Introduction. Gonadal steroid hormones exert a profound influence on the hypothalamus. However, actions of sex hormones on single neurons are not easily studied in vivo due to the complexity of the intact (integrated) central nervous system and difficulty in controlling variables. To circumvent these problems, cell culture is used to elucidate hormonally regulated events, but is limited by the inability to obtain isolated neurons and the lack of quantitative data.
Specific Aims and Methods. Recently, it was shown that when cultured in a three-dimensional (3-D) extracellular matrix (ECM), single hypothalamic neurons not in contact with any other cell type rapidly grow morphologically identifiable axons and dendrites. Growth and differentiation of these processes can be reliably quantified in absolute rather than relative terms. This new method will be used to: 1. Determine the effects of the sex steroids, 17 B estradiol, testosterone and dihydrotestosterone, on growth and differentiation of single hypothalamic neurons in a 3-D ECM. 2. Determine if luteinizing hormone releasing hormone (LHRH), vasopressin (VP) and oxytocin (OXY) can be demonstrated in single hypothalamic neurons and whether culture conditions or factors that influence peptide release will alter synthesis-secretion. An image analysis system coupled to a phase contrast microscope and microcomputer will be used to measure the following indices of growth and differentiation over time: axon length in um/neuron; dendrite length in um/neuron; total combined length of all processes in um/neuron; the number of primary processes, branch points, segments and terminals/neuron. A Student's t or Anova test will be used to compare data between treatment groups. Light and electron microscopy will be used to qualitatively evaluate the cultures. Immunocytochemistry and radioimmunoassay will be used to assess LHRH, VP and OXY. Significance. Treatment with and without gonadal steroids or factors affecting peptide release can be investigated in cultures of developing single hypothalamic neurons. Results should provide insight into feedback or other mechanisms, at the cellular level, that regulate growth and differentiation or secretion of hypothalamic neurons important for reproduction.
