Mechanistic insights into ultrasonic neurostimulation of disconnected neurons using single short pulses

Ultrasonic neurostimulation is a potentially potent noninvasive therapy, whose mechanism has yet to be elucidated. We designed a system capable of applying ultrasound with minimal reflections to neuronal cultures. Synaptic transmission was pharmacologically controlled, eliminating network effects, enabling examination of single-cell processes. Short single pulses of low-intensity ultrasound were applied, and time-locked responses were examined using calcium imaging. Low-pressure (0.35 MPa) ultrasound directly stimulated ∼20% of pharmacologically disconnected neurons, regardless of membrane poration. Stimulation was resistant to the blockade of several purinergic receptor and mechanosensitive ion channel types. Stimulation was blocked, however, by suppression of action potentials. Surprisingly, even extremely short (4 μs) pulses were effective, stimulating ∼8% of the neurons. Lower-pressure pulses (0.35 MPa) were less effective than higher-pressure ones (0.65 MPa). Attrition effects dominated, with no indication of compromised viability. Our results detract from theories implicating cavitation, heating, non-transient membrane pores >1.5 nm, pre-synaptic release, or gradual effects. They implicate a post-synaptic mechanism upstream of the action potential, and narrow down the list of possible targets involved. •Single, extremely short (4 μs) ultrasound pulses stimulate neuronal cultures.•Stimulation is resistant to synaptic blockade and independent from membrane poration.•Action potentials are necessary, implicating an upstream post-synaptic mechanism.•Results detract from cavitation, heating, pre-synaptic release, or gradual mechanisms.•TRPA, TRPV, TREK-2, and Piezo channels as well as P2 receptors are precluded.