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

 描述(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，随后这些受损轴突的再生非常有限。这种再生的失败严重阻碍了中枢神经系统功能的恢复，并导致瘫痪、感觉障碍和认知障碍。到目前为止，对脑轴突再生的研究几乎完全依赖于对完整标本中固定组织的尸检分析，这会产生静态图像。这些快照对于评估治疗干预来说是次优的，因为它们往往无法区分再生 损伤部位未受损纤维萌发的轴突或备用轴突。我们已经开发了一个模型系统，在这个系统中，可以用时间推移成像技术在完整的成年小鼠大脑中研究轴突的长距离再生。用对氯苯丙胺(PCA)全身处理成年大鼠，会导致中缝背侧5-羟色胺轴突迅速退化，随后在数周内5-羟色胺能神经缓慢恢复。林登实验室已经将这种PCA方案应用于成年BAC转基因小鼠，在成年BAC转基因小鼠中，5-羟色胺神经元的完整范围被绿色荧光蛋白标记。使用双光子显微镜和颅窗，我们重复成像相同体积的新皮质，从而追踪PCA损伤前和损伤后26周的5-羟色胺能轴突，以提供对已识别的存活、退化和再生纤维的时间推移测量。在此，我建议扩展这一模式系统，并转向机械式询问。目的1.新皮质刺伤后5-羟色胺轴突再生吗？我建议重复5-羟色胺轴突的免疫组织化学和体内延时成像，用胶质瘢痕诱导的刺伤代替PCA治疗。我的目标是拥有两个明确的轴突损伤和再生模型系统，一个是常规的、泛细胞和神经胶质瘢痕形成的，另一个是特定细胞类型的和非瘢痕形成的，以便比较分子干预和功能恢复的候选治疗方法。目的2.中缝背侧的5-羟色胺神经元是否具有独特的基础基因表达谱，从而支持其不寻常的轴突再生能力？或者，这些细胞中的关键基因表达事件是否只有在PCA或刺伤后才变得明显？我建议进行单细胞遗传图谱，以量化轴突损伤前、PCA或刺激性损伤后以及轴突再生后5-羟色胺神经元内的基因表达模式。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.