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.

3rd Faculty of Medicine Charles University Ruská 87 100 00 Prague 10 Czech Republic

Department of Animal Physiology Faculty of Science Charles University Albertov 6 128 00 Prague Czech Republic

Department of Experimental Neurobiology National Institute of Mental Health Topolová 748 25067 Klecany Czech Republic

Department of Physiology and Biochemistry Faculty of Bioscience Shaheed Benazir Bhutto University of Veterinary and Animal Sciences Sakrand 67210 Pakistan

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