This proposal is concerned with cellular and molecular mechanisms underlying the development and regulation of sensitivity of the developing brain to the gonadal steroid hormone, estrogen. The proposed interrelated and multidisciplinary experiments represent novel and unique correlative in vivo and in vitro approaches to understanding the distribution, responses, and control of three key elements involved in estrogen action on the developing brain: the cellular target, receptor system (mRNA and protein), and responsive genes. These experiments advance the novel hypothesis that gonadal steroid action in the developing brain is much less restricted than previously thought and uses the same basic mechanisms as in other (extra-neural) estrogen targets. Using techniques to look at responses of cells in ways never before possible, the proposed experiments address how steroids influence the maturation of brain centers by looking at the basis of the responses cell-by-cell; comparing receptor message and protein. For the first time, it may be possible to understand what estrogens do to promote neuronal differentiation, at a cellular and molecular level. Organotypic cultures of forebrain regions of the fetal and newborn rodent and comparable brain regions in vivo will be studied morphologically in living and fixed preparations by means of neurohistological stains and chemically-specific neuroanatomic techniques: autoradiography (125l-estrogen and 3H-thymidine); immunocytochemistry (neuropeptide/neurotransmitter markers); and in situ hybridization histochemistry (mRNA expression). Morphological findings will be correlated with functional output, using very sensitive quantitative measurements of estrogen receptor content (quantitative autoradiography; nuclear receptor assay) and of receptor mRNA expression (Nucleic acid dot blotting, quantitative in situ hybridization; Ribonuclease T1 protection assay). The experiments are designed to investigate in parallel: phenotypes of estrogen target neurons; time course of expression and spatiotemporal distribution of estrogen receptor mRNA and the encoded protein; epigenetic influences which may regulate differentiation and survival of estrogen receptor-containing neurons and expression of the estrogen receptor system; and mechanisms underlying estrogen-stimulated growth (transcription of an estrogen-regulated gene, the cellular oncogene, c-fos). These studies have significance for understanding the mechanisms underlying the genesis of a wide variety of human abnormalities of considerable clinical, socio-cultural and educational importance which include: the sexually dimorphic childhood disorders of cognition (learning disabilities), infantile autism, delayed speech acquisition, and stuttering; the cognitive deficits associated with Turner's syndrome, aging and Alzheimer's disease and perhaps even schizophrenia; disorders of psychosexual differentiation; and certain types of reproductive infertility. These studies will continue to lead to new fields, requiring new knowledge and new methodologies which, along with the learning opportunities afforded by the collaborative approaches, will greatly benefit and continue to significantly advance professional growth.
