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

描述（由申请人提供）：海马齿状回（DG）是成人大脑中唯一持续表现神经发生的区域，从干细胞和祖细胞形成新的神经元。尽管进行了广泛的研究，但对成人神经发生如何影响海马功能知之甚少。我们认为，通过加深我们对新生神经元如何发育并融入现有脑回路的理解，可以获得关于这一基本问题的见解。目前缺乏在完整大脑中观察新生海马神经元数周发育时间尺度的技术，这阻碍了对一些基本问题的研究，如：新生神经元在生命过程中何时（如果有的话）达到结构稳定性？作为成人神经发生的一部分，细胞增殖、迁移和细胞死亡的大规模事件在多大程度上影响了先前存在的神经元的回路组织？动物的年龄和身体活动水平（两者都是成年神经发生的调节因素）是如何影响这些动态过程的？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的最新技术进步，这是一种新的方法，可以在组织深处成像细胞数周或数月。因此，我们的研究目标是：(1)开发一种慢性小鼠制剂，用于在数周和数月的时间内对DG中的成年海马神经发生进行体内微内窥镜成像。我们的方法使用：(a)光学显微内窥镜成像大脑深处的细胞；(b) DG延时显微内镜小鼠慢性制剂；(c)在神经祖细胞和新生神经元中表达荧光蛋白的转基因小鼠和病毒载体。总之，这些工具将允许纵向，高分辨率的双光子成像细胞的详细形态的祖细胞，新的和成熟的神经元在活的成年DG。(2)表征成体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如何受到动物年龄和身体活动的影响，通过测量新生神经元树突分支尖端的生长或收缩，树突树的分支复杂性作为发育时间的函数，以及细胞从亚颗粒层迁移到颗粒层的距离和速率。我们将在基线条件下和允许自愿运动的小鼠中比较年轻成年小鼠和老年小鼠的这些参数。我们还将评估结构动力学在成熟DG神经元中发生的程度，这取决于动物的年龄和身体活动。因此，我们的研究将初步了解活体成人海马体中发育中的神经元，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期融入DG网络。

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.