Abstract. Ultrasound that is widely used in medical diagnosis has drawn growing interests as a noninvasive means of neuromodulation. Focused pulsed ultrasound (FPUS) effectively modulates neural encoding and transmission in the peripheral nervous system (PNS) with unclear mechanism of action, which is further confounded by contradictory experimental outcomes from recordings of compound action potentials (CAP). To address that, we developed a novel in vitro set up to achieve simultaneous single-unit recordings from individual mouse sciatic nerve axon and systematically studied the neuromodulation effects of FPUS on individual axon. Unlike previous CAP recordings, our single-unit recordings afford superior spatial and temporal resolution to reveal the subtle but consistent effects of ultrasonic neuromodulation. Our results indicate that, 1) FPUS did not evoke action potentials directly in mouse sciatic nerve at all the tested intensities (spatial peak temporal average intensity, ISPTA of 0.91 to 28.2 W/cm 2 ); 2) FPUS increases the nerve conduction velocity (CV) in both fast-conducting A-and slow-conducting C-type axons with effects more pronounced at increased stimulus duration and intensity; and 3) effects of increased CV is reversible and cannot be attributed to the change of local temperature. Our results support existing theories of non-thermal mechanisms underlying ultrasonic neuromodulation with low-intensity FPUS, including NICE, flexoelectricity, and solition models. This work also provides a solid experimental basis to further advance our mechanistic understandings of ultrasonic neuromodulation in the PNS.
IntroductionUltrasound (US) has prevailed the field of medical diagnosis for long but yet to be established as a therapeutic paradigm. Neuromodulatory effects of US were first reported in 1929 when Harvey showed that innervated skeletal muscles from frogs and turtles responded to US stimulation in vitro (1). More recent researches focus on the US effects at the central nervous system (CNS), including the disruption of blood brain barrier, motor and sensory responses, and suppressed or evoked action potentials e.g., (2-12), which culminated in the approval of the Food and Drug Administration (FDA) to treat refractory patients with essential tremor using MRI-guided focused ultrasound (13). In addition, ex vivo studies on hippocampal slice cultures have shown that, low-intensity FPUS can elicit electrical activities as indicated by calcium imaging from mouse (14) and simultaneously enhance (at fiber volley) and suppress (at dendritic layers) compound action potentials (CAP) from rat hippocampal dentate gyrus (15).In contrast, the effects of US on the peripheral nervous system (PNS) is comparatively understudied with inconsistent reports. US appears to evoke or enhance the peripheral neural activities in frog sciatic nerve in vitro (16), sensitize neuron in C. elegans (17), cause deqi sensations (i.e., tingling, numbness, heaviness, and fullness) by stimulation of an acupuncture point (LI4, He Gu) (18), an...