# Noninvasive Focused Ultrasound as a Safe Modulator of Calcium-Dependent Neurochemical Signalling in Primary Cortical Cultures

## TL;DR

This study shows that low-intensity focused ultrasound can safely boost calcium signaling in brain cells without harming them.

## Contribution

The study defines a safe acoustic window for FUS that enhances calcium signaling without compromising neuronal health.

## Key findings

- FUS treatment did not affect cell viability or protein concentration compared to controls.
- Calcium imaging showed a significant increase in intracellular calcium responsiveness after FUS exposure.
- Neuronal morphology remained intact with no signs of damage or death following FUS treatment.

## 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.

The online version contains supplementary material available at 10.1007/s11064-026-04676-z.