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.

Focused ultrasound (US) is an emerging neuromodulation technology that has gained much attention due to its ability to modulate, noninvasively, neuronal activity in a variety of animals, including humans. However, there has been considerable debate about exactly which types of neurons can be influenced and what underlying mechanisms are in play. Are US-evoked motor changes driven indirectly by activated mechanosensory inputs, or more directly via central interneurons or motoneurons?Although it has been shown that US can mechanically depolarize mechanosensory neurons, there are no studies that have yet tested how identified motoneurons respond directly to US and what the underlying mechanism might be. Here, we examined the effects of US on a single, identified motoneuron within a well-studied and tractable invertebrate preparation, the medicinal leech, Hirudo verbana. Our approach aimed to clarify single neuronal responses to US, which may be obscured in other studies whereby US is applied across a diverse population of cells. We found that US has the ability to inhibit tonic spiking activity through a predominately thermal mechanism. USevoked effects persisted after blocking synaptic inputs, indicating that its actions were direct. Experiments also revealed that US-comparable heating blocked the axonal conduction of spontaneous action potentials. Finally, we found no evidence that US had significant mechanical effects on the neurons tested, a finding counter to prevailing views. We conclude that a non-sensory neuron can be directly inhibited via a thermal mechanism, a finding that holds promise for clinical neuromodulatory applications.

This review article highlights the historical developments and current state of knowledge of an important neuromodulation technology: low-intensity focused ultrasound. Because compelling studies have shown that focused ultrasound can modulate neuronal activity non-invasively, especially in deep brain structures with high spatial specificity, there has been a renewed interest in attempting to understand the specific bioeffects of focused ultrasound at the cellular level. Such information is needed to facilitate the safe and effective use of focused ultrasound to treat a number of brain and nervous system disorders in humans. Unfortunately, to date, there appears to be no singular biological mechanism to account for the actions of focused ultrasound, and it is becoming increasingly clear that different types of nerve cells will respond to focused ultrasound differentially based on the complement of their ion channels, other membrane biophysical properties, and arrangement of synaptic connections. Furthermore, neurons are apparently not equally susceptible to the mechanical, thermal and cavitation-related consequences of focused ultrasound application—to complicate matters further, many studies often use distinctly different focused ultrasound stimulus parameters to achieve a reliable response in neural activity. In this review, we consider the benefits of studying more experimentally tractable invertebrate preparations, with an emphasis on the medicinal leech, where neurons can be studied as unique individual cells and be synaptically isolated from the indirect effects of focused ultrasound stimulation on mechanosensitive afferents. In the leech, we have concluded that heat is the primary effector of focused ultrasound neuromodulation, especially on motoneurons in which we observed a focused ultrasound-mediated blockade of action potentials. We discuss that the mechanical bioeffects of focused ultrasound, which are frequently described in the literature, are less reliably achieved as compared to thermal ones, and that observations ascribed to mechanical responses may be confounded by activation of synaptically-coupled sensory structures or artifacts associated with electrode resonance. Ultimately, both the mechanical and thermal components of focused ultrasound have significant potential to contribute to the sculpting of specific neural outcomes. Because focused ultrasound can generate significant modulation at a temperature <5°C, which is believed to be safe for moderate durations, we support the idea that focused ultrasound should be considered as a thermal neuromodulation technology for clinical use, especially targeting neural pathways in the peripheral nervous system.