Characterization and modulation of directly and delayed ultrasound-activated cells under single-pulse exposure

Low-intensity ultrasound offers therapeutic promise through noninvasive, precise neuronal activation. Advancing its clinical translation requires a clear understanding of its biophysical mechanisms. However, due to intercellular communications, directly ultrasound-activated cells inevitably trigger delayed activation in neighboring cells. Accurate identification of these activation types is therefore essential for understanding ultrasound-cell interactions and improving ultrasound neuromodulation. Under continuous-wave ultrasound excitation, using the onset time of intracellular Fluo-4 fluorescence and the change in intracellular propidium iodide, directly ultrasound-activated cells, delayed-activated cells, and ultrasound-permeated cells were accurately characterized. For directly ultrasound-activated cells, the activation efficiency, amplitude, and time-to-peak of intracellular Ca²⁺ transients correlated positively with acoustic pressure but not pulse duration, suggesting acoustic radiation force may play a crucial role in cellular activation. Longer pulse intervals increased the probability of repeated cellular activation, with a ∼ 5-min interval significantly strengthening the temporal coupling between ultrasound pulse and cellular activation. Gd³⁺ and 2-APB treatments reduced activation efficiency and intracellular Ca²⁺ response levels, suggesting involvement of TRP family ion channels in direct activation. For delayed-activated cells, the propagation velocity of intercellular Ca²⁺ wave increased nearly linearly with the proportion and degree of directly ultrasound-activated cells, but decreased with longer time-to-peak of intracellular Ca²⁺ transients in those cells, demonstrating delayed activation originated from directly ultrasound-activated cells. The reduced propagation velocity in delayed-activated cells after Gd³⁺, 2-APB, and BAPTA treatment indicates involvement of gap-junction-dependent pathways and ATP-mediated paracrine signaling in intercellular Ca²⁺ propagation. This information facilitates the differentiation between directly ultrasound-activated cells and delayed-activated cells, advancing understanding of the mechanisms governing each activation type.