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

Abstract

The concomitant release of corticotropin-releasing factor (CRF), vasopressin (AVP) and somatostatin (SRIF) has been followed from primary 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 × 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 µM 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 steadier. 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.

Mechanisms of Norepinephrine Mediated Corticotropin-Releasing Factor-41 Release from Cultured Fetal Hypothalamic Cells

“…Fetal hypothalamic cell culture was carried out using the method of Clarke et al [29] with minor modifications [20], 17-to 19-day pregnant Sprague-Dawley rats were decapitated and the fetuses were removed and placed in sterile saline. The fetal hypothalami were dissected and placed into Ca2+/M g2 + -free Flanks' balanced salt solution (F1BSS) containing 0.1% w/v bovine albumin (BSA fraction V; Sigma), 20 mM Hepes and the antibiotics penicillin (100 IU/ml) and streptomycin (100 ¡ig/ml).…”

Dexamethasone and Aldosterone Modulate Corticotropin-Releasing Factor-41 Release from Cultured Rat Fetal Hypothalamic Cells through Type II and Type I Corticosteroid Receptors Respectively

“…The ex periments presented in the current paper utilized the same model, based on the technique of Clarke et al [33], and were performed on cells isolated from hypothalami taken from rats on day 17-19 of fetal life and after 15-17 days in culture. Basal levels of the released hormones var ied between different batches of cells presumably due to differences in the percentages of cell attached to the cul ture dishes.…”

“…There are no re ports of type II or type 1 receptor binding on specific CRF-containing neurons in cultured hypothalamic cells. The present culture system contains various cell types, in cluding primitive nerve cells, glia, ventricular cells and maturing neurons with elementary axons [22,33], which makes it difficult to characterize binding to specific cells by steroid ligand binding assay.…”

Vasopressin in neuronal cultures from neonatal rat brain.

“…25 These observations are similar to findings Fraction Number FIGURE 6. Arginine vasopressin (A VP) detected by radioimmunoassay for selected high-performance liquid chromatography fractions (1-5, 27-35, 66-70 32 who showed that cell AVP contents of 4-, 7-, and 11-day-old cultures from 18-day-old fetal rat hypothalamic tissues remain quite stable. Moreover, the decrease in cell AVP content of cultures from SHR and captopril-treated SHR after stimulation by 56 mM KCl (7-32 pg) was in agreement with the amount of AVP released (approximately 20 pg) after incubation of such fetal hypothalamic cultures with 56 mM KCl.…”