Studies will continue to focus on the intrinsic organization of the mediobasal hypothalamus. We plan to use a combination of light and electron microscopy to study Golgi impregnated and immunocytochemically stained tissue from the rat medial hypothalamus, concentrating on those areas which play an important role in neuroendocrine regulation. Using several double ultrastructural immunostaining methods developed in our lab, particular attention will be paid to characterizing the transmitters, peptides, and pituitary tropins found in both pre-synaptic and post-synaptic neurons in this part of the brain. Combinations of pre-embedding immunostaining with peroxidase and silver-intensified colloidal gold and post-embedding immunostaining with colloidal gold will be used to localize and characterize neurons and axonal endings containing various neuroactive substances. The question of subcellular localization of several neuroactive substances will be addressed with a quantitative evaluation of post-embedding immunostaining with colloidal gold particles. Cells which send efferent axonal projections to the median eminence will be identified with retrograde transport of cholera toxin conjugated to horseradish peroxidase; the pituitary tropin contained in these cells will be identified with post-embedding immunocytochemistry using 5 nm colloidal gold adsorbed to Protein A or a secondary immunoglobulin as a marker, and the neurotransmitters of synaptic terminals on identified cells will be labeled with 10 nm colloidal gold. This should allow a precise characterization of the neuroactive substances which are in direct synaptic contact with neurons with identified pituitary tropins which project to the median eminence. Golgi silver chromate impregnations and horseradish peroxidase intracellular filling will be combined with ultrastructural and immunocytochemical analyses to study the dendritic trees of immunocytochemically identified neurons; afferent boutons which are in synaptic contact with peroxidase- or Golgi- labeled dendritic trees will be immunocytochemically identified, and the relative synaptic efficacy of identified boutons will be estimated on the basis of a computer-assisted correlation between light and electron microscopy of this material. The hypothesis that estrogen-induced pituitary prolactin cell tumors may result from insult to hypothalamic dopamine neurons will be examined immunocytochemically.
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