In this proposal, cFos immunoreactivity will serve as a marker for stimulated neuronal activity. General principles governing regulation of LHRH neuronal activation will be determined using the steroid-induced LH surge as a model system. Using double-label immunocytochemical techniques, the goal of these studies is to establish the anatomical pattern of cFos immunoreactivity in LHRH neurons and other neurons within the reproductive neuroendocrine axis during an LH surge. Preliminary data indicate there is a very high correlation between the number of LHRH neurons expressing cFos and the intensity of staining, and the magnitude of the LH surge. For each of the experiments in the initial phases of the proposal, a similar strategy will be applied: first, the relationship between cFos activation in LHRH neurons and LH release will be determined (the precise time of onset of cFos expression, characterization of which LHRH neurons express cFos, and the relationship between cFos expression in LHRH neurons and LH release). Plasma LH levels (determined by radioimmunoassay) will be correlated with indices of the cFos immunoreactivity within LHRH neurons (numbers of LHRH neurons expressing cFos and intensity of cFos expression). Secondly, attention will be directed toward comparing the patterns of cFos immunoreactivity within the brainstem, hypothalamus, and preoptic area with patterns of cFos immunoreactivity in LHRH neurons during the LH surge to determine which neuronal systems express cFos when LHRH neurons are activated (and when possible, their phenotype). Thirdly, tract-tracing techniques will determine whether the identified neurons project to the sites where LHRH neurons lie. Next, the emphasis of experiments will shift from determining which neuronal pathways are activated during the LH surge, to assessing how known effectors of LH secretion (norepinephrine, neuropeptide Y, and excitatory amino acids) affect cFos expression in the reproductive neuroendocrine axis. Lastly, in situ hybridization will be used to address the possibility that cFos induction in LHRH neurons reflects increased synthetic activity. The studies described in this proposal, for the first time, will characterize activation of LHRH neurons at the cellular level, by identifying the location of these neurons and determining the degree of stimulation (both number of neurons and the intensity of cFos staining) and by quantifying LHRH mRNA levels. These studies will also determine whether other neuronal systems expressing cFos are part of the reproductive neuroendocrine axis involved in gonadotropin regulation.
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