Optogenetic approaches to study post-stroke recovery mechanisms

 描述（由申请人提供）：中风是一种严重的急性神经损伤，会扰乱大脑功能并导致神经元死亡。中风后可恢复失去的功能，这归因于大脑重组和神经可塑性。在本提案中，我们将使用 光遗传学和成像技术研究脑回路动力学和轴突可塑性在中风后恢复。光遗传学允许以毫秒级的精度激活或抑制大脑中的特定细胞群和回路，因此是研究神经系统的有价值的工具。 参与中风后恢复的回路。我们最近证明，使用光遗传学在同侧运动皮层（iM 1）中选择性神经元刺激可以激活可塑性机制并促进恢复。在目标1中，我们将使用光遗传学功能MRI（ofMRI）来研究在非刺激中风小鼠和重复刺激中风小鼠中中风后恢复期间由选择性光遗传学刺激诱发的脑回路激活的全局变化。在目标2A中，我们将使用两种活性报告小鼠（神经元激活报告小鼠（FosTRAP）和突触活性报告小鼠（ArcTRAP））在3-D全脑中在细胞水平上检查脑回路激活。我们将确定这些激活的神经元的细胞类型（兴奋性与抑制性）。在目标2B中，我们将研究刺激如何改变结构 可塑性通过轴突发芽使用顺行示踪剂（生物素标记的葡聚糖胺）。目标2将使用MITHITY技术，以可视化三维全脑中回路活动和轴突发芽的细胞分辨率。在目标3中，我们将使用光遗传学技术来确定在中风后恢复过程中对侧皮层（中风的对侧）的作用。一些研究表明，对侧皮层激活是必要的恢复，而其他研究表明，对侧皮层激活可能是适应不良和恶化恢复。为了确定对侧病灶皮层在恢复期间是否发挥有益或有害的作用以及是否存在时间依赖性作用，我们将在中风后不同阶段（早期与晚期）操纵对侧病灶皮层中的特定神经回路，并检查它们对功能恢复的影响。功能恢复将通过一组在我们实验室建立良好的感觉运动行为测试进行评估。将在表现出功能恢复的组中检查活动依赖性神经营养因子表达和结构可塑性（轴突发芽）。我们的研究结果将促进对中风后恢复过程中神经回路和大脑重组的理解。

 DESCRIPTION (provided by applicant): Stroke is a major acute neurological insult that disrupts brain function and causes neuron death. Recovery of lost function can occur after stroke and is attributed to brain reorganization and neuroplasticity. In this proposal, we will use optogenetics and imaging techniques to study brain circuit dynamics and axonal plasticity during post-stroke recovery. Optogenetics allows activation or inhibition of specific cell groups and circuits in the brain with millisecond-scale precision, thus is a valuable tool for studying neural circuits involved in post-stroke recovery. We recently demonstrated that selective neuronal stimulation in the ipsilesional motor cortex (iM1) using optogenetics can activate plasticity mechanisms and promote recovery. In Aim 1 we will use optogenetic functional MRI (ofMRI) to study global changes in brain circuit activation evoked by selective optogenetic stimulations during post-stroke recovery in non-stimulated stroke mice and repeatedly-stimulated stroke mice. In Aim 2A we will examine brain circuit activation at the cellular level in 3-D whole brain using two lines of activity reporter mice: neuronal activation reporter mice (FosTRAP) and synaptic activity reporter mice (ArcTRAP). We will determine the cell type of these activated neurons (excitatory vs inhibitory). In Aim 2B we will investigate how stimulations alter structural plasticity through axonal sprouting using the anterograde tracer (biotinylated-labeled dextranamine). The CLARITY technique will be used for Aim 2 to visualize cellular resolution of circuit activities and axonal sprouting in 3-D whole brains. In Aim 3 we will use the optogenetics technique to determine the role of the contralesional cortex (side opposite to stroke) during post-stroke recovery. Some studies indicate that contralesional cortex activation is necessary for recovery, whereas other studies suggest that contralesional cortex activation may be maladaptive and worsen recovery. To determine whether the contralesional cortex exerts beneficial or deleterious effects during recovery and whether there is a time-dependent role, we will manipulate specific neural circuits in the contralesional cortex at different post- stroke phases (early vs late) and examine their effects on functional recovery. Functional recovery will be evaluated by a panel of sensorimotor behavior tests that are well established in our lab. Activity-dependent neurotrophin expression and structural plasticity (axonal sprouting) will be examined in the groups that exhibit functional recovery. Our findings will advance the understanding of neural circuits and brain reorganization during post-stroke recovery.