Low Intensity Pulsed Ultrasound Activates Excitatory Synaptic Networks in Cultured Hippocampal Neurons

Fenfang Li, Hao Jiang, Jiawei Lin, Chaofeng Qiao, George J. Augustine

Ultrasound in Medicine & Biology 2025, 51, 1250-1260 · 10.1016/j.ultrasmedbio.2025.04.011

in vitro cellhealthyinvasive electrophysiologycellular imaging

Abstract

Objective Ultrasound can noninvasively penetrate deep into the brain for neuromodulation, demonstrating good potential for clinical application. However, the underlying mechanisms are unclear. So far most in vitro studies have focused on the activation of individual neurons by ultrasound with calcium imaging. As the focal region of ultrasound is typically millimeter or submillimeter size, it is important to investigate yet so far unclear how the mechanical effects of ultrasound would influence the synaptic circuit activity of neurons. Methods Low-intensity pulse ultrasound was used to stimulate cultured hippocampal neurons. Postsynaptic currents were recorded in individual cells with the whole-cell patch-clamp technique. We also simultaneously imaged intracellular calcium, along with neuronal electrical signals, to resolve neuronal network dynamics during ultrasound stimulation. Results Excitatory postsynaptic currents (EPSCs) were evoked by ultrasound in high-density neuronal cultures with increased frequency and amplitude, indicating enhanced glutamatergic synaptic transmission. The probability of evoking responses and the total charge of EPSCs increased with ultrasound intensity. Mechanistic analysis reveals that extracellular calcium influx, action potential firing and synaptic transmission are necessary for the responses to ultrasound in high-density culture. In contrast, EPSCs were not enhanced in low-density culture. Simultaneous calcium imaging of neuronal network activity indicates that recurrent excitatory network activity is recruited during ultrasound stimulation in high-density cultures, which lasts over tens to hundreds of seconds. Conclusion Our study provides insights into the mechanisms involved in the response of the brain to ultrasound and illuminates the potential to use ultrasound to regulate synaptic function in neurological disorders.

Abstract via europepmc.

Speciesmouse

Subjectsnot reported cultures

Sessions per subjectnot applicable

Randomisednot applicable

Blindingnot applicable

Sham / controlinactive transducer

Auditory controlnot reported

Readout timingboth

Anaesthesianot applicable

Readoutsinvasive electrophysiology, cellular imaging — Whole-cell voltage-clamp patch-clamp recording of postsynaptic currents/action potentials; fluo-4 calcium imaging of network activity; particle image velocimetry (PIV) of ultrasound-induced acoustic streaming.

Direction of effectexcitatoryLIPUS evoked barrages of excitatory postsynaptic currents (EPSCs) and, at higher intensities, action potentials in high-density hippocampal cultures, via recurrent activation of an excitatory glutamatergic network; responses were abolished by TTX, kynurenic acid, or calcium-free medium, and were greatly attenuated in low-density or single-neuron (autaptic) cultures.

Adverse eventsnot applicableTemperature increases associated with LIPUS were negligible (within 0.1-0.2 degrees by thermocouple; a fraction of a degree or less by infrared camera after 20 s of LIPUS), and LIPUS intensities as high as 1.61 W/cm2 had little effect on membrane holding current, indicating no evidence of thermal or damage-related artefact.

Exposures

Exposure 1: LIPUS stimulation of high-density (and low-density/autaptic) cultured hippocampal neurons

Target: hippocampus — “cultured hippocampal neurons”
Device: Olympus / Panametrics · Olympus · V324-N-SU-F0.5IN ✓

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	25,000	✓✓✓
Pulse duration (ms)	10	✓✓✓⚑
Pulse repetition frequency (Hz)	5	✓✓✓
Duty cycle (%)	5pulse duration × PRF gives 5%	✓✓✓
Sonication duration (s)	20	✓✓✓

Pressure and intensity, by domain
Free-field pressure (kPa)	590, 910swept	✓✓✓
Free-field Isppa (W/cm²)	not reported
Free-field Ispta (W/cm²)	0.35, 1.61swept	✓✓✓⚑
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

Protocol, in the paper’s words

The 25 MHz transducer was operated in pulse mode: PRF 5 Hz, duty cycle 5%, giving a 10-ms pulse duration, for a total sonication (burst train) duration of 20 s. Ultrasound intensity (ISPTA) was varied across experiments from 0.35 to 1.61 W/cm2 (grouped as lower 0.35-0.58, medium 0.72-1.03, and higher 1.21-1.61 W/cm2) to characterise the dose-response of evoked synaptic activity; at least 100 s separated successive stimuli to avoid refractory responses.

Consistency checks: f0 out of range.

Flags from extraction

n_subjects — Paper reports per-figure numbers of recorded neurons/experiments (e.g. N=28 patched neurons, N=26 calcium-imaging experiments, N=4-10 per intensity bin) rather than a single total count of independent cultures/animals; no overall subject total is stated.
exposures[0].free_field.ispta_w_cm2 — 0.35-1.61 W/cm2 combines the hydrophone-characterised range (0.58-1.41 W/cm2, Fig. 1c) with the wider functional threshold/dose-response range reported later in Results (0.35 to 1.41 W/cm2, extending to 1.61 W/cm2 for the highest tested bin); domain is a water-coupled dish/chamber setup with no skull or tissue path, so values are recorded under free_field.
exposures[0].timing.pulse_duration_ms — Pulse duration is directly stated by the authors ('we selected a pulse repetition frequency of 5 Hz with a 5% duty cycle to achieve a 10 ms pulse duration'), resolving the duty-cycle/PRF relationship explicitly rather than by the extractor's own arithmetic.