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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD022806-03
Application #
3322691
Study Section
Reproductive Biology Study Section (REB)
Project Start
1987-09-01
Project End
1992-08-31
Budget Start
1989-09-01
Budget End
1992-08-31
Support Year
3
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Texas Tech University
Department
Type
Schools of Medicine
DUNS #
609980727
City
Lubbock
State
TX
Country
United States
Zip Code
79430