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

描述(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，随后这些受损轴突的再生非常有限。这被认为严重制约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措施来说是次优的：它们往往无法区分再生轴突和病变部位未受损纤维或备用轴突的萌芽。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。成年大鼠全身注射对氯苯丙胺(PCA)后，中缝背侧5-羟色胺轴突迅速退化，随后数周内5-羟色胺能神经缓慢恢复。我们已经将这种PCA方案应用于成年BAC转基因小鼠，在这些转基因小鼠中，5-羟色胺神经元的完整范围被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质，从而追踪PCA损伤前和损伤后13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。在这里，我们建议对该模型系统进行开发和扩展。目的1.前列腺癌损伤后5-羟色胺轴突再生的快速动态事件能预测稳定的再生轴突形态特征吗？到目前为止，我们已经进行了一次每周测量的低时间分辨率调查。这提供了一个概述，但没有考虑到以分钟到天的时间尺度来检查轴突。我们建议在两个关键时期增加我们的数据集，每10分钟测量一次，每隔一天测量一次：紧跟在PCA之后以捕捉回归，以及在PCA之后约7-8周，此时许多开创性的纤维进入视野。目的2.新皮质热损伤后再生5-羟色胺轴突的关键短期和长期结构动力学是什么？我们建议重复体内5-羟色胺轴突的时间推移成像，用局灶性热损取代PCA治疗。我们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，以比较分子干预和功能恢复的候选治疗方法。目的3.热损伤或前列腺癌损伤后新皮质轴突再生的所有阶段是否都需要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，这是一种形成允许生长底物层粘连蛋白受体的蛋白质。我们将交叉小鼠品系：将整合素1与5-羟色胺转运体驱动的Cre杂交，选择性地删除5-羟色胺神经元中的整合素1，并结合对PCA和热损伤反应的体内5-羟色胺轴突的延时成像。

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.