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Cholinergic fast time-scale modulation of cortical physiology is critical for cognition, but direct local measurement of neuromodulators in vivo is challenging. Choline oxidase (ChOx)-based electrochemical biosensors have been used to capture fast cholinergic signals in behaving animals. However, these transients might be biased by local field potential and O 2 -evoked enzymatic responses. Using a novel Tetrode-based Amperometric ChOx (TACO) sensor, we performed highly sensitive and selective simultaneous measurement of ChOx activity (COA) and O 2 . In vitro and in vivo experiments, supported by mathematical modeling, revealed that non-steady-state enzyme responses to O 2  give rise to phasic COA dynamics. This mechanism accounts for most of COA transients in the hippocampus, including those following locomotion bouts and sharp-wave/ripples. Our results suggest that it is unfeasible to probe phasic cholinergic signals under most behavioral paradigms with current ChOx biosensors. This confound is generalizable to any oxidase-based biosensor, entailing rigorous controls and new biosensor designs.

Phasic oxygen dynamics confounds fast choline-sensitive biosensor signals in the brain of behaving rodents