Ultrasound Modulates Calcium Activity in Cultured Neurons, Glial Cells, Endothelial Cells and Pericytes

Objective: Ultrasound is being researched as a method to modulate the brain. Studies of the interaction of sound with neurons support the hypothesis that mechanosensitive ion channels play an important role in ultrasound neuromodulation. The response of cells other than neurons (e.g., astrocytes, pericytes and endothelial cells) have not been fully characterized, despite playing an important role in brain function. Methods: To address this gap in knowledge, we examined cultured murine primary cortical neurons, astrocytes, endothelial cells and pericytes in an in vitro widefield microscopy setup during application of a 500 ms burst of 250 kHz focused ultrasound over a pressure range known to elicit neuromodulation. We examined cell membrane health in response to a range of pulses and used optical calcium indicators in conjunction with pharmacological antagonists to selectively block different groups of thermo- and mechanosensitive ion channels known to be responsive to ultrasound. Results: All cell types experienced an increase in calcium fluorescence in response to ultrasound. Gadolinium (Gad), 2-aminoethoxydiphenyl borate (2 -APB) and ruthenium red (RR) reduced the percentage of responding neurons and magnitude of response. The percentage of astrocytes responding was significantly lowered only by Gad, whereas both 2 -APB and Gad decreased the amplitude of the fluorescence response. 2 -APB decreased the percentage of responding endothelial cells, whereas only Gad reduced the magnitude of responses. Pericytes exposed to RR or Gad were less likely to respond to stimulation. RR had no detectable effect on the magnitude of the pericyte responses while 2 -APB and Gad significantly decreased the fluorescence intensity, despite not affecting the percentage responding. Conclusion: Our study highlights the role of non -neuronal cells during FUS neuromodulation. All of the investigated cell types are sensitive to mechanical ultrasound stimulation and rely on mechanosensitive ion channels to undergo ultrasound neuromodulation.