Hippocampal neurodifferentiation studied in young and aged animals by in vivo mi

描述(申请人提供)：海马齿状回(DG)是成人大脑中唯一持续表现出神经发生的区域，即从干细胞和前体细胞形成新的神经元。尽管进行了广泛的研究，但对成年神经发生如何影响海马体功能知之甚少。我们认为，通过加深对新生神经元如何发育并整合到现有大脑电路中的理解，可以获得对这一根本问题的洞察。目前缺乏在完整的大脑中可视化新生海马神经元数周发育时间尺度的技术，这阻碍了对基本问题的研究，如：新生神经元在其生命过程中何时达到结构稳定？作为成人神经发生的一部分，大量的细胞增殖、迁移和细胞死亡事件在多大程度上影响先前存在的神经元的回路组织？动物的年龄和体力活动水平，这两个成年神经发生的调节者，如何影响这些动态过程？在这项工作中，我们将解决这一迫切需要的方法，能够成像成人海马神经发生在活的大脑。我们计划利用延时光学显微内窥镜技术的最新进展，这是一种新的方法，可以在组织内数周和数月内对细胞进行成像。因此，我们的研究目标是：(1)开发一种慢性小鼠准备，用于在体内进行延时显微内窥镜成像，以观察DG中成年海马神经发生的持续数周和数月。我们的方法使用：(A)光学显微内窥镜，用于成像大脑深处的细胞；(B)慢性小鼠，用于DG的延时显微内窥镜检查；(C)转基因小鼠和病毒载体，在神经前体细胞和新生神经元中表达荧光蛋白。这些工具结合在一起，将允许纵向高分辨率双光子成像的细胞的详细形态的前体细胞，以及新的和成熟的神经元在活的成年DG。(2)观察成年DG神经元的发育和结构可塑性。我们将评估新生细胞的发育和结构动力学是如何受到动物年龄和体力活动的影响的，方法是测量新生神经元树突支尖的生长或收缩，树突树的分支复杂性作为发育时间的函数，以及细胞从亚颗粒到颗粒层的距离和速度。我们将在基线条件下和允许自愿锻炼的小鼠中比较年轻成年小鼠和老年小鼠的这些参数。我们还将评估在多大程度上成熟的DG神经元发生结构动力学，并取决于动物的年龄和体力活动。因此，我们的研究将初步了解活的成人海马体中发育的神经元，并解决一些关键的悬而未决的问题，即新生神经元如何在成年期和衰老过程中融入DG网络。 与公共健康相关：海马体是一种对学习和记忆至关重要的大脑结构，是人类成年大脑中唯一持续展示神经发生的区域，即从干细胞和祖细胞形成新的神经元。成年海马区的神经发生随着年龄的增长而下降，神经发生缺陷与许多神经精神疾病有关，包括癫痫、抑郁症、精神分裂症和阿尔茨海默病。我们工作的目标是开发一种新的成像技术来直接显示活着的小鼠的成年海马神经发生，然后使用这项技术来检查新生神经元发育在年轻成年鼠和小鼠的海马区之间的差异，以确定特定的发育阶段作为未来干预的潜在治疗靶点。

DESCRIPTION (provided by applicant): The hippocampal dentate gyrus (DG) is the only area in the adult human brain that continually exhibits neurogenesis, the formation of new neurons from stem and progenitor cells. In spite of extensive research, little is known about how adult neurogenesis contributes to hippocampal function. We contend that insight regarding this fundamental issue can be gained by deepening our understanding of how newborn neurons develop and integrate into existing brain circuitry. The present lack of techniques for visualizing in the intact brain the newborn hippocampal neurons over their developmental time scales of weeks has stymied the investigation of fundamental questions such as: When, if ever, in their life course do newborn neurons attain structural stability? To what extent do the massive events of cell proliferation, migration, and cell death that are part of adult neurogenesis impact the circuit organization of the previously existing neurons? How do an animal's age and physical activity level, both regulators of adult neurogenesis, influence these dynamic processes? In this work we will address this pressing need for a method capable of imaging adult hippocampal neurogenesis in the live brain. We plan to capitalize on recent technological advances in time-lapse optical microendoscopy, a novel methodology for imaging cells over weeks and months deep within tissue. The goals of our research are thus to: (1) Develop a chronic mouse preparation for time-lapse in vivo microendoscopy imaging of adult hippocampal neurogenesis in the DG over weeks and months. Our approach uses: (a) Optical microendoscopes for imaging cells deep in the brain; (b) A chronic mouse preparation for time-lapse microendoscopy in the DG; (c) Transgenic mice and viral vectors to express fluorescent proteins in neural progenitors and newborn neurons. Together, these tools will permit longitudinal, high-resolution two-photon imaging of cells' detailed morphologies for progenitor cells, and new and mature neurons in the live adult DG. (2) Characterize the development and structural plasticity of neurons in the adult DG. We will assess how newborn cells' development and structural dynamics are affected by an animal's age and physical activity, by measuring the growth or retraction of newborn neurons' dendritic branch tips, the branching complexity of their dendritic trees as a function of developmental time, and distances and rates of cell migration from the sub-granular to the granular layer. We will compare these parameters between young adult and aged mice, under baseline conditions and in mice permitted voluntary exercise. We will also assess to what extent structural dynamics occurs in mature DG neurons and depends on an animal's age and physical activity. Our study will thus yield initial glimpses of developing neurons in the live adult hippocampus and address some key unanswered questions about how newborn neurons integrate into the DG network in adulthood and aging. PUBLIC HEALTH RELEVANCE: The hippocampus, a brain structure important for learning and memory, is the only area in the human adult brain that continually exhibits neurogenesis, the formation of new neurons from stem and progenitor cells. Adult hippocampal neurogenesis declines during aging, and defects in neurogenesis have been implicated in many neuropsychiatric disorders, including epilepsy, depression, schizophrenia, and Alzheimer's disease. The goals of our work are to develop a novel imaging technology for directly visualizing adult hippocampal neurogenesis in living mice, and then to use this technology to examine how newborn neuron development differs between the hippocampi of young adult and mice, towards identifying specific developmental stages as potential therapeutic targets for future interventions.