Time lapse imaging of serotonin axon regeneration in the neocortex of adult mouse

成年小鼠新皮质中血清素轴突再生的延时成像

• 批准号: 8429778
• 负责人: DAVID J. LINDEN
• 金额: $ 20.25万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8429778
• 关键词: Address; Adult; Amphetamines; Animals; Autopsy; Axon; Beds; Biological Models; Brain; Cell Culture Techniques; Cells; Cellular biology; Cephalic; Chemicals; Cicatrix; Coupled; Data Set; Development; Dorsal; Event; Fiber; Fishes; Functional disorder; Genes; Goals; Growth; Growth Inhibitors; Hybrids; Image; Immunohistochemistry; Impaired cognition; Impairment; Individual; Injury; Integrins; Intervention; Label; Laminin; Laminin Receptor; Lasers; Lesion; Measurement; Measures; Microscope; Microscopy; Midbrain structure; Modeling; Molecular; Molecular Analysis; Monitor; Mood Disorders; Morphology; Mouse Strains; Movement; Mus; Mutant Strains Mice; Natural regeneration; Neocortex; Nervous System Physiology; Neuraxis; Neuroglia; Neuronal Injury; Neurons; Neuropil; Neurotoxins; Paralysed; Pathway interactions; Phase; Physiologic pulse; Preparation; Process; Proteins; Protocols documentation; Rana; Rattus; Recovery; Recovery of Function; Research Personnel; Resolution; Sensory; Serotonin; Signal Transduction; Site; Somatosensory Cortex; Spinal Cord; Stroke; Surveys; System; Testing; Therapeutic Intervention; Time; Tissues; Transgenic Mice; Trauma; Traumatic Brain Injury; Varicosity; axon growth; axon regeneration; cell type; hindbrain; in vivo; nerve supply; neuronal cell body; novel therapeutics; promoter; raphe nuclei; receptor; response; serotonin transporter; therapy development; tissue fixing; two-photon

DESCRIPTION (provided by applicant): Following local trauma, damaged axons in the adult mammalian central nervous system (CNS) regress, and subsequent regeneration of these damaged axons is very limited. This is thought to strongly constrain 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: 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. We have 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 can repeatedly image the same volume of neocortex and thereby track serotonergic axons before and e 13 weeks after lesion with PCA to provide time-lapse measurements of identified surviving, regressing and regenerating fibers. Here, we propose to develop and extend this model system. Aim 1. Do rapid dynamic events in regenerating serotonin axons following lesion with PCA predict features of stable regenerated axon morphology? To date, we have performed a low temporal resolution survey with weekly measurements. This provides an overview but has not allowed for examination of axons on a minutes-to-days timescale. We propose to augment our dataset with measurements at 10 min and daily intervals during two crucial periods: immediately following PCA to capture regression and ~ 7-8 weeks following PCA, when many pioneering fibers are entering the field of view. Aim 2. What are the key short and long-term structural dynamics of regenerating 5HT axons following a thermal lesion of the neocortex? We propose to repeat in vivo time-lapse imaging of serotonin axons, replacing PCA treatment with focal thermal lesions. Our 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 3. Is expression of integrin ¿1 in serotonin neurons required for all phases of axonal regeneration in the neocortex following thermal or PCA lesion? Serotonin neurons express high levels of integrin ¿1, a protein that forms part of the receptor for the permissive growth substrate laminin. We shall cross mouse strains: floxed integrin ¿1 with serotonin transporter-driven Cre, to selectively delete integrin ¿1 in serotonin neurons coupled with in vivo time-lapse imaging of serotonin axons in response to PCA and thermal lesions. PUBLIC HEALTH RELEVANCE: Following stroke or traumatic brain injury in adults, recovery of function is often very limited, resulting in persistent paralysis, sensory dysfunction, cognitiv impairment and disorders of mood. Researchers have attempted to develop therapies to promote recovery of function by stimulating the regrowth of axons, the long information-sending extensions of nerve cells in the brain. We have developed a new system for making time- lapse images of regenerating axons in the brain of this mouse, with the ideas that this will provide an ideal test- bed for developing new therapeutic measures.

描述（由申请人提供）：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，这些受损轴突的后续再生非常有限。这被认为强烈限制了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：它们通常无法区分再生轴突与损伤部位未受损纤维或备用轴突的发芽。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）对成年大鼠进行全身性治疗会导致中缝背核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。我们已经适应了这个PCA协议的成年BAC转基因小鼠，其中的5-羟色胺神经元的完整程度与EGFP标记。使用双光子显微镜和颅窗，我们可以重复成像相同体积的新皮层，从而跟踪PCA损伤前和损伤后13周的轴突，以提供识别的存活，退化和再生纤维的延时测量。在这里，我们建议开发和扩展这个模型系统。目标1. PCA损伤后5-羟色胺轴突再生的快速动力学事件能否预测稳定再生轴突形态的特征？到目前为止，我们已经进行了低时间分辨率的调查，每周测量。这提供了一个概述，但还没有允许在几分钟到几天的时间尺度上检查轴突。我们建议在两个关键时期以10分钟和每日间隔的测量来增强我们的数据集：PCA后立即捕获回归和PCA后约7-8周，此时许多先锋纤维正在进入视野。目标二。新皮层热损伤后再生5 HT轴突的关键短期和长期结构动力学是什么？我们建议重复在体内的5-羟色胺轴突的延时成像，取代PCA治疗与局灶性热损伤。我们的目标是有两个明确定义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3. 5-羟色胺神经元中整合素1的表达是否是热损伤或PCA损伤后新皮层轴突再生所有阶段所必需的？5-羟色胺神经元表达高水平的整合素1，整合素1是一种形成允许生长底物层粘连蛋白受体的蛋白质。我们将杂交小鼠品系：floxed integrin 1与5-羟色胺转运蛋白驱动的Cre，选择性删除整合素1在5-羟色胺神经元加上在体内的时间推移成像的5-羟色胺轴突响应PCA和热损伤。 公共卫生相关性：在成年人中风或创伤性脑损伤后，功能恢复通常非常有限，导致持续性瘫痪、感觉功能障碍、认知障碍和情绪障碍。研究人员试图通过刺激轴突的再生来开发促进功能恢复的疗法，轴突是大脑中神经细胞的长信息发送延伸。我们已经开发了一种新的系统，用于制作这只老鼠大脑中再生轴突的延时图像，其想法是，这将为开发新的治疗措施提供一个理想的试验台。