Rats were maintained under standardized conditions of food and water ad libitum and a lighting schedule of 12 h light/we h dark for seven days. Animals were sacrificed at 0500 and 1700 h and tissues were removed and washed. Isolated intact adrenal and pituitary cells were prepared by collagenase treatment. Using a sequential incubation procedure, the release of pituitary ACTH by hypothalamic acid extracts (CRF) was assayed by stimulation of corticosteroid secretion from isolated adrenal cells. Maximum stimulation of adrenal steroids was achieved with hypothalamic extracts and pituitary cells from rats sacrificed at 1700 h, and minimum values were obtained at 0500 h. Intermediate levels were obtained when hypothalamic extracts at one time point were incubated with pituitary cells at the other. The results substantiate the late afternoon peak level of hypothalamic and pituitary secretion occurring prior to the peak activity pattern of the rat.
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