Focused ultrasound excites action potentials in mammalian peripheral neurons in part through the mechanically gated ion channel PIEZO2

Neurons of the peripheral nervous system (PNS) are tasked with diverse roles, from encoding touch, pain, and itch to interoceptive control of inflammation and organ physiology. Thus, technologies that allow precise control of peripheral nerve activity have the potential to regulate a wide range of biological processes. Noninvasive modulation of neuronal activity is an important translational application of focused ultrasound (FUS). Recent studies have identified effective strategies to modulate brain circuits; however, reliable parameters to control the activity of the PNS are lacking. To develop robust noninvasive technologies for peripheral nerve modulation, we employed targeted FUS stimulation and electrophysiology in mouse ex vivo skin-saphenous nerve preparations to record the activity of individual mechanosensory neurons. Parameter space exploration showed that stimulating neuronal receptive fields with high-intensity, millisecond FUS pulses reliably and repeatedly evoked one-to-one action potentials in all peripheral neurons recorded. Interestingly, when neurons were classified based on neurophysiological properties, we identified a discrete range of FUS parameters capable of exciting all neuronal classes, including myelinated A fibers and unmyelinated C fibers. Peripheral neurons were excited by FUS stimulation targeted to either cutaneous receptive fields or peripheral nerves, a key finding that increases the therapeutic range of FUS-based peripheral neuromodulation. FUS elicited action potentials with millisecond latencies compared with electrical stimulation, suggesting ion channel–mediated mechanisms. Indeed, FUS thresholds were elevated in neurons lacking the mechanically gated channel PIEZO2. Together, these results demonstrate that transcutaneous FUS drives peripheral nerve activity by engaging intrinsic mechanotransduction mechanisms in neurons [B. U. Hoffman, PhD thesis, (2019)].

The nervous system is a central command center that governs homeostasis in physiological and pathophysiological states. Virtually all tissues, including the skin, heart, lungs, and gut, and immune organs, such as the bone marrow, spleen, and lymph nodes, are innervated by neurons of the peripheral nervous system (PNS). These specialized neurons serve both afferent functions, sending sensory information to the brain, and efferent roles, delivering neural signals to organs to alter their physiological outputs (1). For example, in the case of injury or infection, PNS neurons represent an essential component of immune responses (2). The intersection between the PNS and effector organs thus represents an ideal target for therapeutic development. Indeed, peripheral neuromodulation devices are approved by the US Food and Drug Administration (FDA) or in clinical trials to treat wide-ranging diseases from depression to rheumatoid arthritis (3). These devices rely on implanted electrodes, which require surgical procedures that inherently carry risk (4, 5). Thus, noninvasive strategies to modulate PNS activity are an appealing alternative to treat chronic diseases.Focused ultrasound (FUS) enables noninvasive neuromodulation of deep brain tissue and has shown promise as a therapeutic tool (6). More than 60 y ago, William Fry and colleagues demonstrated the reversible inhibitory effects of ultrasound on the central nervous system (CNS) of frogs, monkeys, and cats (7–9). Since that pioneering work, stimulation of the CNS with ultrasound has been shown to elicit action potentials in hippocampal slices, noninvasively stimulate intact motor circuits, and display therapeutic potential for seizure disruption in mammals (6, 10–13). Compared to the CNS, the effects of ultrasound stimulation on peripheral nerves are less clear. Ultrasound has been reported to both suppress and augment electrically evoked activity in the mammalian and invertebrate PNS (14–20). Notably, human psychophysical studies revealed that transdermal sonication induced somatic sensations such as touch, thermoreception, and pain, suggesting that ultrasound activates sensory neurons (15, 21–23). In addition, noninvasive sonication of the mouse sciatic nerve elicited muscle activity, indicating that FUS excites motor neurons (24, 25). Moreover, one report showed that sonication of a cat Pacinian corpuscle evoked neural activity consistent with receptor or action potentials (21). Despite these tantalizing studies, a systematic analysis of FUS-activated action potentials in mammalian peripheral neurons is lacking. This gap in knowledge is an impediment to the therapeutic development of PNS ultrasound neuromodulation, as protocols to reliably control neuronal activity have yet to be established despite decades of research efforts.To address this gap, we sought to determine reliable FUS parameters that excite action potentials in mammalian peripheral neurons in intact tissue. We focused on mechanosensory neurons of mouse dorsal root ganglia, whose peripheral axons, or afferents, densely innervate skin and internal organs to convey sensory information to the CNS. Activation of primary sensory neurons gives rise to distinct sensations, including touch, pain, itch, warmth, and cold. These distinct percepts are initiated by an impressive array of somatosensory neuronal subtypes, including multiple classes of mechanoreceptors, thermoreceptors, and nociceptors (or pain-sensing neurons). Peripheral sensory neurons can be further classified based on neurophysiological properties, including conduction velocity (CV), receptive field (RF; the area of skin they innervate), sensory threshold, and firing pattern (26). Thus, these well-studied neurons provide a robust platform for examining the excitatory effects of FUS in intact mammalian tissue.Here, we show that millisecond, high-intensity stimulation of sensory neurons with FUS is sufficient to elicit action potentials in all mechanosensory neurons studied. Moreover, the mechanically gated ion channel PIEZO2 sets the threshold for FUS activation of sensory neurons in peripheral tissues. These results define a parameter space to noninvasively excite sensory neurons in intact tissue and reveal molecular mechanisms that enable the transduction of sonication to neural activation—insights that have the potential to inform the development of neuromodulatory therapeutics.