DISTINCTIVE SPATIOTEMPORAL MAP OF TARGET ACTIVATION BY YOUNG NEURONS OF THE ADULT

描述（由申请人提供）：成人齿状回不断产生特定学习和记忆任务所需的颗粒细胞（DGCs），但新神经元对海马回路信息处理的确切贡献尚不清楚。在过去，我们已经证明了围产期大脑中发生的关键发育过程，如兴奋性成熟、传入突触发生和功能，都在成人神经发生过程中重演。我们现在提出了一个中心假设，即新生细胞在发育过程中建立了功能输出，而年轻神经元接触的突触后靶细胞主要是抑制性的，因此不同于成熟颗粒细胞激活的混合兴奋/抑制网络。因此，会有一个时间窗口，年轻的DGCs主要激活前馈和/或反馈抑制回路，而不刺激锥体细胞，对齿状回输出施加严格的抑制控制。在目的1中，我们将建立一个由成年齿状回的年轻发育神经元靶激活的时空图。我们将利用逆转录病毒转导在年轻成年雌性小鼠新生DGCs （C57Bl6/J）中表达光激活阳离子通道Channelrhodopsin-2。我们将在不同的时间献祭动物，并准备急性脑切片进行电生理记录。通过用短暂的光脉冲刺激整个海马体切片，所有逆转录病毒转导的神经元都会出现尖峰。我们将在整个门部和CA3区域随机搜索活跃的突触后靶细胞，并通过结合松散斑块和全细胞记录来识别和表征响应神经元。在Aim 2中，我们将研究新的苔藓纤维突触在gaba能和谷氨酸能靶点上的功能成熟。我们计划利用全细胞记录来测试在gaba能中间神经元目标上形成的突触是否比在锥体细胞上形成的突触成熟得更快，正如我们之前的结构研究所表明的那样。我们还将研究短期和长期可塑性的突触前机制，这些机制将塑造活动依赖性竞争和突触后回路的激活。该项目将解决有关新生细胞的连接和激活（尖峰）的基本问题，这将有助于理解成人神经发生在预先存在的海马网络中的确切影响，以及成人大脑中神经元连接的规则。确定神经元以安全和功能相关的方式整合到现有网络中的规则，对于开发未来的大脑修复疗法也至关重要。将与盖奇实验室合作开发新的逆转录病毒工具，以增强通道视紫红质-2的表达，从而提高新生细胞的光激活能力。反过来，这里获得的实验数据将被用于盖奇实验室正在开发的关于未成熟神经元在信号处理中的作用的理论模型。拟议项目的成功依赖于加强Leloir研究所（布宜诺斯艾利斯）的Schinder实验室和La Jolla Salk研究所的Gage实验室之间的密切互动。Schinder实验室的成员将有机会在Salk接受有关新型逆转录病毒载体的生成和表征方面的培训，从而提高该外国机构开发先进分子工具的能力。我们预计，在勒卢瓦尔研究所进行的导致设计、生成和使用新型逆转录病毒工具的能力建设将对当地科学界产生巨大影响。因此，这一合作努力将成为一种驱动力，以增加阿根廷研究人员的临界质量，这些研究人员将研究神经退行性疾病的竞争性技术纳入其研究重点。进行再生工程的化验室数目的增加，肯定会提高市民对这些及相关问题的认识。

DESCRIPTION (provided by applicant): The adult dentate gyrus continuously generates granule cells (DGCs) that are needed for specific learning and memory tasks, but the precise contribution of new neurons to information processing in the hippocampal circuitry remains unknown. In the past, we have demonstrated that key developmental processes occurring in the perinatal brain such as maturation of excitability, afferent synaptogenesis and function are all recapitulated during adult neurogenesis. We now propose the central hypothesis that newly born cells establish functional outputs as they develop, and the population of postsynaptic target cells contacted by young neurons is predominantly inhibitory, therefore different from the mixed excitatory/inhibitory network activated by mature granule cells. Thus, there would be a time window in which young DGCs primarily activate feedforward and/or feedback inhibitory circuits without exciting pyramidal cells, exerting a tight inhibitory control over the dentate gyrus output. In Aim 1 we will build a spatio-temporal map of target activation by young developing neurons of the adult dentate gyrus. We will use retroviral transduction to express the light- activated cation channel Channelrhodopsin-2 in newborn DGCs of young-adult female mice (C57Bl6/J). We will sacrifice the animals at different times and prepare acute brain slices to carry out electrophysiological recordings. By stimulating the whole hippocampal slice with brief light pulses, all retrovirally transduced neurons will spike. We will search randomly for active postsynaptic target cells throughout the hilus and CA3 regions, and identify and characterize responsive neurons by combining loose patch and whole-cell recordings. In Aim 2 we will investigate the functional maturation of new mossy fiber synapses made onto GABAergic and glutamatergic targets. We plan to utilize whole-cell recordings to test whether synapses forming onto GABAergic interneuron targets mature faster than those made onto pyramidal cells, as suggested by our previous structural studies. We will also study presynaptic mechanisms of short- and long-term plasticity that will shape both activity-dependent competition and activation of postsynaptic circuits. This project will address fundamental questions about connectivity and activation (spiking) of newborn cells that will contribute to understanding the precise impact of adult neurogenesis in the preexisting hippocampal network and the rules of neuronal connectivity in the adult brain. Identifying the rules by which neurons integrate in the existing network in a manner that is both safe and functionally relevant is also crucial for developing future brain repair therapies. Novel retroviral tools will be developed in collaboration with the Gage lab to enhance Channelrhodopsin-2 expression, thus improving the capability of light activation of newborn cells. In turn, experimental data obtained here will be used to feed into the theoretical model being developed by the Gage lab on the role of immature neurons in signal processing. The success of the proposed project relies on strengthening the close interaction between the Schinder laboratory at the Leloir Institute (Buenos Aires) and the Gage laboratory at the Salk Institute of La Jolla. Members of the Schinder lab will have the opportunity to train at Salk on the generation and characterization of novel retroviral vectors, improving the capabilities to develop advanced molecular tools at the foreign institution. We anticipate that capacity building leading to the design, generation and use of novel retroviral tools at the Leloir Institute will have an enormous impact on the local scientific community. This collaborative effort will therefore serve as a driving force to increase the critical mass of Argentine investigators that incorporate competitive technologies for the study of neurodegenerative disorders within their research focus. An increase in the number of laboratories carrying out regeneration- related projects will certainly enhance awareness to these and related problems to our community. PUBLIC HEALTH RELEVANCE: There is a lot of expectation in our society on the potential of neural progenitor cells as powerful tools for brain repair, and we will need to understand how adult-born neurons establish their connectivity in the adult healthy brain before attempts can be made in case of neurodegeneration or trauma. Solid research on functional integration of newborn neurons of the adult hippocampus will certainly contribute to the development of regenerative therapies for the treatment of disorders occurring in non-neurogenic areas. In particular, it is critical to identify the rules by which neurons integrate in the adult brain circuits in a manner that is both safe and functionally relevant. This will be a major outcome of the proposed project.