Cholinergic modulation of limbic cortico-striatal circuitry

执行项目摘要：该项目寻求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质中乙酰胆碱(ACh)的释放。这项技术代表着测量大脑胆碱能传递的范式转变，到目前为止，这种测量依赖于微透析和酶生物传感器等粗略采样技术，这些技术固有的不稳定和短暂。在第一年，该学生将与伯林格-英格尔海姆大学(BI)的神经科学家和工程师合作，优化基于GPCR激活的ACH传感器，用于体外超灵敏ACh检测。在接下来的几年里，该项目将设在剑桥，记录体内ACh释放的亚秒级波动。该项目取决于BI和剑桥大学的技术专长和可用资源。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，需要BI的专门投入。剑桥大学在将神经化学采样技术与行为相结合方面有着成熟的记录。科学背景：内侧前额叶皮质(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。在过去，体内ACh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行为相关的单项试验ACH事件。最近，BI合作者李玉龙发表了一种适合体内的基于gpr激活的ACh传感器，称为GRABACH3.0(Jing et.al，PMID：32989318)。简而言之，为了创造这种新型的GRAB传感器，李玉龙的团队将3型毒鼠强ACh受体(M3R)与循环排列的GFP结合起来。由此产生的基因编码传感器将ACh诱导的M3R构象变化转换为可用光学记录方法检测的荧光。勃林格-英格尔海姆(BI)拥有使用这种新型传感器的早期特权。在这里，我们建议将GRABACh3.0引入任务执行大鼠的mPFC，其中胆碱能信号将使用纤维光度法记录。目标1(BI第1年)：优化基于GPCR激活的ACh传感器，用于体外超灵敏ACh检测。这一阶段的研究将在测量传感器GFP响应的同时，验证GRABACh3.0对应用或诱发ACh的敏感性。这些实验将建立GRABACh3.0对ACh的敏感性，并显示当被引入大鼠大脑时它是有功能的。Aim 2(剑桥大学2-4年)：记录体内ACh释放的亚秒波动。实验将重点放在训练持续视觉注意任务的大鼠身上ACh传感器的整合。最初的实验旨在通过针对基底前脑胆碱能胞体的干预措施(例如，使用选择性免疫毒素192-Ig-Saporin)以及mPFC中的胆碱能终末(例如，河豚毒素)来验证体内传感器信号。然后，传感器将在执行任务的大鼠身上进行测试，以测量ACh释放的亚秒级波动，并使用计算模型来使行为数据与阶段(快速)释放事件保持一致。最后一组实验将调查认知增强药物对胆碱能传递和视觉注意表现的影响。

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.