Towards an ion-channel-centric approach to ultrasound neuromodulation.

Ultrasound neuromodulation is a promising technology that could revolutionize study and treatment of brain conditions ranging from mood disorders to Alzheimer's disease and stroke. An understanding of how ultrasound directly modulates specific ion channels could provide a roadmap for targeting specific neurological circuits and achieving desired neurophysiological outcomes. Although experimental challenges make it difficult to unambiguously identify which ion channels are sensitive to ultrasound in vivo, recent progress indicates that there are likely several different ion channels involved, including members of the K2P, Piezo, and TRP channel families. A recent result linking TRPM2 channels in the hypothalamus to induction of torpor by ultrasound in rodents demonstrates the feasibility of targeting a specific ion channel in a specific population of neurons.

Spike frequency-dependent inhibition and excitation of neural activity by high-frequency ultrasound.

Ultrasound can modulate action potential firing in vivo and in vitro, but the mechanistic basis of this phenomenon is not well understood. To address this problem, we used patch-clamp recording to quantify the effects of focused, high-frequency (43 MHz) ultrasound on evoked action potential firing in CA1 pyramidal neurons in acute rodent hippocampal brain slices. We find that ultrasound can either inhibit or potentiate firing in a spike frequency-dependent manner: at low (near-threshold) input currents and low firing frequencies, ultrasound inhibits firing, while at higher input currents and higher firing frequencies, ultrasound potentiates firing. The net result of these two competing effects is that ultrasound increases the threshold current for action potential firing, the slope of frequency-input curves, and the maximum firing frequency. In addition, ultrasound slightly hyperpolarizes the resting membrane potential, decreases action potential width, and increases the depth of the after-hyperpolarization. All of these results can be explained by the hypothesis that ultrasound activates a sustained potassium conductance. According to this hypothesis, increased outward potassium currents hyperpolarize the resting membrane potential and inhibit firing at near-threshold input currents but potentiate firing in response to higher-input currents by limiting inactivation of voltage-dependent sodium channels during the action potential. This latter effect is a consequence of faster action potential repolarization, which limits inactivation of voltage-dependent sodium channels, and deeper (more negative) after-hyperpolarization, which increases the rate of recovery from inactivation. Based on these results, we propose that ultrasound activates thermosensitive and mechanosensitive two-pore-domain potassium (K2P) channels through heating or mechanical effects of acoustic radiation force. Finite-element modeling of the effects of ultrasound on brain tissue suggests that the effects of ultrasound on firing frequency are caused by a small (<2°C) increase in temperature, with possible additional contributions from mechanical effects.

Activation of Piezo1 but Not NaV1.2 Channels by Ultrasound at 43 MHz.

Ultrasound (US) can modulate the electrical activity of the excitable tissues, but the mechanisms underlying this effect are not understood at the molecular level or in terms of the physical modality through which US exerts its effects. Here, we report an experimental system that allows for stable patch-clamp recording in the presence of US at 43 MHz, a frequency known to stimulate neural activity. We describe the effects of US on two ion channels proposed to be involved in the response of excitable cells to US: the mechanosensitive Piezo1 channel and the voltage-gated sodium channel NaV1.2. Our patch-clamp recordings, together with finite-element simulations of acoustic field parameters indicate that Piezo1 channels are activated by continuous wave US at 43 MHz and 50 or 90 W/cm2 through cell membrane stress caused by acoustic streaming. NaV1.2 channels were not affected through this mechanism at these intensities, but their kinetics could be accelerated by US-induced heating.

Dynamic response of model lipid membranes to ultrasonic radiation force.

Low-intensity ultrasound can modulate action potential firing in neurons in vitro and in vivo. It has been suggested that this effect is mediated by mechanical interactions of ultrasound with neural cell membranes. We investigated whether these proposed interactions could be reproduced for further study in a synthetic lipid bilayer system. We measured the response of protein-free model membranes to low-intensity ultrasound using electrophysiology and laser Doppler vibrometry. We find that ultrasonic radiation force causes oscillation and displacement of lipid membranes, resulting in small (<1%) changes in membrane area and capacitance. Under voltage-clamp, the changes in capacitance manifest as capacitive currents with an exponentially decaying sinusoidal time course. The membrane oscillation can be modeled as a fluid dynamic response to a step change in pressure caused by ultrasonic radiation force, which disrupts the balance of forces between bilayer tension and hydrostatic pressure. We also investigated the origin of the radiation force acting on the bilayer. Part of the radiation force results from the reflection of the ultrasound from the solution/air interface above the bilayer (an effect that is specific to our experimental configuration) but part appears to reflect a direct interaction of ultrasound with the bilayer, related to either acoustic streaming or scattering of sound by the bilayer. Based on these results, we conclude that synthetic lipid bilayers can be used to study the effects of ultrasound on cell membranes and membrane proteins.

Gating modes in AMPA receptors.

AMPA receptors are ligand-gated ion channels that show multiple conductance levels, indicating that gating of individual AMPA subunits is to some extent independent of the other subunits. To study AMPAR subunit interactions during activation gating, we recorded from single channels in the absence of channel block and desensitization and at negative and positive membrane potentials. In saturating glutamate, the relative occupancies of the various conductance levels are consistent with complete subunit independence. In contrast, the relative occupancies in subsaturating glutamate indicate that the channel switches between a low open probability mode and a high open probability mode in which the behavior of the channel is identical to that in saturating glutamate. These gating modes occur at both negative and positive potentials, with the high open probability mode becoming more prominent at positive potentials. The switch between gating modes and its modulation by voltage and other factors may constitute a novel mechanism regulating AMPAR-mediated synaptic activity.