A tissue culture model of the hypophysiotrophic CRF producing neuronal system
Details
Serval ID
serval:BIB_0D929D654A9E
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
A tissue culture model of the hypophysiotrophic CRF producing neuronal system
Journal
Neuroendocrinology
ISSN
0028-3835 (Print)
Publication state
Published
Issued date
04/1993
Volume
57
Number
4
Pages
716-28
Notes
Journal Article
Research Support, Non-U.S. Gov't --- Old month value: Apr
Research Support, Non-U.S. Gov't --- Old month value: Apr
Abstract
To investigate functional and chemical properties of anatomically characterized corticotropin-releasing factor-41 (CRF-41) producing neurons in vitro, hypothalamic slices of 6-day-old rats were maintained in culture for up to 6 weeks using a modified roller culture technique. This technique yields thick (100 microns) slices that contained an average of 300-400 CRF-41-immunostained neurons. The majority of CRF-41-positive cells were of small size (12-15 microns in diameter), and contained CRF-41-labeled dense core vesicles of 100 nm diameter as detected by electron microscopic postembedding immunocytochemistry. These cells represented the only CRF-41-positive cell population in the culture. Light microscope double immunolabelling of colchicine-treated cultures kept in a serum-containing media (SCM) indicated that about 60% of these CRF-41-positive neurons contains detectable levels of vasopressin-associated neurophysin (VP-NP). Culturing slices in serum-free, chemically defined media (SFM) resulted in an increased VP-NP immunostaining: parvicellular neurons labeled for both CRF-41 and VP-NP could be detected without colchicine treatment, and practically all CRF-41-positive neurons expressed VP-NP immunoreactivity. At the electron microscopic level there was a significant increase in VP-NP labeling density in the dense core vesicle compartment of CRF-41-positive varicosities. Adding dexamethasone (10 nM) to the SFM restored the staining pattern originally observed in SCM. Hence, the increased VP-NP and CRF-41 immunostaining after culturing CRF-41 neurons in SFM is most likely due to the absence of inhibitory glucocorticoids. The capacity of cultured paraventricular cells to release CRF-41 was assessed using an immunoassay. Unstimulated (basal) secretion of CRF-41 was not altered by five successive samplings at 2-hour intervals and stimulation of the same culture with 56 mmol K+ significantly increased (2-3 times) the CRF-41 content in the medium. The presence of dexamethasone (10 nM) in SFM induced a 6-fold reduction of K(+)-stimulated CRF-41 release and a 5 times reduction in tissue content in relation to cultures maintained in SFM without dexamethasone. In summary, we have demonstrated that cultured CRF-41 cells display morphological and biochemical features, as well as responsiveness to glucocorticoids, that is reminiscent to the situation in vivo. Thus, the model is well suited for studies of hypophysiotrophic CRF-41 cell functions.
Keywords
Animals
Colchicine/pharmacology
Corticotropin-Releasing Hormone/*biosynthesis
Culture Media
Culture Techniques
Dexamethasone/pharmacology
Fluorescent Antibody Technique
Hypothalamus/drug effects/*metabolism
Immunohistochemistry
Microscopy, Electron
*Models, Biological
Neurons/*metabolism
Neurophysins/analysis
Rats
Rats, Sprague-Dawley
Vasopressins/metabolism
Pubmed
Web of science
Create date
15/02/2008 17:57
Last modification date
20/08/2019 13:34