Development And Regulation Of The Luteinizing Hormone Releasing Hormone System

National Institute Of Neurological Disorders And Stroke

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Abstract

GnRH-1 (also known as LHRH) neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain where they become integral members of the hypothalamic-pituitary-gonadal axis. We study mechanism(s) underlying GnRH-1 neuronal differentiation, migration and axonal targeting in normal/transgenic animals, and nasal explants. Using these same models, our work also addresses the mechanisms regulating (intrinsic and trans-synaptic) GnRH gene expression, peptide synthesis and secretion in GnRH-1 neurons. Multiple approaches are used to identify and understand the multitude of molecules and factors which play a role in directing the GnRH-1 neurons to their final location in the CNS. These include differential screening of libraries obtained from migrating versus non-migrating cells, examination of molecules differentially expressed at key locations along the migratory route, morphological examination of the development of the GnRH-1 system in knockout mice, and perturbation of molecules in vitro and subsequent monitoring of GnRH-1 neuronal movement. As GnRH-1 neurons migrate they also mature and the two processes may in fact be linked. To investigate the maturation of GnRH-1 neurons we use calcium imaging, electrophysiology and biochemical measures to examine GnRH-1 neuronal activity and peptide secretion. Over the past year several studies were finished: 1) Kisspeptins, the natural ligands of the G-protein-coupled receptor (GPR)-54, are the most potent stimulators of GnRH-1 secretion and as such are critical to reproductive function. However, the mechanism by which kisspeptins enhance calcium-regulated neuropeptide secretion is not clear. In the present study, we used GnRH-1 neurons maintained in mice nasal explants to examine the expression and signaling of GPR54. Under basal conditions, GnRH-1 cells exhibited spontaneous baseline oscillations in intracellular calcium concentration (Ca2+i), which were critically dependent on the operation of voltage-gated, tetrodotoxin (TTX)-sensitive sodium channels and were not coupled to calcium release from intracellular pools. Activation of native GPR54 by kisspeptin-10 initiated Ca2+i oscillations in quiescent GnRH-1 cells, increased the frequency of calcium spiking in oscillating cells that led to summation of individual spikes into plateau-bursting type of calcium signals in a subset of active cells. These changes predominantly reflected the stimulatory effect of GPR54 activation on the plasma membrane oscillator activity via coupling of this receptor to phospholipase C signaling pathways. Both components of this pathway, inositol 1,3,4-trisphosphate and protein kinase C, contributed to the receptor-mediated modulation of baseline Ca2+i oscillations. TTX and 2-aminoethyl diphenylborinate together abolished agonist-induced elevation in Ca2+i in almost all cells, whereas flufenamic acid was less effective. Together these results indicate that a plasma membrane calcium oscillator is spontaneously operative in the majority of prenatal GnRH-1 neurons and is facilitated by kisspeptin-10 through phosphatidyl inositol diphosphate hydrolysis and depolarization of neurons by activating TTX-sensitive sodium channels and nonselective cationic channels. 2) Pulsatile release of GnRH-1 is critical to stimulate gonadotropes of the anterior pituitary. This secretory pattern seems to be inherent to GnRH-1 neurons however, the mechanisms underlying such episodic release remains unknown. In monkey nasal explants, the GnRH-1 population exhibit synchronized calcium events (1) with the same periodicity as GnRH-1 release (2), suggesting a link though the sequence of events was unclear. GnRH-1 neurons in mouse nasal explants also exhibit synchronized calcium events. In the present work, GnRH-1 release was assayed in mouse nasal explants using radioimmunology and its relationship with calcium signaling analyzed. GnRH-1 neurons generated episodic release as early as 3 days in vitro (div) and maintained such release throughout the period studied (3-21 div). The pulse frequency remained constant suggesting that the pulse generator is operative at an early developmental stage. In contrast, pulse amplitude increased 2-fold between 3-7 div and again between 7-14 div, suggesting maturation in synthesizing and/or secretory mechanisms. To evaluate these possibilities, total GnRH-1 content was measured. Only a small increase in GnRH-1 content was detected between 7-14 div while a large increase occurred between 14-21 div. These data indicate that GnRH-1 content was not a limiting factor for the amplitude of the pulses at 7 div but that the secretory mechanisms matures between 3-14 div. The application of kisspeptin-10 revealed the ability GnRH-1 neurons to integrate signals from natural ligands into a secretory response. Finally, simultaneous sampling of medium and calcium imaging recordings indicated that the synchronized calcium events and secretory events are congruent. New investigations using Cre-lox-mice to specifically remove molecules of interest from GnRH-1 cells during development have been initiated this year. Studies continue on the early development of the GnRH-1 neurons and the location of their progenitor cells in relation to nasal and anterior pituitary placodal cells as well as neural crest. In progress examine the role of NELF (a migrational molecule), cytokines, and growth factors in GnRH-1 development as well as in situ characterization of the migration of GnRH-1 neurons (real time microscopy). In addition, we continue to study the role of estrogen on GnRH-1 neuronal activity and have recently start monitoring GnRH-1 neuronal activity in nasal explants generated from estrogen receptor knockout mice. Other studies include examining/identifying 1) the maturation of electrical properties associated with GnRH-1 neuronal activity, 2) molecules that modulate GnRH-1 neuronal activity that participate in reproductive functions such as NPY, 3) midline cues which influence olfactory axon outgrowth and 4) GABAergic signals during development of the GnRH-1 system.