Project 2 Serotonin as a Modulator Of Axon Guidance Signals

除了在神经传递中的作用外，5-HT似乎还调节基本的发育过程 影响大脑回路的组装。在人类中，SERT和5 HT受体多态性是 与心理健康相关的神经发育障碍有关，如自闭症。在动物模型中， 早期5-HT作用包括对神经元分化、存活和神经突生长的微妙影响， 对感觉地图的形成有显著的影响。遗传学研究表明，5-HT受体的破坏， 发育的限制期导致长期的行为功能障碍。的分子机制 5-羟色胺对神经发育的影响的基础是未知的，由于大量的5-羟色胺 受体和有限的理解，他们的表达模式和信号转导能力。在 在初步研究中，Levitt的研究小组已经确定了5 HT在调节经典的 轴突导向分子在项目2中：大脑中轴突引导的5-羟色胺调制 发展，帕特莱维特和同事提出了三个目标，以利用康特中心 合作，并研究5 HT在参与心理健康的两个关键回路组装中的作用 疾病、丘脑皮质轴突（TCA）和中缝背核（DR）轴突。在目标I中，他们建议使用 外植体分析系统和一种新的子宫内基因电穿孔策略来建立机制 特异性5 HT受体亚型通过Netrin-1调节TCA引导。他们将确定 5-HT调节对介导Netrin-1的轴突导向受体膜表达的影响 吸引和排斥以及环核苷酸在介导调节中的作用。长期影响 通过siRNA或过表达的5 HT受体敲低破坏子宫内的TCA地形图也将是 考察TCA地形的详细分析将在Pet-1缺失小鼠中进行，与 项目1。在目标II中，将研究指导含5-HT的DR轴突的机制。 原位杂交研究将确定DR神经元的导向受体的表达模式， 引导分子沿着他们的途径（与项目六）。将使用外植体测定来确定 DR轴突对这些引导线索的响应性，以及5 HT调节响应性的能力。 将在其功能测定中检查在项目I DR转录组谱分析中鉴定的候选物。在 目的III，他们将确定SERT遗传变异对TCA和DR轴突对以下反应的影响： 5-HT对轴突导向分子生物活性的调节作用。与项目3合作 （Blakely），他们将确定如何通过功能性SERT变体改变引导线索的5 HT调制， 以及SERT活性如何独立于5-HT影响轴突导向。SERT相互作用的作用 蛋白ITGB 3和SYN 1A作为SERT调节轴突导向的调节剂，将通过原位杂交来研究。 杂交作图、使用ITGB 3缺失小鼠的轴突引导测定和SYN 1A功能的破坏。

In addition to its role in neurotransmission, 5HT appears to regulate fundamental developmental processes that influence the assembly of brain circuitry. In humans, SERT and 5HT receptor polymorphisms are associated with mental health-related neurodevelopmental disorders, such as autism. In animal models, early 5HT actions include subtle effects on neuronal differentiation, survival and neurite outgrowth, and pronounced effects on sensory map formation. Genetic studies show that disruption of 5HT receptors during a restricted period of development results in long-term behavioral dysfunction. The molecular mechanisms that underlie the 5HT impact on neurodevelopment are unknown, owing to the large number of serotonin receptors and limited understanding of their expression patterns and signal-transducing capabilities. In preliminary studies, Levitt's group has identified a novel role for 5HT in modulating the signaling of classic axon guidance molecules. In Project 2: Serotonin Modulation of Axon Guidance During Brain Development, Pat Levitt and colleagues propose three aims to take advantage of Conte Center collaborations and investigate the role of 5HT in the assembly of two key circuits involved in mental health disorders, thalamocortical axons (TCAs) and dorsal raphe (DR) axons. In Aim I, they propose use of an explant assay system and a novel in utero gene electroporation strategy to establish the mechanisms through which specific 5HT receptor subtypes modulate TCA guidance by Netrin-1. They will identify the impact of 5-HT modulation on the membrane expression of axon guidance receptors that mediate Netrin-1 attraction and repulsion and the role of cyclic nucleotides in mediating modulation. The long term impact of disrupting TCA topography in utero by 5HT receptor knockdown by siRNA or over-expression also will be examined. Detailed analysis of TCA topography will be done in the Pet-1 null mice, in collaboration with Project 1. In Aim II, the mechanisms that govern the guidance of 5-HT-containing DR axons will be studied. In situ hybridization studies will determine the expression patterns of guidance receptors by DR neurons and guidance molecules along their pathway (with Project VI). Explant assays will be used to determine the 'sponsiveness of DR axons to these guidance cues, and the ability of 5HT to modulate responsiveness. andidates identified in Project I DR transcriptome profiling will be examined in their functional assay. In Aim III, they will determine the influence of SERT genetic variants on the responseof TCAs and DR axons to the modulatory effects of 5HT on axon guidance molecule bioactivity. In collaboration with Project 3 (Blakely), they will determine how 5HT modulation of guidance cues is altered by functional SERT variants, and how SERT activity, independent of 5-HT, influences axon guidance. The role of SERT-interacting proteins ITGB3 and SYN1A as modulators of SERT modulation of axon guidance will be studied by in situ hybridization mapping, axon guidance assays using ITGB3 null mice, and disruption of SYN1A function.