Focused Ultrasound Neuromodulation and the Confounds of Intracellular Electrophysiological Investigation

Abstract

Focused ultrasound (US) 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 US 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 US. Here, we present evidence from the medicinal leech, Hirudo verbana, that researchers should apply caution when using US in conjunction with single-cell electrophysiological recording techniques, including sharp-electrode intracellular recording. Although we found that US 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 US is known to induce resonance of recording electrodes, we aimed to determine how similarly US-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 before the application of focused US, they show a statistically significant change in their membrane potential as compared with the homologous cells that received US with no initial impalement. Future investigations into US's cellular effects should attempt to control for potential electrode resonance or use alternative recording strategies.

Sonogenetic control of mammalian cells using exogenous Transient Receptor Potential A1 channels

“…However, these responses are significantly smaller than those observed in hsTRPA1-expressing neurons, and ultrasoundevoked action potentials were rarely detected at the frequency and pressures tested. Additionally, the infrequent responses in control neurons in vitro could be a result of membrane deflection as we recently showed 64 , or a result of interactions with the substrate, or with the patch pipette in electrophysiology 65 . Control neurons have been previously shown to generate action potentials upon ultrasound stimuli at lower fundamental frequencies, however, these responses required repeated pulses 4 .…”

Sonogenetic control of mammalian cells using exogenous Transient Receptor Potential A1 channels

“…However, these responses are significantly smaller than those observed in hsTRPA1-expressing neurons, and ultrasoundevoked action potentials were rarely detected at the frequency and pressures tested. Additionally, responses in control neurons in vitro could be likely an artefact of the 2-dimensional cultures, interactions with the substrate, or interactions with the patch pipette in electrophysiology 65 .…”

The Inhibitory Thermal Effects of Focused Ultrasound on an Identified, Single Motoneuron

“…Transducer output was characterized by hydrophone (ONDA HNR-0500), as described previously ( Collins and Mesce, 2020 ). Vertical and horizontal cross-sections of linearly interpolated hydrophone measurements at peak amplitudes overlaid with scaled preparation dimensions are shown in Figure 1 E .…”

“…We investigated how US inhibited neural activity thermally at relatively low temperatures (< 5 °C). As a proxy for US-associated heat, we utilized the laser, as it was the most compatible with our intracellular recording electrode, and did not generate electrode resonance as does US, which can obscure the fidelity of intracellular recording data (Collins and Mesce, 2020). During heat application, we observed a continuation of spikes recorded in the soma with a loss of spikes distal to the stimulus (Fig.…”