Cholinergic modulation of limbic cortico-striatal circuitry

执行项目总结：本项目旨在开发一种最先进的传感器来测量执行任务的大鼠前额叶皮层中乙酰胆碱（ACh）的释放。这项技术代表了脑内胆碱能传输测量的范式转变，迄今为止，胆碱能传输的测量依赖于微透析等粗采样技术，以及固有不稳定且寿命短的酶生物传感器。在第一年，学生将与勃林格殷格翰（BI）的神经科学家和工程师合作，优化基于gpcr激活的乙酰胆碱传感器，用于体外超灵敏的乙酰胆碱检测。在接下来的几年里，该项目将以剑桥为基地，记录体内乙酰氨基酚释放的亚秒波动。该项目独特地依赖于BI和剑桥大学的技术专长和资源。因此，传感器的开发和验证，包括提供工具化合物和药理学专业知识，都需要BI的专门投入。剑桥大学在将神经化学取样技术与行为结合方面有着良好的记录。科学背景：内侧前额叶皮层（mPFC）中的乙酰胆碱（ACh）与注意力、学习和记忆等认知功能密切相关。过去，体内乙酰胆碱动力学通常是通过电生理学和药理学配对或体内微透析来测量的。然而，这些技术缺乏足够的特异性和/或时间分辨率来捕获与行为相关的单次试验乙酰胆碱事件。最近，BI合作者李玉龙发表了一种体内适用的基于gpcr激活的ACh传感器，名为GRABACh3.0 （Jing et.al, PMID: 32989318）。简而言之，为了制造这种新型的GRAB传感器，李玉龙团队将3型毒蕈碱ACh受体（M3R）与环状排列的GFP结合在一起。由此产生的基因编码传感器将ach诱导的M3R构象变化转化为荧光，用光学记录方法检测。勃林格殷格翰（BI）很早就获得了这种新型传感器的特权。在这里，我们建议将GRABACh3.0引入任务执行大鼠的mPFC，使用纤维光度法记录胆碱能信号。目标1 (BI年1)：优化基于gpcr激活的乙酰胆碱传感器，用于体外超灵敏的乙酰胆碱检测。这一阶段的研究将验证GRABACh3.0在已知作用的胆碱能药物存在的情况下对施加或诱发的乙酰胆碱的敏感性，同时测量传感器的GFP响应。这些实验将建立GRABACh3.0对乙酰氨基酚的敏感性，并证明其在引入大鼠大脑后的功能。目标2（剑桥2-4年级）：记录体内乙酰胆碱释放亚秒波动。实验将集中在持续视觉注意力任务训练大鼠乙酰胆碱传感器的整合上。最初的实验旨在通过干预基底前脑的胆碱能体（例如，使用选择性免疫毒素192 igg皂苷）以及mPFC中的胆碱能末端（例如河豚毒素）来验证体内传感器信号。然后将在执行任务的大鼠身上测试传感器，以测量乙酰胆碱释放的亚秒波动，并使用计算模型将行为数据与阶段性（快速）释放事件相结合。最后一组实验将研究认知增强药物对胆碱能传递和视觉注意力表现的影响。

Executive project summary: This project seeks to develop a state-of-the-art sensor to measure acetylcholine (ACh) release in the prefrontal cortex of task-performing rats. This technology represents a paradigm shift in the measurement of cholinergic transmission in the brain, which hitherto has relied on coarse sampling techniques such as microdialysis, and enzymatic biosensors that are inherently unstable and short-lived. During year 1, the student will work with neuroscientists and engineers at Boehringer Ingelheim (BI) to optimise GPCR-activation-based ACh sensors for ultra-sensitive ACh detection in-vitro. In subsequent years, the project will be based in Cambridge to record sub-second fluctuations in ACh release in-vivo. The project is uniquely contingent on the technical expertise and resources available at BI and Cambridge. Thus, sensor development and validation, including the provision of tool compounds and pharmacological expertise requires dedicated input from BI. Cambridge has a proven track record in coupling neurochemical sampling techniques with behaviour.Scientific background: Acetylcholine (ACh) in the medial prefrontal cortex (mPFC) is strongly implicated in cognitive functions such as attention, learning and memory. In the past, in vivo ACh dynamics were typically measured with paired electrophysiology and pharmacology or by in-vivo microdialysis. However, these techniques lack sufficient specificity and/or temporal resolution to capture behaviourally relevant single trial ACh events. Recently, the BI collaborator Yulong Li published an in vivo suitable GPCR-activation-based ACh sensor called GRABACh3.0 (Jing et.al, PMID: 32989318). Briefly, to create this novel GRAB sensor Yulong Li's group combined the type 3 muscarinic ACh receptor (M3R) with circularly permutated GFP. The resulting genetically encoded sensor converts ACh-induced M3R conformational changes into fluorescence detectable with optical recording methods. Boehringer Ingelheim (BI) has early privileged access to this novel sensor. Here, we propose to bring GRABACh3.0 into the mPFC of task-performing rats where cholinergic signals will be recorded using fibre photometry. Aim 1 (BI year 1): Optimise GPCR-activation-based ACh sensors for ultra-sensitive ACh detection in-vitro. This phase of the study will validate GRABACh3.0 sensitivity to applied or evoked ACh in the presence of cholinergic agents with known effects while the sensor GFP response is measured. These experiments will establish GRABACh3.0 sensitivity to ACh and show it is functional when introduced into the rat brain.Aim 2 (Cambridge years 2-4): Record sub-second fluctuations in ACh release in-vivo. Experiments will focus on the integration of the ACh sensors in rats trained on a sustained visual attentional task. Initial experiments will aim to validate in-vivo sensor signals with interventions targeting cholinergic soma in the basal forebrain (e.g., using the selective immunotoxin 192 IgG-saporin) as well as cholinergic terminals in the mPFC (e.g., tetrodotoxin). Sensors will then be tested in task-performing rats to measure sub-second fluctuations in ACh release, with computational modelling used to align behavioural data with phasic (fast) release events. A final set of experiments will investigate the effects of cognitive enhancing drugs on cholinergic transmission and visual attentional performance.