HYPOPHYSIOTROPIC NEURON DIFFERENTIATION--TARGET FEEDBACK

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The broad, long-term objective of the proposed research is to define how target hormonal signals from the anterior pituitary affect differentiation of hypothalamic neurons that regulate pituitary function. The studies are designed to elucidate the influence of growth hormone (GH) and prolactin (PRL) on survival and morphology, transcriptional activation, and restriction in gene expression in hypophysiotropic neurons. The health relatedness of the project is in understanding the mechanism by which target signals or intermediate neurotrophic factors affect programmed neuronal cell death, synaptic connectivity and transmitter expression, not only in development, but in genetic disease, following trauma, or in the process of aging. The studies will be conducted in an animal model, the dwarf mouse, that exhibits genetic deficiency of GH and PRL, providing for dirt assessment of the effect of absent target signals, and in which effects of hormone treatment will be physiological, but not complicated by the presence of endogenous hormone. The specific aims are to determine 1) whether hypophysiotropic neuron programmed cell death is exaggerated, or abnormal axon morphology occurs, in the absence of GH and PRL, 2) the sites and mechanisms of GH and PRL influence on hypophysiotropic neurons, and 3) whether the abnormalities in GH and PRL-regulating neurons arise from a genetic defect inherent in the hypothalamus, or occur in response to a primary pituitary mutation. The overall experimental design is comparison of untreated and hormone-treated dwarf and normal sibling mice, during postnatal development and as adults. The methods to be used for Specific Aim I are a) neuronal tract-tracing and b) identification (by immunocytochemistry; ICC) of protein markers of cell viability (metabolic enzyme) and cell death (immediate-early genes), in untreated developing mice and in mice treated with neurotrophic factors including GH and PRL. For Specific Aim 2, the method will be localization of PRL, GH and IGF-I receptors by ICC and in situ hybridization (ISHH) in hypothalamus and phenotypic identification of neurons which express c-fos in response to GH or PRL treatment. Specific Aim 3 will be addressed by examining expression (by ISHH) and structure (by restriction analysis and sequencing) of candidate genes for the Ames dwarf mutation, in a) hypothalamus and b) pituitary.