Optimization of GPCR-based fluorescent sensors for large-scale multiplexed in vivo imaging of neuromodulation

基于 GPCR 的荧光传感器的优化，用于神经调节的大规模多重体内成像

• 批准号: 10700803
• 负责人: Samuel Andrew Hires
• 金额: $ 90.25万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10700803
• 关键词: Acetylcholine; Affinity; Attention; Behavior; Brain; Brain region; Calcium; Chronic; Cognition; Collaborations; Color; Complex; Cre driver; Deposition; Dimensions; Dopamine; Engineering; Fiber; Fluorescence; Functional disorder; G-Protein-Coupled Receptors; Generations; Genetic; Goals; Image; In Vitro; Injections; Lead; Libraries; Ligands; Light; Measures; Membrane Proteins; Memory; Mission; Molecular; Moods; Motivation; Mus; Neuromodulator; Norepinephrine; Opsin; Performance; Photometry; Photons; Plasmids; Play; Public Health; Reporter; Research; Research Personnel; Resolution; Role; Serotonin; Signal Transduction; Site; Sleep; Slice; Structure; Tissues; United States National Institutes of Health; Validation; Vertebral column; addiction; confocal imaging; cost effective; experimental study; genetic approach; improved; in vivo; in vivo imaging; invention; multiplexed imaging; mutant; nervous system disorder; neural circuit; neuroregulation; non-invasive imaging; optogenetics; photoactivation; prevent; red fluorescent protein; response; screening; sensor; somatosensory; temporal measurement; tool; two-photon; uptake; virus genetics; voltage

Neuromodulators regulate addiction, attention, cognition, mood, memory, motivation, sleep, and more through their influence on brain circuits. Classic tools for measuring neuromodulation in the brain have poor spatial and temporal resolution. This has hampered the discovery of the diverse and complex functions neuromodulation plays during behavior. Over the past few years, new indicators for imaging neuromodulator dynamics have begun to dismantle these barriers. However, all existing neuromodulator indicators have significant limitations. The goal of this proposal is to optimize our GPCR-activation-based (GRAB) genetically-encoded fluorescent indicators of four major neuromodulators: dopamine (DA), acetylcholine (ACh), norepinephrine (NE), and serotonin (5-HT). We will make their responses bigger and more specific, create red versions for multiplexed imaging, and make them easier for end-users to successfully deploy in vivo. In Aim 1, we will optimize GRAB indicators for DA, ACh, NE, and 5-HT by iteratively screening libraries via high-content confocal imaging and FACS. We will vary insertion site, linkers, cpGFP, FP-GPCR protein surface interface, and thermostabilizing GPCR residues on a range of chimeric GCPR sensor backbones. Library generation will be prioritized by computational prediction of function from GPCR structures. The dimensions of optimization will be brightness, dF/F0, ligand selectivity, affinity, and non-disruption of endogenous signals. Top hits will be validated following long-term expression in mammalian brain slice and behaving mice. Our targeted performance levels are: 1000x ligand selectivity across all neuromodulators (3rd gen), >5x SNR improvement over 2nd generation indicators in vitro and in vivo (3rd gen), and reliable single-trial subcellular resolution of graded responses with in vivo 2-photon imaging of cortex during behavior for all neuromodulators (4th gen). In Aim 2, we will use the same approach as Aim 1 to develop and validate in vivo 1st and 2nd generation red GRABs for the same neuromodulators to enable simultaneous imaging of multiple signals. Our targeted performance levels for second generation, spectrally orthogonal red GRABs are 10x dF/F in vitro, >50% dF/F in vivo responses. We will also engineer out any photoactivation of red GRAB fluorescence, demonstrate multiplexed imaging and optogenetic stimulation with zero opsin excitation crosstalk from imaging light. In Aim 3, we will optimize GRAB packaging and distribution for maximum end-user ease of use. We will quantify the best FPs for in vivo coexpression with GRABs, engineer viral-genetic strategies for robust, brain- wide GRAB expression from systemic AAV injection, and make cre-reporter mouse lines for the best green GRAB of each neuromodulator. Optimized plasmids, AAVs, and mice will be broadly disseminated. Successful completion of our Aims will yield an optimized suite of powerful molecular tools packaged for maximum utility and ease of use. Since these probes are well-suited for a large number of investigators, they will have a multiplicative impact on our understanding of neural circuit function and dysfunction.

