Remote Excitation of Neuronal Circuits Using Low-Intensity, Low-Frequency Ultrasound
William J. Tyler, Yusuf Tufail, Michael Finsterwald, Monica L. Tauchmann, Emily J. Olson, Cassondra Majestic
PLoS ONE 2008, 3, e3511 · 10.1371/journal.pone.0003511
Abstract
Possessing the ability to noninvasively elicit brain circuit activity yields immense experimental and therapeutic power. Most currently employed neurostimulation methods rely on the somewhat invasive use of stimulating electrodes or photon-emitting devices. Due to its ability to noninvasively propagate through bone and other tissues in a focused manner, the implementation of ultrasound (US) represents a compelling alternative approach to current neuromodulation strategies. Here, we investigated the influence of low-intensity, low-frequency ultrasound (LILFU) on neuronal activity. By transmitting US waveforms through hippocampal slice cultures and ex vivo mouse brains, we determined LILFU is capable of remotely and noninvasively exciting neurons and network activity. Our results illustrate that LILFU can stimulate electrical activity in neurons by activating voltage-gated sodium channels, as well as voltage-gated calcium channels. The LILFU-induced changes in neuronal activity were sufficient to trigger SNARE-mediated exocytosis and synaptic transmission in hippocampal circuits. Because LILFU can stimulate electrical activity and calcium signaling in neurons as well as central synaptic transmission we conclude US provides a powerful tool for remotely modulating brain circuit activity.
Abstract via europepmc.
Exposures
Exposure 1: LILFU-1 (primary waveform; hippocampal slice cultures and ex vivo whole brains)
Target: hippocampus, brain slice — “hippocampal CA1 pyramidal neurons in organotypic slice cultures; dorsal surface of ex vivo whole brain”
Device: custom-built
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 440 | ✓✓✓ |
| Pulse duration (ms) | 22.7 | ✓✓✓⚑ |
| Pulse repetition frequency (Hz) | 0, 100swept | ✓✓✓⚑ |
| Duty cycle (%) | not reported | |
| Sonication duration (s) | 5 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | 2.9 | ✓✓✓ |
| Free-field Ispta (W/cm²) | 0.023 | ✓✓✓ |
| 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 |
LILFU waveforms used as stimuli were generated by repeating pulse trains of US tone bursts at a pulse repetition frequency until a desired number of tone bursts had been generated. Our primary LILFU waveform (LILFU-1) had the following properties: f = 0.44 MHz, TBD = 22.7 ms, c/tb = 10, PRF = 5 sec sweep 0-100 Hz, and Ntb = 250. A related brief waveform (f = 0.44 MHz, TBD = 0.18 msec, c/tb = 80, PRF = 10 Hz, and Ntb = 3) was also used in some Ca2+ imaging experiments to elicit faster-kinetic transients.
Exposure 2: 0.67 MHz LILFU waveform (spH synaptic vesicle release comparison, Figure 5C/D)
Target: hippocampus, brain slice — “hippocampal CA1 stratum radiatum (CA3-CA1 synapses) in organotypic slice cultures”
Device: custom-built
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 670 | ✓✓✓ |
| Pulse duration (ms) | 74.5 | ✓✓✓ |
| Pulse repetition frequency (Hz) | 10 | ✓✓✓ |
| Duty cycle (%) | not reportedpulse duration × PRF gives 74.5% | |
| Sonication duration (s) | 0.5 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| 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 |
For example, a LILFU waveform composed of different US tone bursts (f= 0.67 MHz, TBD = 74.5 msec, c/tb = 50,000; Figure 5C) delivered at PRF = 10 Hz with Ntb = 5 also stimulated synaptic vesicle release. (D) Individual (black) and averaged (green) spH signals obtained in response to stimulation with the LILFU tone burst shown in (C) delivered at a PRF = 10 Hz for 0.5 s to produce Np = 5.
Flags from extraction
exposures[0].timing.pulse_duration_ms— Text states TBD = 22.7 ms for a c/tb = 10 burst at f = 0.44 MHz; 10 cycles at 0.44 MHz corresponds to ~22.7 microseconds, not milliseconds. Likely a unit typo/OCR error in the source (ms vs µs), but recorded verbatim per instructions rather than corrected.exposures[0].timing.pulse_repetition_frequency_hz— LILFU-1 uses a linearly swept PRF (0 to 100 Hz over 5 s), not a fixed PRF value, so it cannot be represented as a single number; recorded as not_reported and described fully in protocol_description. A related brief waveform used a fixed PRF of 10 Hz (mentioned in protocol_description) but was not modelled as a separate list value.exposures[0].target— This exposure aggregates experiments performed in organotypic hippocampal slice cultures and separately in ex vivo whole mouse brains (imaged from the dorsal surface while LILFU-1 was transmitted from the ventral surface); both used the same LILFU-1 waveform and are combined here rather than split into a third exposure.n_subjects— Sample sizes are reported per-assay in terms of cells/boutons and slices (e.g., n=24 cells from 6 slices; n=148 release sites from 15 slices) rather than as a single subject count for the study; not reduced to one number.auditory_control— The schema does not offer a null/not-applicable option for auditory_control (unlike anaesthesia, randomised, blinding); set to ['not_reported'] even though this is in vitro/ex vivo tissue work where auditory confounds are not physiologically applicable.
Notes: This is an in vitro/ex vivo study (organotypic hippocampal slice cultures, ex vivo whole mouse brains, and some whole-cell patch-clamp recordings); anaesthesia, auditory_control, randomised and blinding are set to null as not applicable to tissue/cell work per the schema. The paper also states an aggregate safety figure ('The peak rarefactional pressure used in our studies was <1 MPa') describing the whole set of LILFU waveforms tested, which is not assignable to a single exposure and was not entered as a field value.