Mechanisms of Serotonin Axon Regeneration Revealed by In Vivo 2-photon Microscopy in the Mouse

 描述（由申请人提供）：在局部创伤后，成年哺乳动物中枢神经系统（CNS）中受损的轴突退化，并且这些受损轴突的后续再生非常有限。这种再生的失败强烈阻碍CNS功能的恢复，并导致瘫痪、感觉功能障碍和认知障碍。迄今为止，脑轴突再生的研究几乎完全依赖于从完整的准备，产生静态图像的固定组织的死后分析。这些快照对于评估治疗干预是次优的，因为它们通常不能区分再生 损伤部位未受损纤维或备用轴突的出芽形成轴突。我们已经开发了一个模型系统，其中轴突的长距离再生可以在完整的成年小鼠大脑中进行延时成像研究。用对氯苯丙胺（PCA）对成年大鼠进行系统性治疗，导致中缝背核5-羟色胺轴突迅速消退，随后在数周内缓慢恢复多巴胺能神经支配。林登实验室已经将这种PCA方案应用于成年BAC转基因小鼠，其中5-羟色胺神经元的完整范围用EGFP标记。使用双光子显微镜和颅窗，我们反复成像相同体积的新皮层，从而跟踪神经元轴突损伤前和= 26周后PCA提供的时间推移测量确定的存活，退化和再生纤维。在这里，我建议扩展这个模型系统，并转向机械调查。目标1。5-羟色胺轴突在新皮层刺伤后再生吗？我建议重复免疫组化和在体内的5-羟色胺轴突的延时成像，取代PCA治疗与胶质瘢痕诱导刺伤。我的目标是有两个明确的模型系统轴突损伤和再生，一个传统的，泛细胞和胶质瘢痕形成和其他细胞类型特异性和非瘢痕形成，以比较分子干预和候选疗法的功能恢复。目标2.中缝背核的5-羟色胺神经元是否具有独特的基础基因表达谱，从而成为其轴突再生能力的基础？或者这些细胞中的关键基因表达事件只有在PCA或刺伤后才变得明显？我建议进行单细胞遗传分析，以量化基因表达模式5-羟色胺神经元轴突损伤前，立即PCA或刺伤后，轴突再生。这将为改变再生的操作提供候选基因。

 DESCRIPTION (provided by applicant): Following localized trauma, damaged axons in the adult mammalian central nervous system (CNS) regress, and subsequent regeneration of these damaged axons is very limited. This failure of regeneration strongly impedes recovery of CNS function and contributes to paralysis, sensory dysfunction and cognitive impairment. To date, the study of brain axon regeneration has almost exclusively relied upon postmortem analysis of fixed tissue from intact preparations, which yields static images. These snapshots are suboptimal for evaluating therapeutic interventions, as they often fail to distinguish regenerating axons from sprouting of undamaged fibers or spared axons at the lesion site. We have developed a model system in which long-distance regeneration of axons can be studied with time-lapse imaging in the intact adult mouse brain. Systemic treatment of adult rats with p-chloro-amphetamine (PCA), causes rapid regression of dorsal raphe serotonin axons, followed by a slow return of serotonergic innervation over many weeks. The Linden lab has adapted this PCA protocol to adult BAC transgenic mice in which the complete extent of serotonin neurons is labeled with EGFP. Using a two-photon microscope and a cranial window, we have repeatedly imaged the same volume of neocortex and thereby tracked serotonergic axons before and = 26 weeks after lesion with PCA to provide time-lapse measurements of identified surviving, regressing and regenerating fibers. Here, I propose to extend this model system and shift towards mechanistic inquiry. Aim 1. Do serotonin axons regenerate following a stab injury to the neocortex? I propose to repeat immunohistochemistry and in vivo time-lapse imaging of serotonin axons, replacing PCA treatment with a glial-scar inducing stab injury. My goal is to have two well-defined model systems for axonal damage and regeneration, one conventional, pan-cellular and glial- scar-forming and the other cell-type-specific and non-scar forming in order to compare molecular interventions and candidate therapies for functional recovery. Aim 2. Do serotonin neurons of the dorsal raphe have a unique basal gene expression profile that underlies their unusual capacity for axonal regeneration? Or might the crucial gene expression events in these cells only become evident following PCA or stab- evoked injury? I propose to perform single-cell genetic profiling to quantify gene expression patterns within serotonin neurons prior to axonal damage, immediately following PCA or stab-evoked injury, and after axonal regeneration. This will provide candidate genes for manipulation to alter regeneration.