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

神经调节剂通过以下方式调节成瘾、注意力、认知、情绪、记忆、动机、睡眠等 它们对大脑回路的影响。传统的测量大脑神经调节的工具在空间和空间上都很差 时间分辨率。这阻碍了对神经调节的多样化和复杂功能的发现 在行为过程中播放。在过去的几年里，神经调节器动力学成像的新指标已经 开始拆除这些障碍。然而，现有的所有神经调节剂指标都有明显的局限性。 这项提议的目标是优化我们的基于GPCR激活(GRAB)的基因编码的荧光 四种主要神经调节剂的指示物：多巴胺(DA)、乙酰胆碱(ACh)、去甲肾上腺素(NE)和 5-羟色胺(5-HT)。我们将使他们的回应更大、更具体，为多路传输创建红色版本 成像，并使最终用户更容易在体内成功部署。 在目标1中，我们将通过迭代筛选文库来优化DA、ACh、NE和5-羟色胺的GRAB指标 高含量共聚焦成像和流式细胞仪。我们将改变插入位点、连接子、cpGFP、FP-GPCR蛋白表面 界面，并在一系列嵌合的GCPR传感器骨架上热稳定GPCR残基。图书馆 生成将通过对GPCR结构的功能进行计算预测来确定优先级。它的尺寸 优化将是亮度、df/f0、配基选择性、亲和力和内源信号的非中断。顶部 HITS将在哺乳动物脑片和表现良好的小鼠中长期表达后得到验证。我们的目标是 性能水平是：所有神经调节剂(第三代)的配体选择性提高1000倍，信噪比提高5倍 超过第二代指标的体外和体内(第三代)，和可靠的单次试验亚细胞分辨率 所有神经调节剂(第4代)行为期间的皮质双光子成像分级反应。 在目标2中，我们将使用与目标1相同的方法在体内开发和验证第一代和第二代RED 抓取相同的神经调节剂，以实现多个信号的同时成像。我们的目标是 第二代，光谱正交红色抓取的性能水平是体外10倍dF/F，&gt；50%df/F 活体反应。我们还将设计出任何光激活的红色抓取荧光，演示 来自成像光的零光激发串扰的多路成像和光发生刺激。 在目标3中，我们将优化Grab的包装和分发，以最大限度地提高最终用户的易用性。我们会 量化与GRABS在体内共表达的最佳FP，设计病毒遗传策略，以实现强大的大脑- 从全身注射AAV获得广泛的表达，并使Cre-Report小鼠品系获得最佳绿色 抓取每个神经调节剂。优化的质粒、AAVs和小鼠将得到广泛传播。 成功完成我们的目标将产生一套优化的功能强大的分子工具套件， 最大的实用性和易用性。由于这些探头非常适合大量的调查人员，他们 将对我们对神经回路功能和功能障碍的理解产生倍增的影响。

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.