title: Simultaneous measurement of cholinergic tone and neuronal network dynamics in vivo in the rat brain using a novel choline oxidase based electrochemical biosensor.
abstract: Acetylcholine (ACh) modulates neuronal network activities implicated in cognition, including theta and gamma oscillations but the mechanisms remain poorly understood. Joint measurements of cholinergic activity and neuronal network dynamics with high spatio-temporal resolution are critical to understand ACh neuromodulation. However, current electrochemical biosensors are not optimized to measure nanomolar cholinergic signals across small regions like hippocampal sub-layers. Here, we report a novel oxidase-based electrochemical biosensor that matches these constraints. The approach is based on measurement of H2O2 generated by choline oxidase (ChOx) in the presence of choline (Ch). The microelectrode design consists of a twisted pair of 50µm diameter Pt/Ir wires (sensor and sentinel), which is scalable, provides high spatial resolution and optimizes common mode rejection. Microelectrode coating with ChOx in chitosan cross-linked with benzoquinone is simple, mechanically robust and provides high sensitivity (324±46nAµM(-1)cm(-2)), a limit of detection of 16nM and a t50 response time of 1.4s. Local field potential (LFP)-related currents dominate high-frequency component of electrochemical recordings in vivo. We significantly improved signal-to-noise-ratio compared to traditional sentinel subtraction by a novel frequency domain common mode rejection procedure that accounts for differential phase and amplitude of LFP-related currents on the two channels. We demonstrate measurements of spontaneous nanomolar Ch fluctuations, on top of which micromolar Ch increases occurred during periods of theta activity in anesthetized rats. Measurements were not affected by physiological O2 changes, in agreement with the low biosensor Km for O2 (2.6µM). Design and performance of the novel biosensor opens the way for multisite recordings of spontaneous cholinergic dynamics in behaving animals.
authors.affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal; Bernstein Center for Computational Neuroscience and Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians Universität München, Grosshaderner Strasse 2, 82152 Planegg-Martinsried, Germany.
authors.forename: Ricardo M
authors.initials: RM
authors.lastname: Santos
authors.affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal; Faculty of Pharmacy, University of Coimbra, Health Campus, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal.
authors.forename: João
authors.initials: J
authors.lastname: Laranjinha
authors.affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal; Faculty of Pharmacy, University of Coimbra, Health Campus, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal. Electronic address: rbarbosa@ff.uc.pt.
authors.forename: Rui M
authors.initials: RM
authors.lastname: Barbosa
authors.affiliation: Bernstein Center for Computational Neuroscience and Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians Universität München, Grosshaderner Strasse 2, 82152 Planegg-Martinsried, Germany. Electronic address: sirota@biologie.uni-muenchen.de.
authors.forename: Anton
authors.initials: A
authors.lastname: Sirota
mesh_terms: D000109:Acetylcholine / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000493:pharmacokinetics*; D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation*; D001921:Brain / Q000378:metabolism*; D001931:Brain Mapping / Q000295:instrumentation; D059329:Cholinergic Neurons / Q000502:physiology; D003217:Conductometry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D008839:Microelectrodes; D008904:Miniaturization; D009415:Nerve Net / Q000502:physiology*; D018377:Neurotransmitter Agents / Q000378:metabolism; D051381:Rats; D020318:Rats, Long-Evans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D009435:Synaptic Transmission / Q000502:physiology
chemical_list: D018377:Neurotransmitter Agents; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D000109:Acetylcholine
keywords: Acetylcholine; Hippocampus; In vivo electrochemistry; LFP oscillations; Microbiosensor; Neuromodulation