Two-Photon Imaging Reveals a Power Threshold of Neuronal Activation by Electrochemical Sensing Waveforms

Electrochemical sensing enables high-temporal-resolution measurements of neurochemical dynamics, yet the impact of sensing waveforms on local neural excitability has not been systematically examined. We combined in vivo two-photon Ca2+ imaging in Thy1-GCaMP6s mice with sensing waveform application through carbon fiber microelectrodes implanted in cortex to determine how sensing waveforms affect neuronal activation. Standard fast-scan cyclic voltammetry (FSCV) waveforms for dopamine and serotonin, as well as square-wave voltammetry and amperometry waveforms, produced Ca2+ activity indistinguishable from no-stimulation controls even over 30 min, confirming established parameters are physiologically silent. We then systematically explored parameters often proposed to increase sensitivity, enhance specificity, or detect new analytes. Increasing the anodic switching potential of the dopamine FSCV waveform produced strong neuronal activation beginning at ∼2.1 V with rapid saturation in activation area beyond ∼2.3 V. Anodic excursions produced stronger activation than cathodic excursions, and slower scan rates and higher scan frequencies increased activation. Unlike conventional microstimulation, the response to sensing waveforms was delayed by 52 ± 26 s and persisted after waveform cessation, consistent with the diffusion of electrochemical reaction byproducts rather than direct membrane depolarization. These results define a parameter space, corresponding to a power dose of <0.5 mW, where FSCV does not perturb neural activity. This establishes a practical threshold to guide waveform development and help avoid unintended neural activation during electrochemical sensing.