Project 2 Serotonin as a Modulator Of Axon Guidance Signals

除了在神经传递中的作用外，5-羟色胺似乎还调节基本的发育过程 会影响大脑回路的组装。在人类中，SERT和5-羟色胺受体的多态是 与精神健康相关的神经发育障碍有关，如自闭症。在动物模型中， 早期的5-羟色胺作用包括对神经元分化、存活和轴突生长的微妙影响，以及 对感觉地图的形成有明显的影响。遗传学研究表明，5-羟色胺受体在 受限的发育期会导致长期的行为障碍。分子机制 由于存在大量的5-羟色胺，5-羟色胺对神经发育的影响尚不清楚。 受体和对其表达模式和信号转导能力的了解有限。在……里面 初步研究，Levitt的研究小组已经确定了5-羟色胺在调节经典的信号转导中的一个新角色 轴突引导分子。项目2：5-羟色胺对脑内轴突引导的调节 开发，Pat Levitt和他的同事提出了利用Conte中心的三个目标 5-羟色胺在参与心理健康的两个关键回路的组装中的作用 疾病，丘脑皮质轴突(TCA)和中缝背侧(DR)轴突。在Aim I中，他们建议使用 外植体分析系统和一种新的宫内基因电穿孔策略来建立机制 特定的5-羟色胺受体亚型通过它来调节Netrin-1对TCA的引导。他们将确定 5-羟色胺对介导Netrin-1的轴突引导受体膜表达的影响 吸引和排斥以及环核苷酸在调节中的作用。的长期影响 通过siRNA或过度表达5HT受体来破坏子宫内的TCA地形图也将是 检查过了。将在Pet-1基因缺失的小鼠中进行TCA地形的详细分析，与 项目1.在AIM II中，将研究支配含有5-羟色胺的DR轴突的引导机制。 原位杂交研究将确定DR神经元和 引导分子沿着它们的路径(项目VI)。外植体分析将用于确定 “DR轴突对这些引导信号的自发性，以及5HT调节反应的能力。” 在项目I DR转录组分析中确定的ANDATE将在其功能分析中进行检查。在……里面 目的III，他们将确定SERT基因变异对TCA和DR轴突反应的影响 5-羟色胺对轴突导向分子生物活性的调节作用。与Project 3协作 (Blakely)，他们将确定功能性SERT变体是如何改变引导线索的5HT调制的， 以及独立于5-羟色胺的SERT活动如何影响轴突引导。SERT相互作用的作用 作为调节轴突引导SERT的蛋白质ITGB3和SYN1A将被原位研究 杂交图谱，使用ITGB3缺失小鼠的轴突引导分析，以及SYN1A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.