Challenges and Lessons Learned in Attempts to Develop In Vitro Models for Ultrasound Neuromodulation using Local Field Potential Measurements in Hippocampal Tissue.

Ultrasound neuromodulation is a rapidly developing tool for non-invasive control of brain activity. An in vitro model recapitulating the effects of ultrasound on neural tissue in vivo would be extremely valuable in guiding the development of this tool for optimal implementation. Yet, there are relatively few studies of ultrasound on neural activity in vitro. Here we describe our attempts to measure neuromodulatory outcomes using local field potential measurements in two in vitro models of hippocampal activity. First, we measured the effects of ultrasound at 1 MHz and 100-600 kPa on a rat hippocampal in vitro model of sharp wave ripples. Our primary protocol involved brief ultrasound pulses delivered at intervals shorter than the mean interval between sharp wave ripple events, with other stimulus protocols tested with small sample size. No set of parameters produced detectable effects on the amplitude or frequency of sharp wave ripples. We considered whether missing synaptic connections or the relatively small volume in brain slices might account for the lack of effect in our experimental setup. To test these hypotheses, and to examine ultrasound's effects in another system, we measured the effects of ultrasound on theta oscillations in the intact mouse hippocampus in vitro. We found that ultrasound at 1 MHz and 500 kPa, applied continuously for 2 seconds, produced no detectable effects on the amplitude or frequency of in vitro theta oscillations. Finally, we considered a novel mechanism for ultrasound's effects on neural activity, in which acoustic pressure causes microscale phase transitions in the pores of ion channels, such as nicotinic receptor channels, that exhibit hydrophobic gating. To test this hypothesis, we repeated our experiments on the intact hippocampus in the presence of 5 μM nicotine; however, as with the other experimental systems, we found no detectable effects of ultrasound in our experimental set-up.