Optogenetic approaches to study post-stroke recovery mechanisms

 描述(申请人提供)：中风是一种严重的急性神经侮辱，扰乱大脑功能并导致神经元死亡。丧失的功能可在中风后恢复，并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中风后康复过程中脑回路动力学和轴突可塑性。光遗传学允许以毫秒级的精度激活或抑制大脑中特定的细胞组和电路，因此是研究神经的宝贵工具 参与中风后恢复的回路。我们最近利用光遗传学证明了选择性刺激损毁运动皮质(IM1)中的神经元可以激活可塑性机制，促进康复。在目标1中，我们将使用光遗传功能磁共振(OfMRI)来研究选择性光遗传刺激在卒中后恢复过程中非刺激性卒中小鼠和反复刺激性卒中小鼠脑回路激活的整体变化。在目标2A中，我们将使用两种活动报告鼠：神经元激活报告鼠(FosTRAP)和突触活动报告鼠(ArcTRAP)，在三维全脑细胞水平上检测脑电路激活。我们将确定这些激活神经元的细胞类型(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 通过使用顺行示踪剂(生物素标记的右旋糖胺)的轴突萌发来实现可塑性。Clarity技术将用于Aim 2，以可视化3-D整个大脑中电路活动和轴突萌发的细胞分辨率。在目标3中，我们将使用光遗传学技术来确定对侧皮层(卒中的对侧)在卒中后康复过程中的作用。一些研究表明，对侧皮质的激活是恢复所必需的，而另一些研究表明，对侧皮质的激活可能是不适应的，从而使恢复更差。为了确定对侧皮质在恢复过程中是有益还是有害的影响以及是否存在时间依赖的作用，我们将在卒中后的不同阶段(早期和晚期)操纵对侧皮质的特定神经回路，并检查它们对功能恢复的影响。功能恢复将通过我们实验室建立的感觉运动行为测试小组进行评估。神经营养因子的活性依赖表达和结构可塑性(轴突萌发)将在表现出功能恢复的组中进行检测。我们的发现将促进对中风后康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.