神经调节器调节成瘾，注意力，认知，情绪，记忆，动机，睡眠等 它们对脑电路的影响。测量大脑神经调节的经典工具的空间不良， 时间分辨率。这阻碍了发现多样化和复杂功能神经调节的发现 在行为期间发挥作用。在过去的几年中，成像神经调节剂动力学的新指标具有 开始拆除这些障碍。但是，所有现有的神经调节剂指标都有重大局限性。 该建议的目的是优化我们的基于GPCR激活（GRAB）遗传编码的荧光 四个主要神经调节剂的指标：多巴胺（DA），乙酰胆碱（ACH），去甲肾上腺素（NE）和 5-羟色胺（5-HT）。我们将使他们的响应更大，更具体，为多路复用创建红色版本 成像，并使最终用户更容易在体内成功部署。 在AIM 1中，我们将通过迭代筛选库来优化DA，ACH，NE和5-HT的抓取指标 高含有共聚焦成像和FACS。我们将改变插入位点，接头，CPGFP，FP-GPCR蛋白表面 界面，以及一系列嵌合GCPR传感器骨架上的GPCR残基。图书馆 GPCR结构的功能预测将优先考虑生成。尺寸 优化将是亮度，DF/F0，配体选择性，亲和力以及内源信号的不中断。顶部 在哺乳动物脑切片和行为小鼠中长期表达后，将验证命中。我们的目标 性能水平为：所有神经调节剂（第三代）的1000倍配体选择性，> 5x SNR改进 超过第二代指标体外和体内（第三代），以及可靠的单审判亚细胞分辨率 在所有神经调节剂（第四代）行为过程中，在体内2光子成像的分级反应。 在AIM 2中，我们将使用与AIM 1相同的方法来开发和验证体内第一代和第二代红色 抓取相同的神经调节剂，以同时对多个信号进行成像。我们的目标 第二代的性能水平，频谱正交红抓取为10x df/f体外，> 50％df/f 体内反应。我们还将设计出任何红grab荧光的光活化，证明 从成像光中使用零OPSIN激发串扰的多路复用成像和光遗传学刺激。 在AIM 3中，我们将优化抓取包装和分发，以最大程度地使用最终用户。我们将 量化体内共表达的最佳fps，用抓取，工程师的病毒遗传策略来稳健，大脑 从系统性AAV注入中宽抓取表达，并制作Cre-Reporter鼠标线，以达到最佳绿色 抓住每个神经调节剂。优化的质粒，AAV和小鼠将被广泛传播。 成功完成我们的目标将产生一套针对包装的强大分子工具的优化套件 最大效用和易用性。由于这些探针非常适合大量研究人员 将对我们对神经回路功能和功能障碍的理解产生乘法影响。

项目成果

Responses and functions of dopamine in nucleus accumbens core during social behaviors.

伏隔核多巴胺在社会行为过程中的反应和功能

• DOI: 10.1016/j.celrep.2022.111246
• 发表时间: 2022-08-23
• 期刊: CELL REPORTS
• 影响因子: 8.8
• 作者: Dai, Bing;Sun, Fangmia;Tong, Xiaoyu;Ding, Yizhuo;Kuang, Amy;Osakada, Takuya;Li, Yulong;Lin, Dayu
• 通讯作者: Lin, Dayu

Spatiotemporal dynamics of noradrenaline during learned behaviour.

学习行为过程中去甲肾上腺素的时空动态

• DOI: 10.1038/s41586-022-04782-2
• 发表时间: 2022-06
• 期刊: Nature
• 影响因子: 64.8

A repurposed vaccine grants durable anterograde access to neural circuits.

一种经过改造的疫苗可以持久地顺行进入神经回路。

• DOI: 10.1038/s41592-021-01345-7
• 发表时间: 2021
• 期刊: Nature methods
• 影响因子: 48
• 作者: Treweek,JenniferB

Genetically encoded sensors for measuring histamine release both in vitro and in vivo.

用于测量体外和体内组胺释放的基因编码传感器。

• DOI: 10.1016/j.neuron.2023.02.024
• 发表时间: 2023
• 期刊: Neuron
• 影响因子: 16.2
• 作者: Dong,Hui;Li,Mengyao;Yan,Yuqi;Qian,Tongrui;Lin,Yunzhi;Ma,Xiaoyuan;Vischer,HenryF;Liu,Can;Li,Guochuan;Wang,Huan;Leurs,Rob;Li,Yulong
• 通讯作者: Li,Yulong

Memory-enhancing properties of sleep depend on the oscillatory amplitude of norepinephrine.

睡眠增强记忆的特性取决于去甲肾上腺素的振荡幅度

• DOI: 10.1038/s41593-022-01102-9
• 发表时间: 2022-08
• 期刊: NATURE NEUROSCIENCE
• 影响因子: 25
• 作者: Kjaerby, Celia;Andersen, Mie;Hauglund, Natalie;Untiet, Verena;Dall, Camilla;Sigurdsson, Bjorn;Ding, Fengfei;Feng, Jiesi;Li, Yulong;Weikop, Pia;Hirase, Hajime;Nedergaard, Maiken
• 通讯作者: Nedergaard, Maiken