Mechanistic insights into ultrasonic neurostimulation of disconnected neurons using single short pulses
Eyal Weinreb, Elisha Moses
Brain Stimulation 2022 · 10.1016/j.brs.2022.05.004
Abstract
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.
Abstract via europepmc.
Exposures
Exposure 1: Single short pulses to cultures
Target: cultured neurons — “dissociated rat hippocampal neural cultures”
Device: not reported
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 500 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | not applicable | |
| Sonication duration (s) | 0.000004, 0.04swept | ✓✓✓⚑ |
| Free-field pressure (kPa) | 350, 1,320swept | ✓✓✓⚑ |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable |
Cultures were exposed to a single ultrasound pulse per stimulation (4 ms or 40 ms; 0.35 to 1.32 MPa peak pressure) with calcium images acquired for 5 s before and 10 s after; repeated stimulations were separated by recovery periods.
Flags from extraction
exposures[0].timing.sonication_duration_s— Paper says a 2-cycle pulse at 500 kHz corresponds to '4 ms'; 2 cycles at 500 kHz is 4 microseconds, so the unit may be a typo. Recorded as written (4 ms and 40 ms).exposures[0].free_field.pressure_kpa— Peak pressures of 0.35 to 1.32 MPa; the text says output was verified with a hydrophone in a large water tank. Other text gives 0.65, 0.67 MPa for the higher pressure.n_subjects— 78 is the number of cultures (n^c) in the generally-active-cell count; the equals sign was lost in conversion. Total cultures across experiments is not stated.exposures[0].device.family— Transducer maker and model not given in the main text (methods are in the supplementary).exposures[0].timing.waveform— Single short pulses, one per stimulation; recorded as continuous per the single-burst rule.