The overall goal of the proposed study is to determine if blood- borne signals (insulin, glucose, amino-acids, fatty acids and ketones) act directly on hypothalamic tissue to modulate the release of LHRH In hypothalamic explants during perifusion. Such effect could explain the pathophysiology underlying clinical syndromes observed in anorexic and athletic women associated with altered reproductive function. This objective will be approached through use of a unique, custom-designed, computer- controlled perifusion system developed at The University of Michigan, and the use of a multi-sensor micro-probe for online monitoring of tissue's electrical multi-unit activity, temperature and oxygen tension. This system allows the user to provide a defined, yet dynamically-changing, pattern of nutrients and stimuli to cultured cells as well as balancing oxygen tension and flow rate to reduce central hypoxia and minimize peripheral oxygen toxicity in the explant. Hypothalamic tissue will be obtained from mature, ovariectomized female rats, placed in the perifusion system, and periodically exposed to higher or lower concentrations of the desired signal. In particular, attempts will be made to determine the effect on the oscillatory activity of the hypothalamic pulse generator by monitoring the following response parameters: (1) the tissue's electrical activity (evoked potentials) measured at multiple sites in the medial basal-preoptic area of the hypothalamus and examined for periodic changes in frequency of electrical activity, transition time from one frequency to another and signal amplitude (using potentiometry); and (2) LHRH release to the surrounding media, quantified by radioimmunoassay (RIA).
