Development And Regulation Of The Luteinizing Hormone Releasing Hormone System

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. [unreadable] [unreadable] 1) Over the past year we have investigated the role of the brain-gut peptide, CCK, as a modulator of GnRH-1 neurnal activity. Functional analysis indicated that CCK inhibits GnRH-1 neuronal activity via CCK-1 receptors. CCK is a neuropeptide abundantly expressed in the brain, which is implicated in activation of female reproductive behaviors and release of anterior pituitary hormones. Using dual-label immunocytochemistry coupled to confocal analysis, GnRH-1 neurons in adult mouse brain were found to express CCK-1 receptors (CCK-1R) and CCK fibers were detected contacting GnRH-1 axons. To address the function of CCK on GnRH-1 neurons, calcium imaging was used to monitor patterns of activity of GnRH-1 neurons maintained in an in vitro system known to retain many characteristics of GnRH-1 cells in vivo. Endogenous receptors for CCK (CCK-1R and CCK-2R) were blocked with selective antagonists. Results indicate that CCK-1R but not CCK-2R antagonist treatment increased the number of calcium peaks/GnRH-1 cell, mean peak amplitude and percentage of GnRH-1 cells displaying high activity. The increased activity in GnRH-1 neurons observed after application of CCK-1R antagonist was blocked by co-incubation with exogenous CCK. This study provides evidence that CCK acts directly on GnRH-1 neurons to attenuate GnRH-1 neuronal activity via CCK-1R activation.[unreadable] [unreadable] 2) Studies continued to identify the role of GnRH-1 in developing incisor. Morphological changes in the incisor of GnRH-1 mutant mice were analyzed and indicate GnRH-1 participated in tooth maturation and biomineralization.[unreadable] [unreadable] 3) A collaborative study examined the development of the GnRH systems in zebrafish to verify that GnRH-1 cells arise in association with the nasal placode. Using morphological studies as well as tagged GnRH-1 cells in live embryos, GnRH-1 cells were observed migration from the nasal placode region into the developing forebrain. [unreadable] [unreadable] 4) Another collaborative study examined the role hepatocyte growth factor (HGF) and its receptor Met during development of the GnRH-1 system. GnRH-1 cells express Met during their migration and downregulate its expression once they complete this process. Tissue-type plasminogen activator (tPA), a known HGF activator, is also detected in migratory GnRH-1 neurons. Consistent with in vivo expression, HGF is present in nasal explants and GnRH-1 neurons express Met. HGF neutralizing antibody was applied to explants to examine the role of the endogenous growth factor. Migration of GnRH-1 cells and olfactory axon outgrowth was significantly reduced, in line with disruption of a guidance gradient. Exogenous application of HGF to explants increased the distance that GnRH-1 cells migrated suggesting that HGF also acts as a motogen to GnRH-1 neurons. Functional experiments, performed on organotypic slice cultures, show that creation of an opposing HGF gradient inhibits GnRH-1 neuronal migration. Finally, tPA-/- : uPA-/- knock-out mice exhibit strong reduction of the GnRH-1 cell population. Together, these data indicate that HGF signaling via Met receptor influences the development of GnRH-1.[unreadable] [unreadable] 5) Pubertal development is impaired in mice lacking the basic helix-loop-helix transcription factor Nhlh2. The mechanisms underlying changes in reproduction in Nhlh2-/- mice were unclear. In this study, we show that hypothalamic GnRH-1 content is reduced in adult Nhlh2-/- mice as is the number of GnRH-1 neurons localized to mid- and caudal hypothalamic regions. This reduction was detected postnatally after normal migration of GnRH-1 neurons within nasal regions had occurred. Phenotype rescue experiments showed that female Nhlh2-/- mice were responsive to estrogen treatment. In contrast, puberty could not be primed in female Nhlh2-/- mice with a GnRH-1 regimen. The adenohypophysis of Nhlh2-/- mice was hypoplastic although it contained a full complement of the five anterior pituitary cell types. GnRH-1 receptors (GnRHRs) were reduced in Nhlh2-/- pituitary gonadotropes as compared to wild type. In vitro assays indicated that Nhlh2 expression is regulated in parallel with GnRHR expression. However, direct transcriptional activity of Nhlh2 on the GnRHR promoter was not found. These results indicate that Nhlh2 plays a role in the development and functional maintenance of the hypothalamic-pituitarygonadal axis at least at two levels: 1) in the hypothalamus by regulating the number and distribution of GnRH-1 neurons and, 2) in the developing and mature adenohypophysis. [unreadable] [unreadable] Studies in progress center on 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 LHRH 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 electrical properties associated with GnRH-1 neuronal activity (combining electrical recording and calcium imaging), 2) midline cues which influence olfactory axon outgrowth, 3) pacemaker molecules in GnRH-1 neurons that participate in establishment/maintenance of rhythmic activity as well as regulator molecules such as Kisspeptin, and 4) GABAergic signals during development of the GnRH-1 system.