PT - JOURNAL ARTICLE AU - Morgan N. Collins AU - Karen A. Mesce TI - Focused ultrasound neuromodulation and the confounds of intracellular electrophysiological investigation AID - 10.1523/ENEURO.0213-20.2020 DP - 2020 Jul 31 TA - eneuro PG - ENEURO.0213-20.2020 4099 - http://www.eneuro.org/content/early/2020/07/31/ENEURO.0213-20.2020.short 4100 - http://www.eneuro.org/content/early/2020/07/31/ENEURO.0213-20.2020.full AB - Focused ultrasound can modulate neuronal activity noninvasively with high spatial specificity. In intact nervous systems, however, efforts to determine its enigmatic mode of efficacy have been confounded by the indirect effects of ultrasound on mechanosensitive sensory cells and the inability to target equivalent populations of cells with precision across preparations. Single-cell approaches, either via cultured mammalian neurons or tractable invertebrate neural systems, hold great promise for elucidating the cellular mechanisms underlying the actions of ultrasound. Here, we present evidence from the medicinal leech, Hirudo verbana, that researchers should apply caution when utilizing ultrasound in conjunction with single-cell electrophysiological recording techniques, including sharp-electrode intracellular recording. Although we found that ultrasound could elicit depolarization of the resting membrane potential of single neurons, a finding with precedent, we determined that this effect and others could be reliably mimicked via subtle manual displacement of the recording electrode. Because focused ultrasound is known to induce resonance of recording electrodes, we aimed to determine how similarly ultrasound-induced depolarizations matched those produced by micro movements of a sharp glass electrode, a phenomenon we believe can account for purported depolarizations measured in this manner. Furthermore, we show that when clonally related homologous neurons, which are essentially isopotential, are impaled prior to the application of focused ultrasound, they show a statistically significant change in their membrane potential as compared to the homologous cells that received ultrasound with no initial impalement. Future investigations into ultrasound’s cellular effects should attempt to control for potential electrode resonance or utilize alternative recording strategies.SIGNIFICANCE STATEMENT Interest in focused ultrasound (US) neuromodulation has soared in recent years, yet researchers have yet to agree on whether ultrasound excites or inhibits neuronal activity, or what mechanisms underly these effects. Basic investigations have attempted to clarify how US affects neuronal membrane properties to understand how it alters firing rates. Several groups have linked ultrasound-induced excitation to depolarization of the resting membrane potential, as measured with intracellular sharp electrodes or membrane patch methods. Here, we replicate this depolarization while recording with intracellular sharp electrodes, but find that the depolarizing effects of US can be replicated by small displacements of the recording electrode. We conclude that intracellular electrophysiological investigations of ultrasound’s neuromodulatory effects are susceptible to artifacts introduced via electrode resonance.