The relationship between pulsatile secretion and calcium dynamics in single, living gonadotropin-releasing hormone neurons

It is well established that pulsatile release of GnRH regulates the reprodu ctive axis, but little is known about the mechanisms underlying this pulsat ility. Recent findings that GT1 cells, a line derived from the mouse embryo nic hypothalamus, release GnRH in a pulsatile manner indicates that this rh ythmic activity is an intrinsic property of GnRH neurons. In several attemp ts to uncover the intracellular basis for this pulsatile phenomenon, it was revealed that intracellular calcium concentrations change in a rhythmic fa shion in GnRH neurons and that cellular depolarization, which triggers a se cretory event, is associated with profound calcium changes in the cells. Th ese findings raised the intriguing possibility that periodic alterations in intracellular calcium concentrations may underlie the phenomenon of pulsat ile secretion in GnRH neurons. To address this, we first adapted the use of FM1-43 fluorescence to monitor changes of secretion in individual GT1-7 ce lls and then combined this approach with simultaneous measurement of intrac ellular free calcium ([Ca2+](i), fura 2 method). In initial validation expe riments, we found that stimulation of exocytosis with K+ (75 mM) or N-methy l-D-aspartate (NMDA, 100 mu M) predictably evoked dynamic increases of both FM1-43 and fura 2 fluorescence. Later measurement of calcium dynamics and exocytotic activity in unstimulated cells revealed that [Ca2+](i) underwent transitions from quiescence to high oscillatory behavior, and that these s hifts were frequently associated with exocytotic events. Moreover, these ca lcium oscillatory transitions and associated changes in secretory activity occurred synchronously among most adjacent cells and at a frequency similar to that reported for pulsatile release of GnRH by entire cultures of GnRH neurons. Taken together, these results indicate that the intrinsic secretor y pulsatility of GnRH neurons appears to be a consequence of coordinated, p eriodic changes in the pattern of calcium oscillations within individual ce lls.