In Vivo Analysis of Cortical Projection Neurons

描述（由申请人提供）：人类行为的一个重要方面是进行熟练和有目的运动的能力。这种能力依赖于完整的新皮层，特别是不同类别的投射神经元，它们将神经信息从新皮层传递到参与行为的下游回路。有两类皮质投射神经元特别令人感兴趣--投射到脊髓的皮质脊髓神经元和双侧投射到纹状体的皮质纹状体神经元。虽然已经从脑切片实验中学到了很多关于皮质脊髓和皮质纹状体神经元的细胞和突触特性的知识，但由于缺乏易于使用的方法来选择性地记录和操纵这些投射神经元的活动，因此有关它们在体内与意志运动有关的活动模式的知识受到阻碍。在这里，基于先前的体外实验，我们提出了一个研究计划，以开发新的方法来解决这一差距。在一个目标中，我们将开发多种互补的方法来记录清醒的运动小鼠中皮质脊髓和皮质纹状体神经元的活动。在一组研究中，我们将使用体内全细胞记录，以高灵敏度和时间分辨率记录运动皮层神经元，并事后鉴定细胞类型。在平行研究中，我们将感染 皮质脊髓和皮质纹状体神经元与携带遗传编码钙指示剂（GECIs）的重组狂犬病毒，并使用双光子钙成像以高分辨率或使用光纤以群体水平检测活动。在我们的第二个目标中，我们将开发方法来操纵清醒的运动小鼠中皮质脊髓和皮质纹状体神经元的活动。通过使用光遗传学或药物遗传学工具上调或下调投射神经元活性，我们将能够开始解决皮质脊髓和皮质纹状体神经元的活性如何与运动参数因果相关的问题。我们相信，拟议的研究计划是创新和重要的，因为它将产生多种新的实验范式，用于探测皮质投射神经元的体内功能，不仅在运动区，而且在所有的新皮质，因为这些工具将很容易适用于神经系统疾病的小鼠模型。

DESCRIPTION (provided by applicant): An essential aspect of human behavior is the ability to perform skilled and purposive movements. This ability relies on an intact neocortex, and in particular on different classes of projection neurons that convey neural information from the neocortex to downstream circuits involved in behavior. Two classes of cortical projection neurons are of particular interest - corticospinal neurons, projecting to the spinal cord, and corticostriatal neurons, projecting to the striatum bilaterally. While much has been learned from brain slice experiments about the cellular and synaptic properties of corticospinal and corticostriatal neurons, knowledge about their activity patterns in vivo in relation to volitional movements has been impeded due to a lack of easy to use methods for selectively recording from and manipulating the activity of these projection neurons. Here, building on prior ex vivo experiments, we propose a research program to develop new approaches to address this gap. In one aim, we will develop multiple complementary approaches to record the activities of corticospinal and corticostriatal neurons in the awake, moving mouse. In one set of studies we will use in vivo whole-cell recordings to record from motor cortex neurons at high sensitivity and temporal resolution, with post-hoc identification of cell types. In parallel studies we will infect corticospinal and corticostriatal neurons with recombinant rabies viruses carrying genetically encoded calcium indicators (GECIs), and detect activity either at high resolution using two- photon calcium imaging or at a population level using optical fibers. In our second aim, we will develop methods to manipulate the activities of corticospinal and corticostriatal neurons in the awake, moving mice. By either up- or down-regulate projection neuron activity using optogenetic or pharmacogenetic tools, we will be able to begin to address the question of how corticospinal and corticostriatal neurons' activities are causally related to movement parameters. The proposed research program is innovative and significant, we believe, because it will generate multiple new experimental paradigms for probing the in vivo functions of cortical projection neurons, not only in motor areas but across all of neocortex, and because these tools will be readily applicable to mouse models of neurological disease.