Development And Regulation Of The Gonadotropin Releasing Hormone System

GnRH-1（也被称为LHRH）神经元，对生殖至关重要，来源于鼻位并迁移到大脑，在那里它们成为下丘脑-垂体-性腺轴的组成部分。我们研究了GnRH-1神经元在正常/转基因动物和鼻腔外植体中的分化、迁移和轴突靶向的机制。使用这些相同的模型，我们的工作还解决了GnRH-1神经元中调节（内在和跨突触）GnRH基因表达、肽合成和分泌的机制。多种方法用于识别和理解在引导GnRH-1神经元到达其在中枢神经系统中的最终位置中发挥作用的众多分子和因素。这些包括从迁移和非迁移细胞中获得的文库的差异筛选，在迁移路线的关键位置检查差异表达的分子，敲除小鼠中GnRH-1系统发育的形态学检查，以及体外分子扰动和随后监测GnRH-1神经元运动。随着GnRH-1神经元的迁移，它们也成熟了，这两个过程实际上可能是相关联的。为了研究GnRH-1神经元的成熟，我们采用钙显像、电生理和生化方法检测了GnRH-1神经元的活性和肽分泌。

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) Single and double mutant mice are used to examine the lineage of Gonadotropin-releasing hormone (GnRH)-1 cells. GnRH is essential for vertebrate reproduction, with either GnRH-1 or GnRH-3 controlling release of gonadotropins from the anterior pituitary depending on the species. It is clear that the neuroendocrine GnRH cells migrate from extra-CNS locations into the forebrain. However, the embryonic origin of GnRH-1 and GnRH-3 cells is controversial and has been suggested to be nasal placode, adenohypophyseal (anterior pituitary) placode or neural crest, again dependent on the species examined. We showed that mutant mice with either missing or disrupted anterior pituitaries (Gli2-/-, Gli1-/-Gli2-/- and Lhx3-/-) exhibit a normal GnRH-1 neuronal population and that these cells are still found associated with the developing vomeronasal organ. These results indicate that in mice, GnRH-1 cells develop independent of the adenohypophyseal placode and are associated early with the formation of the nasal placode. 2) Neuropeptide Y (NPY), a member of the pancreatic polypeptide family, is an orexigenic hormone. Gonadotropin-releasing hormone-1 (GnRH-1) neurons express NPY receptors. This suggests a direct link between metabolic function and reproduction. However, the effect of NPY on GnRH-1 cells has been variable, dependent on metabolic and reproductive status of the animal. This study circumvents these issues by examining the role of NPY on GnRH-1 neuronal activity in an explant model that is based on the extra-CNS origin of GnRH-1 neurons. These prenatal GnRH-1 neurons express many receptors found in GnRH-1 neurons in the brain and use similar transduction pathways. In addition, these GnRH-1 cells exhibit spontaneous and ligand-induced oscillations in intracellular calcium as well as pulsatile calcium-controlled GnRH-1 release. Single cell PCR determined that prenatal GnRH-1 neurons express the G-protein coupled Y1 receptor (Y1R). To address the influence of NPY on GnRH-1 neuronal activity, calcium imaging was used to monitor individual and population dynamics. NPY treatment, mimicked with Y1R agonist, significantly decreased the number of calcium peaks per minute in GnRH-1 neurons and was prevented by a Y1R antagonist. Pertussis toxin blocked the effect of NPY on GnRH-1 neuronal activity, indicating the coupling of Y1R to inhibitory G protein. The NPY-induced inhibition was independent of the adenylate cyclase pathway but mediated by the activation of G-protein coupled inwardly rectifying potassium (GIRK) channels. These results indicate that, at an early developmental stage, GnRH-1 neuronal activity can be directly inhibited by NPY via its Y1R. 3) Oscillations in intracellular calcium levels have been described in gonadotropin-releasing hormone (GnRH-1) neurons in both prenatal and adult cells. However, differences have been reported in the mechanisms underlying these Ca2+i oscillations, dependent on the model used. The goal of this study was to address whether these changes depend on the maturation status of GnRH-1 neurons by assaying prenatal GnRH-1 cells maintained in explants, at two different developmental stages. This report documents an increase in the frequency of Ca2+i oscillations between 1 and 3 weeks of in vitro maturation. During the early stage, Ca2+i oscillations are blocked by TTX and are mainly triggered by excitatory neurotransmitters, GABA and glutamate. In contrast, in the later stage, some cells exhibit residual TTX-insensitive Ca2+i oscillations which are sustained by action potential-independent GABA and glutamate release. The strength of these two excitatory inputs remained relatively constant during the maturation process and the increase in frequency of Ca2+i oscillations observed at the later stage is due to a novel excitatory input carried by cholecystokinin. Together, these data indicate developmentally-regulated release and interactions of neurotransmitters (known regulators of GnRH-1 cells in adults), and point to extrinsic factors regulating GnRH-1 cellular physiology. New investigations using Cre-lox-mice to specifically remove molecules of interest from GnRH-1 cells during development have been continued this year with specific studies on the early development of the GnRH-1 neurons and the location of their progenitor cells in relation to nasal placodal cells and neural crest. In progress are studies examining the role of growth factor receptors (FGFR1, PDGF), NELF (a migrational molecule), and cytokines, 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 are finishing studies monitoring GnRH-1 neuronal activity in nasal explants generated from estrogen receptor knockout mice. Other studies include examining/identifying 1) the maturation of ion transporters associated with GnRH-1 neuronal activity, 2) molecules that modulate GnRH-1 neuronal activity that participate in reproductive functions such as adiponectin, 3) midline cues which influence olfactory axon outgrowth and 4) the dynamics of GnRH-1 neuronal migration.