The main objective of the present study is to characterize the estrogenic regulation of prepro-cholecystokinin (pCCK) messenger RNA (mRNA) in cells of the limbic-hypothalamic circuit. This interconnected circuit, which includes the medial preoptic nucleus (MPN) and the ventromedial nucleus of the hypothalamus (VMH), is implicated in the integration of sensory and hormonal cues that are involved in the CNS control of reproductive behavior. Estrogen stimulates the expression of pCCK mRNA and CCK peptide and reversibly decreases CCK receptor binding levels in the VMH. The effects of exogenous CCK on reproductive behavior are estrogen dependent and site-specific; in the VMH, CCK inhibits and in the MPN, CCK facilitates lordosis behavior. The proposed research is a systematic examination of estrogenic modulation of pCCK mRNA levels in the limbic- hypothalamic circuit. Initially, we will determine whether estrogen activation of preproCCK mRNA expression is differentially regulated in cells projecting to the MPN compared with those that project to the VMH. We hypothesize that estrogen acts trans-synaptically to regulate pCCK mRNA expression and we propose to study the mechanisms through which estrogen upregulates pCCK message and peptide in the CNS. Experiments will determine if pCCK mRNA levels are regulated by estrogen when electrical activity is disrupted (with local infusions of tetrodotoxin) or when protein synthesis is disrupted (with anisomycin implants). These experiments will test also whether elevating second messengers with phorbol ester and forskolin will augment estrogen induced pCCK mRNA levels. Morphological evidence will be obtained for estrogenic stimulation of early response genes in pCCK cells and whether this is a trans-synaptic effect of estrogen. In the MPN, exogenous CCK facilitates lordosis but the type and distribution of CCK receptors has not been determined. a CCK receptor autoradiography study will map and characterize the distribution of CCK binding sites in the medial preoptic area. This experiment will help elucidate the observed facilitatory effects of endogenous CCK in the MPN. CCK-B receptors are found in the VMH, and the use of a selective CCK-B receptor antagonist, will allow us to examine the role of endogenous CCK in the modulation of lordosis behavior. We have shown that pCCK levels, in adulthood, are determined by the gonadal steroid environment. The adult steroid pattern appears at puberty. The last experiments will characterize the effects of estrogen during pubertal development of the limbic-hypothalamic CCK circuit and correlate the changes in expression of pCCK mRNA with induction of lordosis behavior. Together the proposed research will enhance our understanding not only of the limbic-hypothalamic CCK circuit but also contribute to our understanding of mechanisms involved in gonadal steroid trans-synaptic regulation of CNS neurochemistry.
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