Optogenetic approaches to study post-stroke recovery mechanisms

项目摘要 中风是死亡的主要原因，治疗选择非常有限。这种毁灭性的神经系统疾病 越来越多地被视为大脑连接性疾病，因为受损的中风区域可以影响局部和 连接的大脑区域，导致神经元活动和代谢网络范围内的中断。回收 中风后可能发生功能丧失，这归因于邻近或连接到大脑的区域的脑重塑。 梗塞在这项提议中，我们的目标是研究关键脑回路在中风后恢复中的作用， 功能，细胞和分子水平，使用光遗传学，先进的活体成像和高通量RNA 测序技术。以前，我们的实验室已经证明，选择性光遗传神经元刺激， 同侧运动皮层（iM 1）可激活可塑性机制并促进恢复。最近我们 利用光遗传学功能磁共振成像技术系统地绘制了神经回路的全脑变化 中风后我们已经确定了中风改变的关键电路，并证明了两个关键电路的恢复， iM 1刺激。我们的地图数据还揭示了两个未被iM 1刺激恢复的候选电路， 这表明如果我们能通过直接刺激来挽救这些回路，就能实现更大的恢复。 在这个提议中，我们的目标是研究我们从激活图中识别出的关键神经回路，并阐明 在中风后恢复中的作用。在Aim 1中，我们将使用电路特异性光遗传学工具和功能行为 测试询问中风后恢复的关键电路的作用。这一目标将解决这些电路是否 在中风后的恢复过程中发挥有益或不适应的作用。在Aim 2中，我们将检查细胞分辨率， 使用便携式活体钙成像系统在中风后关键回路中的实时神经元活动动力学。 这将阐明细胞水平上关键回路的神经活动动力学（兴奋性和抑制性）， 使我们能够确定中风改变的时间分布和关键神经元群体，以及iM 1 刺激影响这些特征以增强恢复。在Aim 3中，我们将研究以下基因的转录组： 使用RNAseq的关键电路区域，以确定中风改变的关键分子靶点和途径， iM 1刺激。初步的RNAseq分析揭示了由iM 1刺激改变的不同途径。我们 目的是在多个区域进行RNAseq，包括iM 1（刺激部位）和同病灶丘脑（iM 1- 连接区域），以阐明是否涉及类似的途径，如果我们可以确定一个共同的 分子特征来推动康复我们还将在两种性别中进行RNAseq，以确定任何 中风后恢复可能存在的性别特异性差异。这些结果将共同推动：1） 了解中风后恢复期间的神经回路动力学; 2）识别关键神经回路/细胞 类型/分子靶点和用于设计脑刺激策略的最佳时间窗口以及其他 未来临床研究中的治疗干预。

PROJECT SUMMARY Stroke is the leading cause of death with very limited treatment options. This devastating neurological disease is increasingly viewed as a disease of brain connectivity as a damaged stroke area can affect both local and connected brain regions, causing disruptions in neuronal activity and metabolism network-wide. Recovery of lost function can occur after stroke and is attributed to brain remodeling in areas adjacent to or connected to the infarct. In this proposal, we aim to investigate the role of key brain circuits in post-stroke recovery at the functional, cellular and molecular level, using optogenetics, advanced live imaging and high throughput RNA sequencing techniques. Previously our lab has demonstrated that selective optogenetic neuronal stimulation in the ipsilesional motor cortex (iM1) can activate plasticity mechanisms and promote recovery. Recently we have employed the optogenetic functional MRI technique to systematically map brain-wide changes in neural circuits after stroke. We have identified key circuits altered by stroke and demonstrated two key circuits restored by iM1 stimulations. Our map data also revealed two candidate circuits that were not restored by iM1 stimulations, suggesting that greater recovery could be achieved if we can rescue these circuits by directly stimulating them. In this proposal we aim to investigate key neural circuits we identified from our activation maps and elucidate their role in post-stroke recovery. In Aim1 we will use circuit-specific optogenetic tools and functional behavior tests to interrogate the role of key circuits in post-stroke recovery. This aim will address whether these circuits have beneficial or maladaptive role during post-stroke recovery. In Aim2 we will examine cellular resolution of real-time neuronal activity dynamics in key circuits after stroke using a portable live calcium imaging system. This will elucidate the neural activity dynamics (excitatory and inhibitory) of key circuits at the cellular level, allowing us to identify the temporal profile and the key neuronal populations altered by stroke, and how iM1 stimulations affect these characteristics to enhance recovery. In Aim3 we will investigate the transcriptome of key circuit areas using RNAseq, in order to identify key molecular targets and pathways altered by stroke and by iM1 stimulations. Preliminary RNAseq analysis revealed distinct pathways altered by iM1 stimulations. We aim to perform RNAseq in multiple regions including iM1 (stimulation site) and ipsilesional thalamus (iM1- connected region) to elucidate whether similar pathways are involved, and if we can identify a common molecular signature that drive recovery. We will also perform RNAseq in both sexes in order to ascertain any sex-specific differences that may be present in post-stroke recovery. Together these results will 1) advance the understanding of neural circuit dynamics during post-stroke recovery; and 2) identify key neural circuits/cell types/molecular targets and optimal time window for designing brain stimulation strategies and other therapeutic interventions in future clinical studies.