title: The relationship between pulsatile secretion and calcium dynamics in single, living gonadotropin-releasing hormone neurons.
abstract: It is well established that pulsatile release of GnRH regulates the reproductive axis, but little is known about the mechanisms underlying this pulsatility. Recent findings that GT1 cells, a line derived from the mouse embryonic hypothalamus, release GnRH in a pulsatile manner indicates that this rhythmic activity is an intrinsic property of GnRH neurons. In several attempts to uncover the intracellular basis for this pulsatile phenomenon, it was revealed that intracellular calcium concentrations change in a rhythmic fashion in GnRH neurons and that cellular depolarization, which triggers a secretory event, is associated with profound calcium changes in the cells. These findings raised the intriguing possibility that periodic alterations in intracellular calcium concentrations may underlie the phenomenon of pulsatile 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 cells and then combined this approach with simultaneous measurement ofintracellular free calcium ([Ca2+]i, fura 2 method). In initial validation experiments, we found that stimulation of exocytosis with K+ (75 mM) or N-methyl-D-aspartate (NMDA, 100 microM) 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 shifts were frequently associated with exocytotic events. Moreover, these calcium 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 secretory pulsatility of GnRH neurons appears to be a consequence of coordinated, periodic changes in the pattern of calcium oscillations within individual cells.
authors.affiliation: Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston 29425, USA.
authors.forename: L
authors.initials: L
authors.lastname: Núñez
authors.forename: C
authors.initials: C
authors.lastname: Villalobos
authors.forename: F R
authors.initials: FR
authors.lastname: Boockfor
authors.forename: L S
authors.initials: LS
authors.lastname: Frawley
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004622:Embryo, Mammalian; D005089:Exocytosis / Q000187:drug effects; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D007031:Hypothalamus; D051379:Mice; D008856:Microscopy, Fluorescence; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000502:physiology*; D010507:Periodicity; D011188:Potassium / Q000494:pharmacology
chemical_list: D007987:Gonadotropin-Releasing Hormone; D016202:N-Methylaspartate; D011188:Potassium; D002118:Calcium