Development of a non-invasive method to monitor expression and function of optogenetic tools in non-human primates

开发一种非侵入性方法来监测非人类灵长类动物中光遗传学工具的表达和功能

基本信息

批准号：
10633118
负责人：
Adriana Galvan
金额：
$ 22.75万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-15 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10633118
关键词：
Acceleration Animals Applications Grants Area Autopsy Basal Ganglia Binding Brain Brain region Cells Clinic Clinical Communities Corpus striatum structure Data Development Electron Microscopy Electrophysiology (science)Estradiol Estrogen Receptor alpha FPS-FES Oncogene Failure Functional disorder Genes Genetic Goals Histologic Histology Human Image Injections Joints Knowledge Laboratories Lead Ligand Binding Domain Light Literature Macaca mulatta Mediating Methods Mission Monitor Monkeys Motor National Institute of Mental Health National Institute of Neurological Disorders and Stroke Nature Neurological Models Neurons Neurosciences Research Opsin Parkinson Disease Parkinsonian Disorders Pathologic Pathway interactions Pattern Positron Positron-Emission Tomography Prevalence Primates Productivity Proteins Publishing Radiolabeled Research Research Personnel Research Project Grants Rodent Rodent Model Role Site Techniques Technology Time Transfection Translations Validation Viral Vector Virus Visualization analog brain tissue brain volume cost extracellular in vivo innovation interest light microscopy method development nervous system disorder nonhuman primate novel optogenetics parkinsonian non-human primate putamen response tomography tool wasting

项目摘要

PROJECT SUMMARY Optogenetic methods have been used extensively to expand our knowledge of the normal and pathological roles of the basal ganglia and related brain areas involved in Parkinson’s disease (PD). This revolutionary tech- nology has, so far, been restricted to studies in rodent models of parkinsonism, while technical constraints have largely limited the use of optogenetic experimentation in non-human primates (NHPs). The scarcity of optoge- netics studies in NHPs is not limited to PD-related research. A critical limitation to the expansion of optogenetics in primates is the lack of non-invasive methods to monitor opsin expression in vivo. Postmortem histology re- mains the only reliable method to confirm the correct targeting of brain regions and level of opsin expression in NHPs. Evidently, this method does not allow for in vivo corrections of experimental approaches. Expansion of the use of optogenetics in NHPs require the development of methods to monitor the temporal course and level of opsin expression, so that insufficient expression levels can be detected and corrected in vivo, thus avoiding costly experimental failures, and reducing the number of NHPs needed for research. We propose a non-invasive approach to monitor opsin expression repeatedly in vivo, based on positron imaging tomography (PET) imaging in NHPs. We will use an innovative version of the excitatory opsin channelrhodopsin-2 (ChR2), in which the opsin is fused to the ligand binding domain of the human estrogen receptor alpha, creating the novel opsin “ChRERa”. The radiolabeled fluorinated estradiol analog, [18F]-fluoroestradiol ([18F]-FES), binds strongly to ChRERa, and the brain distribution of [18F]-FES can be visualized with PET. We will use ChRERa and re- peated [18F]-FES PET scans to monitor opsin expression in Rhesus monkeys longitudinally. Using viral vectors, we will express ChRERa in the motor portion of the striatum, and then conduct several [18F]-FES PET scans over the next 12 months to describe the time course of PET detectable opsin expression. In parallel, we will examine if the PET-identified pattern of ChRERa expression corresponds to the presence of functional opsins, by conducting extracellular recordings during light activation. Finally, we will examine the correspondence be- tween the opsin expression pattern found in the [18F]-FES PET scans and the opsin distribution identified with post-mortem histology. These studies will establish a fundamental technology to evaluate in vivo the distribution of opsins in the NHP. In alignment with the purpose of this FOA PA-21-219 (“Joint NINDS/NIMH Exploratory Neuroscience Research Grant”), these studies have the long-term goal of using ChRERa and [18F]-FES PET scans in our NHP optogenetic studies of the pathophysiology of parkinsonism. More broadly, validation of a non-invasive in vivo method to monitor opsin expression could help accelerate reliable use of optogenetics for the study of NHP models of neurological disorders and could eventually facilitate the translation of optogenetic techniques to the clinic.

项目摘要光遗传学方法已被广泛使用，以扩大我们对正常和病理的了解帕金森氏病（PD）涉及的巴萨神经节和相关大脑区域的作用。这项革命技术 - 到目前为止在很大程度上限制了非人类素数（NHP）中光遗传学实验的使用。光电的稀缺性 - NHP中的网络学研究不仅限于与PD相关的研究。光遗传学扩展的关键限制在私人中，缺乏在体内监测Opsin表达的非侵入性方法。验尸组织学重新电源是确认大脑区域正确靶向和opsin表达水平的唯一可靠方法 NHP。显然，此方法不允许体内校正实验方法。 NHP中光遗传学使用的扩展需要开发方法以监测临时性 Opsin表达的课程和水平，因此可以在体内检测和校正表达水平不足，因此，避免了昂贵的实验失败，并减少了研究所需的NHP数量。我们建议一种非侵入性方法，可以基于正电子成像在体内反复监测Opsin表达（PET）NHP中的成像。我们将使用兴奋性Opsin ChannelRhopopsin-2（Chr2）的创新版本，在其中，将Opsin融合到人雌激素受体α的配体结合结构域，从而产生新颖 Opsin“ Chrera”。放射标记的氟化雌二醇类似物，[18F] -FluorooreTradiol（[18f] -fes），强烈结合 Chrera和[18F] FES的大脑分布可以用PET可视化。我们将使用Chrera并重新 peated [18f] - fes pet扫描以纵向监测恒河猴在恒河猴中的表达。使用病毒向量，我们将在纹状体的运动部分中表达Chrera，然后进行几次[18F] -FES PET扫描在接下来的12个月中，以描述可检测到的Opsin表达的时间过程。同时，我们将检查Chrera表达的PET识别模式是否对应于功能性Opsins的存在，通过在光激活过程中进行细胞外记录。最后，我们将研究对应关系推文在[18F] -FES PET扫描中发现的OPSIN表达模式和用OPSIN分布确定验尸组织学。这些研究将建立一种基本技术来评估分布的分布 NHP中的Opsins。与此FOA PA-21-219的目的保持一致（“ ninds/nimh探索性神经科学研究赠款”），这些研究的长期目标是使用Chrera和[18f] -fes Pet 在我们对帕金森病病理生理学的NHP光遗传学研究中扫描。更广泛地，验证监测Opsin表达的无创的体内方法可能有助于加速可靠的光遗传学。神经系统疾病的NHP模型的研究，最终可能促进光学遗传学的翻译诊所的技术。