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

小鼠体内 2 光子显微镜揭示的血清素轴突再生机制

• 批准号: 9115478
• 负责人: Sarah Dougherty
• 金额: $ 5.8万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9115478
• 关键词: Adult; Amphetamines; Animals; Autopsy; Axon; Biological Models; Brain; Brain region; Candidate Disease Gene; Cells; Cephalic; Chemicals; Chondroitin Sulfate Proteoglycan; Cicatrix; Clinical; Cluster Analysis; Complementary DNA; Data; Degenerative Disorder; Dorsal; Dose; Event; Failure; Fascicle; Fiber; Functional disorder; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genetic; Glial Fibrillary Acidic Protein; Glutamates; Goals; Growth; Growth Associated Protein 43; Growth Inhibitors; Human; Image; Immunohistochemistry; Impaired cognition; Impairment; Infiltration; Injury; Inorganic Sulfates; Intervention; Label; Lesion; Measurement; Microglia; Microscope; Microscopy; Molecular; Molecular Profiling; Mood Disorders; Mus; Natural regeneration; Neocortex; Nervous System Physiology; Neuraxis; Neurons; Paralysed; Phase; Postdoctoral Fellow; Preparation; Proteoglycan; Protocols documentation; Rattus; Recovery; Recovery of Function; Research; Research Personnel; Role; Sensory; Serotonin; Signal Transduction; Site; Stab Wounds; Staining method; Stains; Stroke; Techniques; Testing; Therapeutic Intervention; Tilia; Time; Tissues; Toxic effect; Training; Transgenic Mice; Trauma; Traumatic Brain Injury; Unspecified or Sulfate Ion Sulfates; Vestibular nucleus structure; axon injury; axon regeneration; axonal degeneration; cell type; density; genetic profiling; in vivo; injured; insight; neocortical; nerve supply; neuronal cell body; public health relevance; raphe nuclei; sensor; serotonin transporter; targeted treatment; therapy development; tissue fixing; two-photon

 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.

 描述（由适用提供）：在局部创伤之后，成人哺乳动物中枢神经系统（CNS）的轴突损坏，随后对这些受损的轴突的再生非常有限。再生的失败极大地阻碍了中枢神经系统功能的恢复，并导致瘫痪，感觉功能障碍和认知障碍。迄今为止，对脑轴突再生的研究几乎完全依赖于完整制剂中固定组织的死后分析，从而产生静态图像。这些快照是评估治疗干预措施的次优，因为它们通常无法区分再生 病变部位的未标记纤维或储物轴突发芽的轴突。我们已经开发了一个模型系统，其中可以通过完整的成年小鼠脑中的延时成像进行研究轴突的长距离再生。全身治疗成年大鼠用p-氯 - 氨基苯丙胺（PCA）导致背侧​​raphe塞列汀轴突的快速消退，随后在数周内塞列汀能神经支配的缓慢恢复。 Linden Lab已将该PCA方案调整为成年BAC转基因小鼠，其中用EGFP标记了血清素神经元的完整程度。使用两光子显微镜和颅窗，我们反复成像相同的新皮层，从而在使用PCA病变后跟踪六周的六周轴突，以提供确定的生存，回升，回升和再生纤维的延时测量。在这里，我建议扩展此模型系统并转向机械询问。目标1。在新皮层刺伤后，5-羟色胺轴突会再生吗？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，用神经胶质体诱导的刺伤损伤代替PCA治疗。我的目标是拥有两个定义明确的模型系统，用于轴突损伤和再生，一种常规的，泛细胞和神经胶质体形成，以及其他细胞型特异性和非量子形成，以比较分子干预措施和候选疗法以进行功能恢复。目标2。背强奸的5-羟色胺神经元具有独特的基本基因表达谱，其轴突再生的异常能力是基础的？还是这些细胞中关键的基因表达事件只能在PCA或刺激损伤后成为证据？我建议进行单细胞基因分析，以量化轴突损伤之前，PCA或刺伤后的损伤以及轴突再生后立即量化5-羟色胺神经元内的基因表达模式。这将提供候选基因来改变再生。