Optogenetic approaches to study post-stroke recovery mechanisms

项目总结 中风是主要的死亡原因，治疗选择非常有限。这种毁灭性的神经疾病 越来越多地被视为大脑连接的一种疾病，因为中风受损区域会影响到局部和 相互连接的大脑区域，导致整个网络的神经元活动和新陈代谢中断。恢复 功能丧失可在中风后发生，可归因于邻近或连接的区域的大脑重塑 脑梗塞。在这项建议中，我们的目标是研究关键的大脑回路在中风后康复中的作用。 功能、细胞和分子水平，使用光遗传学、先进的实时成像和高通量RNA 测序技术。此前，我们的实验室已经证明，选择性光遗传神经元刺激在脑内 损伤性运动皮质(IM1)可激活可塑性机制，促进康复。最近我们有 利用光遗传功能磁共振技术系统地标测全脑神经回路的变化 中风后。我们已经确定了因击键而改变的按键电路，并演示了由 IM1刺激。我们的MAP数据还显示了两个未被iM1刺激恢复的候选电路， 这表明，如果我们能够通过直接刺激这些电路来拯救它们，可能会实现更大的复苏。 在这项建议中，我们的目标是研究我们从激活图中识别的关键神经回路，并阐明 他们在中风后康复中的作用。在Aim1中，我们将使用特定于电路的光遗传工具和功能行为 测试以询问关键回路在中风后恢复中的作用。这个目标将解决这些电路是否 在卒中后康复过程中起到有益或不良适应的作用。在AIM2中，我们将研究细胞分辨率 使用便携式活体钙成像系统研究中风后关键回路中的实时神经元活动动力学。 这将在细胞水平上阐明关键回路的神经活动动力学(兴奋和抑制)， 这使我们能够识别中风后的时间轮廓和关键神经元群体，以及iM1是如何 刺激会影响这些特征，以促进复苏。在Aim3中，我们将研究转录组学 使用RNAseq的关键电路区域，以确定中风和中风后改变的关键分子靶点和通路 通过iM1刺激。初步的RNAseq分析显示，iM1刺激改变了不同的通路。我们 目的在多个区域进行RNAseq，包括iM1(刺激点)和丘脑自身(iM1- 连接区)，以阐明是否涉及类似的通路，以及我们是否可以确定共同的 推动复苏的分子签名。我们还将在两性身上进行RNAseq，以确定 中风后康复中可能存在的性别差异。这些结果加在一起将1)推动 了解卒中后康复过程中的神经回路动力学；以及2)识别关键神经回路/细胞 设计脑刺激策略的类型/分子靶点和最佳时间窗口等 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.