Cholinergic modulation of limbic cortico-striatal circuitry

边缘皮质纹状体回路的胆碱能调节

• 批准号: 2877048
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2877048
• 关键词: Cholinergic; modulation; limbic; cortico; striatal

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.

项目执行摘要：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。这项技术代表了测量大脑中胆碱能传输的范式转变，迄今为止，大脑中胆碱能传输一直依赖于粗糙的采样技术，如微透析和酶生物传感器，这些技术本身就不稳定，寿命短。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ACh传感器，用于体外超灵敏ACh检测。在随后的几年里，该项目将设在剑桥，以记录体内乙酰胆碱释放的亚秒级波动。该项目是唯一的条件下的技术专长和资源，可在BI和剑桥。因此，传感器开发和验证，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。剑桥在将神经化学采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。在过去，体内乙酰胆碱动力学通常是测量配对的电生理学和药理学或体内微透析。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次试验ACh事件。最近，BI合作者Yulong Li发表了一种体内合适的基于GPCR激活的ACh传感器，称为GRABACh3.0（Jing et.al，PMID：32989318）。简而言之，为了创建这种新型GRAB传感器，Yulong Li的研究小组将3型毒蕈碱ACh受体（M3 R）与环状排列的GFP相结合。由此产生的基因编码的传感器将乙酰胆碱诱导的M3 R构象变化转化为荧光检测与光学记录方法。Boehringer Ingelheim（BI）已提前获得该新型传感器的使用权。在这里，我们建议将GRABACh3.0引入执行任务的大鼠的mPFC中，其中将使用纤维光度法记录胆碱能信号。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。该研究的这一阶段将验证GRABACh3.0在具有已知效应的胆碱能药物存在下对施加或诱发的ACh的敏感性，同时测量传感器GFP响应。这些实验将建立GRABACh3.0对ACh的敏感性，并显示当引入大鼠脑中时它是功能性的。目标2（剑桥2-4年）：记录体内ACh释放的亚秒级波动。实验将集中在ACh传感器在大鼠训练的持续视觉注意力任务的整合。最初的实验将旨在验证具有靶向基底前脑中的胆碱能索马的干预的体内传感器信号（例如，使用选择性免疫毒素192 IgG-皂草素）以及mPFC中的胆碱能末端（例如，河豚毒素）。然后将在执行任务的大鼠中测试传感器，以测量ACh释放的亚秒级波动，并使用计算模型将行为数据与阶段性（快速）释放事件相一致。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。