Development And Regulation Of The LHRH System

LHRH神经元，生殖的关键，是从鼻基板和迁移到大脑，在那里他们成为下丘脑-垂体-性腺轴的组成部分。我们在正常/转基因动物和鼻外植体中研究LHRH神经元分化、迁移和轴突靶向的潜在机制。使用这些相同的模型，我们的工作也解决了机制调节（内在的和跨突触）LHRH基因的表达，肽的合成和分泌LHRH神经元。我们实验室的前期工作1）表明GABA是鼻区LHRH神经元迁移的重要因素，2）鉴定了一个新的基因NELF，我们假设该基因通过嗜同性相互作用影响LHRH神经元在嗅觉轴突上的迁移，3）证明体外LHRH的生物合成和分泌模拟体内所见。在过去的一年里，我们一直致力于NELF的作用，通过检查其表达后嗅神经切断术和建立一个系统，以产生全长NELF蛋白的生化和功能研究。为了进一步阐明GABA能信号对LHRH神经元的影响，我们已经表征了LHRH神经元中GABAA受体亚基的表达，并发现两个亚基，α 2和α 6，显示出随发育的逆变化。α 2增加而α 6减少。此外，我们已经进行了GABA能治疗后的LHRH神经元的差异筛选，并已开始研究这些基因在体内和体外的作用。差异表达的一个基因是肽CCK。到目前为止，我们已经表明，CCK是共同表达的LHRH神经元在发育过程中，并影响LHRH神经元的运动和成熟。我们还研究了LHRH细胞中钙通道的表达模式作为发展的函数，以及这些表达模式变化对LHRH细胞迁移和/或调节的功能后果。我们已经发现，虽然存在N和L型通道，但仅N型通道的破坏会改变LHRH运动。最后，基于去年的工作，显示雌激素受体β亚型在LHRH神经元，我们已经检查了LHRH神经元的活动和雌激素对这个参数的影响。首先，我们证明LHRH神经元表现出同步的钙振荡，不依赖于中枢神经系统的线索，这与脉动式LHRH分泌在时间上相关。最近的实验发现，雌激素对LHRH神经元活性有直接影响，并增加参与同步钙脉冲的LHRH细胞的数量。我们推测这种现象与排卵前峰期体内发生的正反馈有关。未来的研究将针对嗅觉和LHRH神经元系统发育的重要分子和线索以及调节LHRH神经元活性的机制。具体的研究进展主要集中在：1）影响嗅轴突生长的中线线索的分离; 2）NELF和其他分子在LHRH迁移中的作用，3）鉴定LHRH神经元中参与节律活动的建立/维持的起搏分子，4）在LHRH神经元中作为GABA能信号的函数差异表达的基因，和5）雌激素改变LHRH神经元活性的机制。

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 LHRH 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) LHRH gene expression, peptide synthesis and secretion in LHRH neurons. Previous work in our lab 1) showed GABA was an important factor in the migration of LHRH neurons in nasal regions, 2) identified a novel gene termed NELF which we hypothesize acts via a homophilic interaction to influence LHRH neuronal migration on olfactory axons and 3) demonstrated that biosynthesis and secretion of LHRH in vitro mimics that seen in vivo. Over the past year, we have worked on the role of NELF by examining its expression after olfactory axotomy and establishing a system to produce full length NELF protein for biochemical and functional studies. To further elucidate the influence of GABAergic signals on LHRH neurons we have characterized the expression of GABAA receptor subunits in LHRH neurons and found two subunits, alpha 2 and alpha 6, show inverse changes over development ? alpha 2 increases while alpha 6 decreases. In addition we have performed a differential screen of LHRH neurons after GABAergic treatment and have begun to examine the role of these genes both in vivo and in vitro. One gene that was differentially expressed was the peptide CCK. To date we have shown that CCK is co-expressed in LHRH neurons during development and influences both movement and maturation of LHRH neurons. We also examined the expression pattern of calcium channels in LHRH cells as a function of development and the functional consequences of changes in these expression patterns with respect to LHRH cell migration and/or regulation. We have found that although N and L type channels are present, disruption of only the N type channel alters LHRH movement. Finally, based on work from last year which showed estrogen receptor beta subtypes in LHRH neurons, we have examined LHRH neuronal activity and the effects of estrogen on this parameter. First, we documented that LHRH neurons exhibit synchronized calcium oscillations, independent of CNS cues, which temporally correlate with pulsatile LHRH secretion. Recent experiments have found that estrogen has a direct effect of LHRH neuronal activity and increases the number of LHRH cells which participate in a synchronized calcium pulse. We hypothesize that this phenomenon is related to the positive feedback that occurs in vivo during the preovulatory surge. Future studies are directed at the molecules and cues important for development of the olfactory and LHRH neuronal systems as well as the mechanisms regulating LHRH neuronal activity. Specific studies in progress focus on: 1) isolation of midline cues which influence olfactory axon outgrowth; 2) the role of NELF and other molecules in LHRH migration, 3) identifying pacemaker molecules in LHRH neurons that participate in establishment/maintenance of rhythmic activity, 4) genes differentially expressed in LHRH neurons as a function of GABAergic signals and 5) the mechanisms by which estrogen alters LHRH neuronal activity.