Development And Regulation Of The LHRH System

对生殖至关重要的LHRH神经元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LHRH神经元在正常/转基因动物和鼻外植体中的分化、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调节LHRH基因表达、肽合成和LHRH神经元分泌的机制（内在和跨突触）。我们实验室之前的工作1)表明GABA是鼻部LHRH神经元迁移的一个重要因素，2)发现了一个新的基因，我们假设它通过一种亲同性相互作用来影响LHRH神经元在嗅轴突上的迁移，3)证明了LHRH在体外的生物合成和分泌与体内的相似。在过去的一年里，我们通过检测NELF在嗅觉切除后的表达，并建立了一个生产全长NELF蛋白的系统，用于生化和功能研究，研究了NELF的作用。为了进一步阐明GABAA能信号对LHRH神经元的影响，我们对LHRH神经元中GABAA受体亚基的表达进行了表征，发现两个亚基α 2和α 6在发育过程中呈反比变化。α 2增加，α 6减少。此外，我们在gaba能治疗后对LHRH神经元进行了差异筛选，并开始研究这些基因在体内和体外的作用。其中一个差异表达的基因是肽CCK。迄今为止，我们已经证明CCK在LHRH神经元的发育过程中共表达，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育的功能，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。我们发现，尽管存在N型和L型通道，但只有N型通道的破坏会改变LHRH的运动。最后，基于去年在LHRH神经元中发现雌激素受体β亚型的工作，我们研究了LHRH神经元的活性以及雌激素对该参数的影响。首先，我们证明LHRH神经元表现出同步的钙振荡，独立于CNS信号，这与LHRH脉冲分泌在时间上相关。最近的实验发现，雌激素对LHRH神经元活动有直接影响，并增加了参与同步钙脉冲的LHRH细胞的数量。我们假设这种现象与体内在排卵前激增期间发生的正反馈有关。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神经元活动的机制。正在进行的具体研究集中在：1)分离影响嗅轴突生长的中线线索；2) NELF等分子在LHRH迁移中的作用，3)确定LHRH神经元中参与节律性活动建立/维持的起搏器分子，4)作为gaba能信号在LHRH神经元中差异表达的基因，5)雌激素改变LHRH神经元活动的机制。

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.