalamic neurones is improved in terms of peptide output when the cultures are maintained in a defined medium. Differential responses of the peptidergic neurones may be seen in the presence of corticosterone and neurotransmitters, illustrating the retention in vitro of specific receptor-mediated responses which have been observed in vivo. This model should prove useful in the further study of the physiological, pharmacological and biochemical maturation and control of peptidergic neurones.
J. Endocr. (1988) 116, 349–356