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.

Abstract via europepmc.

Flags from extraction

• exposures[0].timing.duty_cycle_pct — a ~30% duty cycle is stated explicitly only for the extended 20-min protocol; the acute 100-ms paradigm states pulse duration (313 us) and PRF (1 kHz) separately but never states its duty cycle in the text, so only the one explicit value (30%) is recorded.
• exposures[0].timing.sonication_duration_s — the acute paradigm (100 ms single tone) and the extended paradigm (10-s tone repeated every minute for 20 min total) are merged into one exposure because both target the same cell type and frequency; the two very different sonication_duration values are listed together and described further in protocol_description.
• exposures[0].unspecified_domain.pressure_kpa — 111 kPa and 4 W/cm2 Isppa are explicitly stated only for the extended (20-min) sub-protocol, not for the acute 100-ms dose-response paradigm, whose tested pressures are shown only in figures; domain (free field vs in situ) is not stated by the paper, so recorded as unspecified_domain.
• exposures[1].unspecified_domain.pressure_kpa — 20 kPa (RMS) is stated as only the first/lowest tested pressure in an ascending sweep to loss of impalement; the full sweep is shown only in a figure, not enumerated in the main text.
• sham_type — the extended-protocol control replaced the 20-min US application with an equivalent waiting period (no US); no inactive-transducer or blinded-sham mechanism is described, so classified as 'other'.
• n_subjects — reported as three separate group sizes for three sub-experiments (14 Retzius ganglia for the acute paradigm, of which 12 were analysed after excluding 2 for unstable baseline; 6 N cells; 8 Retzius ganglia for the extended paradigm); paper never states an overall total.
• anaesthesia — leeches were anaesthetized on ice prior to dissection and ganglion isolation; the isolated ganglion preparation itself was not under anaesthesia during ultrasound delivery.