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

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）受损轴突退化，随后这些受损轴突的再生非常有限。这被认为严重限制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对完整制备的固定组织的死后分析，这种分析产生静态图像。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未受损纤维的发芽或病变部位的未受损轴突。我们开发了一个模型系统，在这个模型系统中，轴突的远距离再生可以用延时成像技术在完整的成年小鼠大脑中进行研究。用对氯安非他明（PCA）对成年大鼠进行全身治疗会导致中叶背5 -羟色胺轴突的快速消退，随后在数周内5 -羟色胺能神经支配缓慢恢复。我们已经将这种PCA方案应用于成年BAC转基因小鼠，其中血清素神经元的完整范围用EGFP标记。使用双光子显微镜和颅窗，我们可以重复成像相同体积的新皮层，从而在PCA病变前和13周后跟踪血清素能轴突，以提供确定的存活，退化和再生纤维的延时测量。在此，我们建议对该模型系统进行开发和扩展。目的1。PCA病变后5 -羟色胺轴突再生的快速动态事件能否预测稳定再生的轴突形态特征？到目前为止，我们已经进行了每周测量的低时间分辨率调查。这提供了一个概述，但不允许在分钟到天的时间尺度上检查轴突。我们建议在两个关键时期以10分钟和每天的间隔进行测量来增加我们的数据集：在PCA之后立即捕获回归，以及在PCA之后约7-8周，当许多开创性的纤维进入视野时。目标2。新皮层热损伤后5HT轴突再生的关键短期和长期结构动力学是什么？我们建议重复血清素轴突的体内延时成像，用局灶性热损伤代替PCA治疗。我们的目标是建立两种明确定义的轴突损伤和再生模型系统，一种是传统的泛细胞和胶质瘢痕形成模型，另一种是细胞类型特异性和非瘢痕形成模型，以便比较分子干预和候选功能恢复疗法。目标3。热损伤或PCA损伤后新皮层轴突再生的所有阶段是否需要5 -羟色胺神经元中整合素1的表达？5 -羟色胺神经元表达高水平的整合素1，这是一种蛋白质，它构成了允许生长底物层粘连蛋白受体的一部分。我们将用5 -羟色胺转运蛋白驱动的Cre杂交小鼠菌株，选择性地删除5 -羟色胺神经元中的整合素，并结合体内5 -羟色胺轴突对PCA和热损伤的反应的延时成像。

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.