Steroids exert dramatic, highly reproducible effects on behavior in every species studied to date from insects to man. Behavioral changes are strongly correlated with structural and functional changes in the connections among the cells in the brain, or synapses in the adult brain. Gonadal steroids such as testosterone have been shown to accelerate and induce changes in the strength and number of synapses in areas responsible for learning and memory. The goal of this project is to determine if the presence or absence of testosterone changes synaptic strength among nerve cells in a neural network responsible for social and reproductive behaviors in adults. Synapses will be identified with the latest technology and examined under low and high power microscopes to identify significant changes in the number, function, and source of synapses in the preoptic area, an important integrating structure in this network. Results from these studies will enhance our understanding of the role of gonadal steroids in the regulation of neural function in the adult brain, thus setting the stage for future studies of growth enhancing chemicals and substrates involved in sexual differentiation, aging and injury. This project will offer unique training opportunites in neuroanatomy and will include undergraduates and graduates in the collection, analysis and processing of the results.
ROA Progress Report Project Title: Gestational Programming of the Hypothalamic-Pituitary-Gonadal Axis Specific Aim: The overall goal of the project was to determine how reduced prenatal energy availability affects the development of GnRH circuits in the brain. This goal was accomplished through examining the effects of low protein diet during gestation on the timing of pubertal onset and analyzing offspring brains for GnRH expression using immunohistochemistry. Progress on the Project: Rat Animal Model: A low protein rat animal model of gestational programming was established as a result of this project. This animal model was used to examine the effects of gestational exposure to low protein diet on pubertal timing. For this experiment, nulliparous timed pregnant female rats (Charles River) were given ad libitum access to either normal protein (20%) or low protein diets (6%) from E7-E21. At birth all animals were weighed, measured and cross fostered to normal protein dams. Starting at P20, all offspring were monitored daily for pubertal onset. Offspring (n=7 per group) at P60 were injected with 100 mg/kg sodium pentobarbital and perfused with heparinized saline followed by 4% paraformaldehyde, and these brains from male and female offspring have been sectioned. The brain sections are currently being stained immunohistochemically for GnRH. Sections will be photographed at 4X and 10X and the images will be examined using ImagePro software to determine the density of staining for GnRH in the hypothalamus. Technical issues: Unfortunately, the transgenic rats that expressed GFP driven by the GnRH promoter only expressed GFP in a subset of the GnRH neurons and would not breed reliably. Despite this setback, we were able to generate gestationally programmed offspring born at low body weight that exhibited rapid catch up growth and early onset puberty in female offspring (see figure above). Because of the temporary setback, we are still processing the brains of gestationally restricted offspring for GnRH expression, but anticipate completing the project in the spring of 2013. Presentations: A. Skiles, K. Bleistein, O. Fladmark, E. Keen-Rhinehart, Effects of protein deficiency on size and weight in rats. Landmark Summer Research Symposium, July 22, 2011, Baltimore, MD Katelynn Ondek, Rebecca Frazier & Erin Keen-Rhinehart, Effects of Low Protein Diet during Gestation on Central Energy Balance, Society for Neuroscience, October 15-18, 2012, New Orleans, LA
