Noninvasive Focused Ultrasound as a Safe Modulator of Calcium-Dependent Neurochemical Signalling in Primary Cortical Cultures
Iqra Bano, Pascal Jorratt, Viera Kútna, Jan Pala, Grygoriy Tsenov
Neurochemical Research 2026 · 10.1007/s11064-026-04676-z
Abstract
Focused ultrasound stimulation (FUS) is a promising non-invasive neuromodulation technique that can influence neuronal activity through mechanical stimulation. In this study, primary cortical neurons were isolated from embryonic rat brains and cultured for 14 days in vitro before being divided into Control, FUS 5 V, and FUS 10 V groups. Cells were exposed to low-intensity pulsed FUS (300 kHz, 10 min) using a vertically mounted transducer positioned 5 mm above the culture dish. Post-exposure analyses included cell viability using the MTS assay, total protein quantification by the Bradford method, morphological assessment by Trypan Blue staining, and Fluo-3 AM-based confocal calcium imaging. FUS treatment produced no significant differences in viability or total protein concentration compared with the Control group. Morphological observations confirmed healthy neuronal somata and intact neuritic networks across all groups, with no evidence of cell death or structural damage compared with controls. In contrast, calcium imaging revealed a robust transient elevation in intracellular Ca²⁺ responsiveness when assessed 24 h after FUS exposure, with a significantly higher integrated area under the curve relative to Control. These findings demonstrate that low-intensity FUS safely enhances intracellular calcium signalling while preserving neuronal viability, protein integrity, and morphology, defining a safe acoustic window for non-destructive neuromodulation and providing a framework for mechanistic studies in neurodegenerative disease models.
Abstract via europepmc.
Exposures
Exposure 1: FUS 5 V and 10 V drive, 10 min
Target: cultured neurons — “primary cortical neurons (DIV14)”
Device: custom-built · custom immersion-type 300 kHz transducer driven by A-M Systems model 4100 isolated High-Power stimulator
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 300 | ✓✓✓ |
| Pulse duration (ms) | 500 | ✓✓✓⚑ |
| Pulse repetition frequency (Hz) | 0.05 | ✓✓✓⚑ |
| Duty cycle (%) | not reportedpulse duration × PRF gives 2.5% | ⚑ |
| Sonication duration (s) | 600 | ✓✓✓ |
| 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 |
30 train bursts of 0.5 s every 20 s (10 min total); each burst is a 300 kHz monophasic pulse train with pulse duration half of the event period (150,000 pulses per burst). Drive amplitude +5 V or +10 V; no acoustic calibration was performed.
Flags from extraction
exposures[0].timing.pulse_duration_ms— Burst rule: the 0.5 s 'train burst' is the gated unit repeated every 20 s, so recorded as pulse duration; within it the carrier is a 300 kHz monophasic square wave with pulse duration half the event period.exposures[0].timing.pulse_repetition_frequency_hz— Converted from the stated 20 s train burst period (1/20 s).exposures[0].timing.duty_cycle_pct— Not stated; not computed.exposures[0].free_field.pressure_kpa— Paper states direct acoustic pressure or intensity calibration was not performed; drive voltage only.exposures[0].device.family— Transducer described only as custom immersion-type; maker not named, so classed as custom_built.n_subjects— n = 3 refers to independent culture preparations; MTS used 5 biological replicates.