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

Fast time-scale modulation of synaptic and cellular physiology by acetylcholine is critical for many cognitive functions, but direct local measurement of neuromodulator dynamics in freely-moving behaving animals is technically challenging. Recent in vivo brain measurements using choline oxidase (ChOx)-based electrochemical biosensors have reported surprising fast cholinergic transients associated with reward-related behavioral events. However, in vivo recordings with conventional ChOx biosensors could be biased by phasic local field potential and O ₂ -evoked enzymatic responses. Here, we have developed a Tetrode-based Amperometric ChOx (TACO) sensor enabling minimally invasive artifact-free simultaneous measurement of cholinergic activity and O ₂ . Strikingly, the TACO sensor revealed highly-correlated O ₂ and ChOx transients following spontaneous locomotion and sharp-wave/ripples fluctuations in the hippocampus of behaving rodents. Quantitative analysis of spontaneous activity, in vivo and in vitro exogenous O ₂ perturbations revealed a directional effect of O ₂ on ChOx phasic signals. Mathematical modeling of biosensors identified O ₂ -evoked non-steadystate ChOx kinetics as a mechanism underlying artifactual biosensor phasic transients. This phasic O ₂ -dependence of ChOx-based biosensor measurements confounds phasic cholinergic dynamics readout in vivo, challenging previously proposed ACh role in reward-related learning. The discovered mechanism and quantitative modeling is generalizable to any oxidase-based biosensor, entailing rigorous controls and new biosensor designs.