The ability of the isolated rat hypothalamus, incubated in vitro, to form and secrete corticotrophin releasing factor (CRF) has been assessed. It was found that serotonin (10 ng/ml), when added to the hypothalamus in vitro, caused the release of CRF and an increase in the activity of CRF contained in the tissue. The activity of CRF was estimated by its ability to cause the secretion of ACTH (determined by the cytochemical assay procedure) from anterior pituitary fragments in vitro. Provided a minimum of 10 min 'rest' in serotonin-free medium was allowed between each challenge with the neurotransmitter, the response of the hypothalamus in vitro was maintained for at least 3 h. A detailed examination of the time-course of CRF formation and secretion following a single addition of serotonin showed that the release was well underway within 40 s and continued up to 4 min, but then ceased. On the other hand, the tissue content of CRF was found to be reduced significantly at 20 and 40 s, had returned to basal values at 1 min and continued to increase to reach a maximum at 4 min that was maintained at 6 min. The stimulated release was approximately six times greater than basal and the increase in content approximately ten times basal. Removal of the serotonin-containing medium after 6 min of stimulation was accompanied by a precipitous fall in the CRF contained in the tissue and this was significant 2 min later with CRF content back to basal levels within 4 min. The serotonin-induced increase in CRF content was shown to be greatest in the area of the ventral portion of the medial basal hypothalamus containing the median eminence and its attendant pituitary stalk.
The energy metabolite status of the hypothalamus in vitro under the conditions employed to cause the secretion of CRF was assessed. The rates of oxygen consumption and release of lactate, and the tissue concentrations of K+ and ATP were all determined and found to be comparable to values published for more conventional cerebral slice preparations for up to 2 h in vitro. At 3 h a fall in tissue K+ and an increase in the amount of lactate released into the medium suggested that the preparation was showing signs of becoming compromised metabolically and this trend was more obvious at 4 h when the ATP level had also fallen significantly.
In more limited cognate experiments the changes in ACTH formation and release induced in anterior pituitary fragments in vitro by the addition of hypothalamic extracts were studied. Once again the release of the hormone was accompanied by an increase in the ACTH content of the tissue. The increase in content reached a maximum by 10 min and then declined to basal levels within 10 min of removal of the CRF-containing medium. Thus, it is possible that degrading enzymes play an important role in the control of hormonal output from both tissues.
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