Long-lasting forms of plasticity through patterned ultrasound-induced brainwave entrainment

Ho-Jeong Kim, Tien Thuy Phan, Keunhyung Lee, Jeong Sook Kim, Sang-Yeong Lee, Jung Moo Lee, Jongrok Do, Doyun Lee, Sung-Phil Kim, Kyu Pil Lee, Jinhyoung Park, C. Justin Lee, Joo Min Park

Science Advances 2024, 10 · 10.1126/sciadv.adk3198

rodenthealthyemg mepeeg meginvasive electrophysiologycellular imaginghistology molecularbehaviour

Abstract

Achieving long-lasting neuronal modulation with low-intensity, low-frequency ultrasound is challenging. Here, we devised theta burst ultrasound stimulation (TBUS) with gamma bursts for brain entrainment and modulation of neuronal plasticity in the mouse motor cortex. We demonstrate that two types of TBUS, intermittent and continuous TBUS, induce bidirectional long-term potentiation or depression-like plasticity, respectively, as evidenced by changes in motor-evoked potentials. These effects depended on molecular pathways associated with long-term plasticity, including N -methyl-d-aspartate receptor and brain-derived neurotrophic factor/tropomyosin receptor kinase B activation, as well as de novo protein synthesis. Notably, bestrophin-1 and transient receptor potential ankyrin 1 play important roles in these enduring effects. Moreover, pretraining TBUS enhances the acquisition of previously unidentified motor skills. Our study unveils a promising protocol for ultrasound neuromodulation, enabling noninvasive and sustained modulation of brain function.

Abstract via europepmc.

Speciesmouse (C57BL/6 and Thy1-GCaMP6f)

Subjects6, 6swept animals

Sessions per subjectnot reported

Randomisednot reported

Blindingnot reported

Sham / controlundescribed

Auditory controlnot reported

Readout timingboth

Anaesthesiaanaesthetised

Readoutsemg mep, eeg meg, invasive electrophysiology, cellular imaging, histology molecular, behaviour — Motor-evoked potentials (MEPs); subdermal EEG for theta-gamma phase-amplitude coupling; Neuropixels extracellular recording; GCaMP6f fiber photometry calcium imaging; whole-cell patch-clamp sEPSCs; c-Fos immunofluorescence and GluR4/AMPAR western blot; single-pellet reaching task (SPRT)

Direction of effectbidirectionalIntermittent theta-burst ultrasound (iTBUS) increased MEP amplitude (LTP-like facilitation), while continuous theta-burst ultrasound (cTBUS) decreased MEP amplitude (LTD-like depression); effects required NMDAR activation, BDNF/TrkB signalling, de novo protein synthesis, and TRPA1/Best1 channels.

Adverse eventsnone observedHematoxylin and eosin staining after 10 days of TBUS administration and Evans blue dye assessment after acute 800-pulse TBUS showed no tissue damage, hemorrhaging, or blood-brain-barrier disruption.

Exposures

Exposure 1: Theta burst ultrasound stimulation (iTBUS/cTBUS) to contralateral primary motor cortex (M1)

Target: primary motor cortex — “contralateral M1 hotspot associated with the proximal forelimb muscle movement”
Device: Olympus / Panametrics · Olympus Panametrics · V301 (V301-SU) ✓

Pulse timing
Waveform	theta_burst
Fundamental frequency (kHz)	500	✓✓✓
Pulse duration (ms)	17	✓✓✓
Pulse repetition frequency (Hz)	30	✓✓✓⚑
Duty cycle (%)	51.1pulse duration × PRF gives 51%	✓✓✓
Sonication duration (s)	2, 100swept	✓✓✓

Pressure and intensity, by domain
Free-field pressure (kPa)	299	✓✓✓
Free-field Isppa (W/cm²)	2.79	✓✓✓
Free-field Ispta (W/cm²)	0.0544, 0.272swept	✓✓✓
In-situ estimate	not reported
In-situ pressure (kPa)	not reported
In-situ Isppa (W/cm²)	not reported
In-situ Ispta (W/cm²)	not reported

Protocol, in the paper’s words

TBUS protocol: a 2-s theta-gamma coupling pattern in which ultrasound stimuli (17-ms, 500-kHz tone bursts) were delivered four times with a 33.33-ms interstimulus onset interval (30-Hz gamma), repeated every 200 ms (5-Hz theta). For intermittent TBUS (iTBUS), the 2-s train was delivered 10 times for 400 pulses, with an 8-s break interval between trains. For continuous TBUS (cTBUS), the protocol was performed continuously without break intervals, extending to 100 s for 400 pulses. A second series doubled pulse number to 800 for both iTBUS and cTBUS to test longevity of effects. TBUS was administered at 80% of the single-pulse motor-threshold-referenced intensity used to elicit MEPs.

Flags from extraction

exposures[0].unspecified_domain.isppa_w_cm2 — This ISPPA (2.787 W/cm2) and pressure (0.299 MPa) value is for the 100%-intensity single ultrasound stimulus used to elicit MEPs and calibrate motor threshold; TBUS itself used 80% of this intensity but the paper does not restate the resulting scaled pressure/ISPPA number, only the ISPTA values specific to iTBUS/cTBUS.
exposures[0].unspecified_domain.pressure_kpa — Domain not stated as free field or in situ; intensity was measured with a needle hydrophone at the end of the acoustic collimator (near-field, close to brain), so placed in unspecified_domain.
exposures[0].timing.pulse_repetition_frequency_hz — The paper nests two frequencies (30 Hz gamma pulse-repetition rate and 5 Hz theta train-repetition rate); 30 Hz is recorded as the PRF of the individual 17-ms tone-burst pulses, while the 5 Hz theta nesting and inter-train structure are described in protocol_description.
sham_type — Sham stimulation is mentioned (e.g., for c-Fos/western blot cohorts) but its mechanism (transducer off, blocked, etc.) is not described in the text.
n_subjects — The paper reports many separate small-group sub-experiments (e.g., n=6-10 per pharmacology/behaviour/imaging group) but never states an overall total number of animals used in the study.
n_sessions_per_subject — Most experiments used a single TBUS session per animal, but the single-pellet reaching task used daily TBUS for 10 consecutive days; no single overall number applies across the heterogeneous sub-experiments.