The pulsatile release of GnRH in a small subset of hypothalamic neurons is a driving force for reproductive function in mammals. This secretion appears to be dependent on oscillations in intracellular free calcium that are derived from intermittent calcium influx through voltage- operated membrane calcium channels. Recent evidence also suggests changes in GnRH expression occur in an episodic manner. In order to gain a mechanistic understanding of these dynamic fluctuations, new cellular and molecular strategies corresponding more closely to """"""""real-time"""""""" cellular events are required. The PI and his colleagues have developed strategies to make multiple measurements of GnRH gene expression (via luciferase reporter gene activity imaging), GnRH release (by changes in membrane dye uptake and recycling), calcium oscillations (Fura-2 imaging), and combinations of these in living GT-1 cells. Using these tools, the specific aims are to: 1) elucidate the molecular dynamics of GnRH gene expression in living GT-1 cells are representatives of immortalized GnRH neurons; 2) explore the cellular basis for secretory pulsatility in individual GnRH neurons; and 3) establish possible relationships among GnRH secretion, gene expression, and calcium dynamics in the same, living cells.
