Ivan Lieberburg, MD PhD - US grants

The gonadal steroid hormones interact with the CNS during development and adulthood, producing physiologic and biochemical changes at both ages. In particular, estradiol (E) will cause a permanent and irreversible physiologic and biochemical organization in the rodent hypothalamus during a critical period of the first five days of life. In adulthood, however, E now induces reversible changes in hypothalamic neuronal physiology and biochemistry. Both effects of E are achieved through E receptors and a susbsequent alteration of gene expression in neurons of the hypothalamus and other areas of the limbic system. The proposed project will apply recombinant DNA technology toward the analysis of E dependent events in the CNS, and more generally toward the understanding of the basic difference between permanent and transient changes in the CNS. We plan to (1) clone and identify E modulated gene products in development and adulthood by employing kinetic selection of E induced hypothalamic mRNA species followed by hydroxyapatite chromatography and standard cloning techniques (2) confirm E induction by dot blot and Northern analysis of hypothalamic mRNA (3) compare developmental and adult E induced clones by Southernanalysis and standard sequencing techniques (4) identify the neurons which express these gene products using the technique of in situ hybridization (5) examine the temporal, anatomic, hormonal and developmental expression of these gene products also using in situ hybridization (6) examine the expression of these gene products in other developing neuronal systems such as hypothalamic cultures, the visual cortex, and PC12 cells by dot blot, Northern and in situ hybridization analysis in an attempt to identify a component of a general "trophic" program that may be activated in all developing neurons (7) synthesize small polypeptides predicted by the DNA sequences and use them to generate antibodies against the gene products (8) use these antibodies to identify the subcellular organization and processing of the gene products employing the techniques of cell free translation, immunoprecipitation and immunohistochemistry. In this fashion we hope to address the nature of estrogen interaction with the CNS, thereby linking sexual behavior and development with specific gene products. More generally, we hope to address the very nature of the differences between permanent and transient changes in the CNS in response to an identical stimulus.