Development of cortical circuits using multiscale optical imaging in awake mice

项目总结 神经回路的发展依赖于分子过程和活动依赖过程的结合。这个 活动可以是自发的--例如穿过哺乳动物围产期视网膜的视网膜波--或者 感官刺激。视网膜活动的自发波对于大脑中的电路完善是必要的。虽然 有关视网膜电波产生的视网膜回路已知很多，但目前还不清楚视网膜电波是如何产生的 塑造功能性皮质结构的发展以及它们在功能性皮质结构的出现中所起的作用 在睁开眼睛时已经确定的皮质特性，如视网膜复视和定向选择性。它 也完全不知道哪些神经元亚型参与了视觉皮质中的波，以及它们何时参与 在发展过程中被招募。在这里，我们将确定兴奋性和抑制性的作用和动力学 视网膜波期间视皮层神经元活动的同时细胞分辨率2- 局部回路内遗传定义神经元的光子钙成像和同时广域单视野成像 跨皮质神经元活动的光子成像评估视网膜波在皮质回路中的功能 在清醒的头部固定的小鼠中形成。这种新的多尺度、双成像方法将弥合 为了更好地了解局部细胞与显微和宏观成像的关系 活动和中尺度、大脑皮层的活动。鉴定中间神经元并测量它们在视网膜中的参与程度 在皮层的波活动，我们将表达荧光报告tdTomato在抑制性中间神经元使用 PAN下表达绿色钙指示剂GCaMP6s转基因小鼠的Cre/lox系统 神经元Snap25启动子。我们还将评估Nkx2.1-的时空活动模式的个体发育， 生长抑素(SOM)和血管肠肽表达(VIP)中间神经元与兴奋/抑制 病毒在视皮层视网膜波活动II期和III期中尺度水平的耦合 JRCaMP1b在中间神经元和GCaMP6在兴奋性神经元中的表达我们假设大脑皮层 中间神经元塑造视皮层视网膜波活动的流动，并有助于视网膜移植的出现 和取向选择性。我们将通过使用GiDREADD慢性沉默这些细胞来测试这一点，然后评估 除视网膜定位外，视皮层视网膜波产生活动的时空动力学 以及在睁开眼睛时使用视觉刺激测量定向调谐。此外，我们将确定 Nkx2.1、SOM、VIP中间神经元在视皮层II、III期视网膜波活动中的具体作用 这项工作将开发和应用必要的工具来详细研究特定神经元的影响 关于人脑回路的功能组织，并将描述大脑皮质如何发育 传播自发活动，以及不同亚型大脑皮质之间的功能连接 神经元是由早期的自发活动塑造的。这对于理解正常的大脑发育至关重要。 以及与神经发育障碍相关的功能障碍。

PROJECT SUMMARY Development of neural circuits depends on a combination of molecular and activity-dependent processes. The activity can be spontaneous—such as retinal waves that course across the mammalian perinatal retina—or sensory-evoked. Spontaneous waves of retinal activity are necessary for circuit refinement in the brain. Although much is known about the retinal circuitry responsible for retinal wave generation, it is unclear how retinal waves shape the development of functional cortical architecture and what role they play in the emergence of functional cortical properties that are already established at eye opening, such as retinotopy and orientation selectivity. It is also completely unknown which subtypes of neurons participate in waves in visual cortex and when they are recruited during development. Here we will determine the roles and dynamics of excitatory and inhibitory neuron activity in visual cortex during the period of retinal waves using simultaneous cellular-resolution two- photon calcium imaging of genetically defined neurons within a local circuit and simultaneous wide field single- photon imaging of neuronal activity across cortex to assess the function of retinal waves in cortical circuit formation in awake, head-fixed mice. This novel multiscale, dual-imaging approach will bridge the gap between microscopic and macroscopic imaging in order to better understand the relationship between local cellular activity and mesoscale, cortex-wide activity. To identify interneurons and measure their participation in retinal wave activity in cortex, we will express the fluorescent reporter tdTomato in inhibitory interneurons using the cre/lox system in transgenic mouse lines expressing the green calcium indicator GCaMP6s under the pan- neuronal snap25 promotor. We will also assess the ontogeny of the spatiotemporal activity pattern of Nkx2.1-, somatostatin- (SOM), and vasointestinal peptide-expressing (VIP) interneurons and excitatory/inhibitory coupling on the mesoscale level during stage II and stage III retinal wave activity in visual cortex using viral expression of jRCaMP1b in interneurons and GCaMP6s in excitatory neurons. We hypothesize that cortical interneurons shape the flow of retinal wave activity in visual cortex and contribute to the emergence of retinotopy and orientation selectivity. We will test this by chronically silencing these cells using GiDREADD, then assessing spatiotemporal dynamics of retinal wave generated activity in visual cortex, in addition to mapping retinotopy and measuring orientation tuning using visual stimulation at eye opening. Furthermore, we will determine the specific roles of Nkx2.1, SOM, VIP interneurons during stage II and stage III retinal wave activity in visual cortex. This work will develop and apply essential tools for detailed investigation of the impact of specific neuronal populations on the functional organization of brain circuits, and will describe how the developing cortex propagates spontaneous activity, and how the functional connectivity between different subtypes of cortical neurons is sculpted by early spontaneous activity. This is critical to understanding normal brain development and dysfunction associated with neurodevelopmental disorders.