Cholinergic modulation of limbic cortico-striatal circuitry

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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.