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

神经调质调节成瘾、注意力、认知、情绪、记忆、动机、睡眠等， 对大脑回路的影响用于测量大脑中神经调节的经典工具具有较差的空间和 时间分辨率这阻碍了神经调节功能的多样性和复杂性的发现 在行为中玩耍。在过去的几年里，成像神经调质动力学的新指标， 开始拆除这些障碍。然而，所有现有的神经调质指标都有明显的局限性。 该提案的目标是优化我们的基于GPCR激活（GRAB）的基因编码荧光标记。 四种主要神经调质的指标：多巴胺（DA）、乙酰胆碱（ACh）、去甲肾上腺素（NE）和 血清素（5-HT）。我们将使他们的反应更大，更具体，创造红色版本的多路复用 成像，并使最终用户更容易在体内成功部署。 在目标1中，我们将通过以下方法迭代筛选文库来优化DA、ACh、NE和5-HT的GRAB指标： 高容量共聚焦成像和流式细胞仪。我们将改变插入位点、接头、cpGFP、FP-GPCR蛋白表面 界面，和在一系列嵌合GCPR传感器主链上的热稳定GPCR残基。图书馆 将通过从GPCR结构计算预测功能来优先生成。的尺寸 优化将是亮度、dF/F0、配体选择性、亲和力和内源性信号的不中断。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：所有神经调质的配体选择性为1000倍（第三代），SNR改善> 5倍 超过第2代体外和体内指示剂（第3代），以及可靠的单次试验亚细胞分辨率 在所有神经调质的行为过程中，用体内2-光子成像对皮质进行分级反应（第4代）。 在目标2中，我们将使用与目标1相同的方法来开发和验证体内第1代和第2代红色 GRAB用于相同的神经调质，以实现多个信号的同时成像。我们的目标 第二代光谱正交红色GRAB的体外性能水平为10 x dF/F，>50% dF/F in vivo体内responses响应.我们还将设计出红色GRAB荧光的任何光活化，证明 具有来自成像光的零视蛋白激发串扰的多路复用成像和光遗传学刺激。 在目标3中，我们将优化GRAB的包装和分销，以最大限度地提高最终用户的易用性。我们将 量化与GRAB体内共表达的最佳FP，设计病毒遗传策略， 从全身注射AAV中广泛表达GRAB，并使Cre-reporter小鼠品系为最佳绿色 每种神经调质的GRAB。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优化的强大的分子工具， 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， 将对我们理解神经回路功能和功能障碍产生倍增影响。

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.