Novel fluorescent sensors for imaging neuromodulation

总结 动物的行为是由复杂的神经系统协调的， 由包括脂质和神经肽的神经调质动态调节。Endocannabinoids （eCB）是广泛存在于大脑中的神经脂质，并调节学习和记忆，成瘾， 疼痛感和食物摄入在神经肽中，胆囊收缩素（CCK）参与 营养感测、食物摄入和睡眠调节，以及催产素（OXT）和加压素（AVP） 在社会行为的各个方面发挥着重要作用。然而，如何以及何时脂质和 神经肽传递在大脑中发生的情况在很大程度上还不清楚。现有方法（例如： 微透析）测量大脑化学含量的方法， 分辨率此外，由于神经脂质和神经肽的释放通常需要重复的 神经元放电，可以发生在轴突和树突部位，神经调质的活性， 释放神经元不能可靠地预测神经脂质和神经肽在何处以及何时被释放。 发布 在这里，我们建议开发一套长期监测神经脂质的新工具 和神经肽。我们的策略利用了它们的天然受体，人类G蛋白偶联 受体（GPCR），其与GFP偶联。在神经脂质或神经肽的存在下， 这些基于GPCR激活（GRAB）的传感器将配体结合诱导的 构象变化转化为快速荧光信号。我们的目标是开发和优化 神经脂质和神经肽GRAB传感器，具有>500%的荧光变化（dF/F）和10- 纳摩尔亲和力，并在离体脑切片和小鼠中验证这些新工具 活体行为模式。在目标1中，我们将开发用于内源性大麻素的GRAB传感器， CCK、加压素和OXT通过系统地改变参与配体结合的关键位点， 在目标2中，我们将验证这些传感器在大脑中的性能 使用病毒工具在长期表达后切片。在目标3中，我们将使用三种不同的 成像方法（纤维光度法、落射荧光法和双光子成像结合GRIN 透镜）以测试新型GRAB传感器在不同行为范例中的体内性能。 小鼠目标2-3中的实验反馈将指导目标1中的迭代优化。 成功完成我们的建议将产生一套强大的工具和技术 方法，这将极大地促进神经脂质和神经肽的研究下， 生理和病理条件，帮助揭示疾病机制，提供 治疗指导，最终造福人类健康。

SUMMARY Animal behaviors are orchestrated by the sophisticated nervous system, which is dynamically regulated by neuromodulators including lipids and neuropeptides. Endocannabinoids (eCBs) are neurolipids exist broadly in the brain and regulate learning and memory, addiction, pain sensation, and food intake. Among neuropeptides, cholecystokinin (CCK) is involved in nutrient sensing, food intake, and sleep regulation, and oxytocin (OXT) and vasopressin (AVP) play important roles in various aspects of social behaviors. However, how and when lipid and neuropeptide transmission occur in the brain are largely unclear. Existing methods (e.g. microdialysis) that measures brain chemical content suffer from low temporal and spatial resolution. Additionally, since neurolipid and neuropeptide releases often require repetitive neuronal firing and can occur at both axonal and dendritic sites, activity of the neuromodulator- releasing neurons cannot reliably predict where and when neurolipids and neuropeptides are released. Here we propose to develop a set of new tools for long-term monitoring of neurolipids and neuropeptides. Our strategy taps into their natural receptors, human G protein-coupled receptors (GPCRs), which are coupled to GFP. In the presence of neurolipids or neuropeptides, these GPCR Activation-Based (GRAB) sensors transform ligand binding-induced conformational changes into rapid fluorescent signals. We aim to develop and optimize neurolipid and neuropeptide GRAB sensors with >500% fluorescence change (dF/F) and 10- nanomolar affinity in vitro and validate these novel tools in brain slices ex vivo and mouse behavioral paradigms in vivo. In Aim 1, we will develop GRAB sensors for endocannabinoids, CCK, vasopressin, and OXT by systematically varying key sites involved in ligand binding, conformational change, etc. In Aim 2, we will validate the performance of these sensors in brain slice following long-term expression using viral tools. In Aim 3, we will use three different imaging methods (fiber photometry, epifluorescence and 2-photon imaging coupled with GRIN lens) in different behavioral paradigms to test in vivo performance of the novel GRAB sensors in mice. Feedback from experiments in Aims 2-3 will guide iterative optimization in Aim 1. Successful completion of our proposal will yield a suite of powerful tools and technical approaches, which will greatly facilitate studies of neurolipids and neuropeptides under both physiological and pathological conditions, helping reveal disease mechanisms, providing therapeutic guidance, and eventually benefiting human health.