There is an increased prevalence of eating disorders and a growing appreciation of their health costs. In order to effect human health, a greater understanding of the mechanisms modulating ingestive behaviors is needed. The lateral hypothalamus has long been associated with feeding behavior and the neurotransmitter glutamate appears to play a unique, initiatory role at this region. In this proposal the low-flow push-pull perfusion method will be used to: 1.determine the precise location of an endogenous glutamatergic surge that is coincident with the initiation of a meal, 2.pharmacologically manipulate lateral hypothalamic sub areas to verify the link between ingestive behaviors and glutamate signaling. Because our new in vivo sampling method employs perfusion flow rates among the lowest of any in vivo sampling technique, the approximate 200-#m spatial resolution is significantly better than existing methods and no tissue damage is seen by light microscopy. Also, with low-flow push-pull sampling analyte recoveries are high and will be useful for recovery of large, slower diffusing molecules. We will determine optimal low-flow push-pull conditions for maximal protein recovery and the protein content will be determined for the most abundant cerebrospinal fluid proteins. Protease activity in perfusates will be measured and minimized by the use of capillary wall coatings. The developmental studies in this R21 proposal highlight the particular advantages that will be afforded by the use of the low-flow push-pull perfusion method and will lead to sampling conditions appropriate for future studies of neuropeptides implicated in feeding behavior. The unique capabilities of low-flow push-pull perfusion complement other sampling methodologies and are widely applicable to in vivo sampling studies.
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