Low intensity pulsed ultrasound activates excitatory synaptic networks in cultured hippocampal neurons

Ultrasound can non-invasively penetrate deep into the brain for neuromodulation and has demonstrated good potential for clinical application. Excitation or inhibition of neurons by ultrasound has been reported, but the underlying mechanisms are largely unknown. So far most in vitro studies have focused on the activation of individual neurons by ultrasound with calcium imaging. As the focal region of ultrasound is typically millimeter or submillimeter size, it is important to investigate yet so far unclear how the mechanical effects of ultrasound would influence on the synaptic circuit activity of neurons. Low-intensity pulse ultrasound was used to stimulate cultured hippocampal neurons. Postsynaptic currents were recorded in individual cells with the whole-cell patch-clamp technique. We also simultaneously imaged intracellular calcium, along with neuronal electrical signals, to resolve neuronal network dynamics during ultrasound stimulation. Excitatory postsynaptic currents (EPSCs) were evoked by ultrasound in high-density neuronal cultures. Both the frequency and amplitude of EPSCs increased, indicating enhanced glutamatergic synaptic transmission. The probability of evoking responses, as well as the total charge of EPSCs evoked by ultrasound, increased with ultrasound intensity. Mechanistic analysis reveals that extracellular calcium influx, action potential firing and synaptic transmission are necessary for the responses to ultrasound in the high-density culture. In contrast, EPSCs were not enhanced in cultures with low densities of neurons. Simultaneous calcium imaging of neuronal network activity indicates that recurrent excitatory network activity is recruited during ultrasound stimulation in high-density cultures, which lasts over tens to hundreds of seconds. Our study provides insights into the mechanisms involved in the response of the brain to ultrasound and illuminates the potential to use ultrasound to regulate synaptic function in neurological disorders.