Ultrasound has been used to non-invasively manipulate neuronal functions in humans and other animals. However, this approach is limited as it has been challenging to target specific cells within the brain or body. Here, we identify human Transient Receptor Potential A1 (hsTRPA1) as a candidate that confers ultrasound sensitivity to mammalian cells. Ultrasound-evoked gating of hsTRPA1 specifically requires its N-terminal tip region and cholesterol interactions; and target cells with an intact actin cytoskeleton, revealing elements of the sonogenetic mechanism. Next, we use calcium imaging and electrophysiology to show that hsTRPA1 potentiates ultrasound-evoked responses in primary neurons. Furthermore, unilateral expression of hsTRPA1 in mouse layer V motor cortical neurons leads to c-fos expression and contralateral limb responses in response to ultrasound delivered through an intact skull. Collectively, we demonstrate that hsTRPA1-based sonogenetics can effectively manipulate neurons within the intact mammalian brain, a method that could be used across species.

Our understanding of the nervous system has been fundamentally advanced by light- and small molecule-sensitive proteins that can be used to modify neuronal excitability. However, optogenetics requires invasive instrumentation while chemogenetics lacks temporal control. Here, we identify a candidate channel that confers sensitivity to non-invasive ultrasound on millisecond timescales. Using a functional screen, we find that human Transient Receptor Potential A1 (hsTRPA1) increases ultrasound-evoked intracellular calcium levels and membrane potentials. Ultrasound, but not agonist, -evoked, gating of hsTRPA1, requires the N-terminal tip region, intact actin cytoskeleton, and cholesterol, implicating these features in the sonogenetic mechanism. We then use calcium imaging and electrophysiology to confirm that ultrasound-evoked responses of primary neurons are potentiated by hsTRPA1. We also show that unilateral expression of hsTRPA1 in mouse layer V motor cortical neurons leads to ultrasound-evoked contralateral limb responses to ultrasound delivered through an intact skull. Finally, ultrasound induces c-fos in hsTRPA1-expressing neurons, suggesting that our approach can be used for targeted activation of neural circuits. Together, our results demonstrate that hsTRPA1-based sonogenetics can effectively and non-invasively modulate neurons within the intact mammalian brain, a method that could be extended to other cell types across species.

Ultrasound has been used to manipulate cells in both humans and animal models. While intramembrane cavitation and lipid clustering have been suggested as likely mechanisms, they lack experimental evidence. Here, high‐speed digital holographic microscopy (kiloHertz order) is used to visualize the cellular membrane dynamics. It is shown that neuronal and fibroblast membranes deflect about 150 nm upon ultrasound stimulation. Next, a biomechanical model that predicts changes in membrane voltage after ultrasound exposure is developed. Finally, the model predictions are validated using whole‐cell patch clamp electrophysiology on primary neurons. Collectively, it is shown that ultrasound stimulation directly defects the neuronal membrane leading to a change in membrane voltage and subsequent depolarization. The model is consistent with existing data and provides a mechanism for both ultrasound‐evoked neurostimulation and sonogenetic control.

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Ultrasound has been used to manipulate cells in both humans and animal models. While intramembrane cavitation and lipid clustering have been suggested as likely mechanisms, they lack experimental evidence. Here we use high-speed digital holographic microscopy (to 100-kHz order) to visualize the cellular membrane dynamics. We show that neuronal and fibroblast membranes deflect about 150 nm upon ultrasound stimulation. Next, we develop a biomechanical model that predicts changes in membrane voltage after ultrasound exposure. Finally, we validate our model predictions using whole-cell patch clamp electrophysiology on primary neurons. Collectively, we show that ultrasound stimulation directly defects the neuronal membrane leading to a change in membrane voltage and subsequent depolarization. Our model is consistent with existing data and provides a mechanism for both ultrasound-evoked neurostimulation and sonogenetic control.

Ultrasound neuromodulation has rapidly developed over the past decade, a consequence of the discovery of strain‐sensitive structures in the membrane and organelles of cells extending into the brain, heart, and other organs. A key limitation to its use in the brain is the formation of standing waves within the skull. In standing acoustic waves, the maximum ultrasound intensity spatially varies from near zero to double the mean in one‐half of a wavelength, and has led to localized tissue damage and disruption of normal brain function while attempting to evoke a broader response. This phenomenon also produces a large spatial variation in the actual ultrasound exposure in tissue, leading to heterogeneous results. One approach to overcome this limitation is presented here: transducer‐mounted diffusers that result in spatiotemporally incoherent ultrasound. It is shown through experiment and analysis that adding a diffuser to the transducer leads to a twofold increase in ultrasound responsiveness of transient receptor potential ankyrin 1 (TRPA1)‐transfected human embryonic kidney cells. Furthermore, it is shown that the diffuser produces a uniform spatial distribution of pressure within the rodent skull. The approach offers uniform ultrasound delivery into irregular cavities for sonogenetics.

Rapid-acting antidepressants like ketamine hold promise to change the approach to treatment of major depressive disorder (MDD), but their cellular and molecular targets remain unclear. Passivity induced by behavioral futility underlies learned helplessness, a process that becomes maladaptive in MDD. Antidepressants decrease futility-induced passivity (FIP) in rodent models such as the forced swimming or tail suspension tasks, but these models lack the throughput and accessibility for screening compounds and investigating their effects on the brain in vivo. Therefore, we adapted a recently discovered FIP behavior in the small and optically accessible larval zebrafish to create a scalable behavioral assay for antidepressant action. We found that rapid-acting antidepressants with diverse pharmacological targets demonstrated a suppression of FIP conserved between fish and rodents. While fast-acting antidepressants are thought to primarily target neurons, using brain-wide imaging in vivo we found, surprisingly, that ketamine, but not psychedelics or typical antidepressants, drove cytosolic calcium elevation in astroglia lasting many minutes. Blocking neural activity did not prevent ketamine's effects on FIP or astroglial calcium, suggesting an astroglia-autonomous mechanism of ketamine's action. Chemogenetic and optogenetic perturbations of astroglia reveal that the aftereffects of calcium elevation are sufficient to suppress FIP by inhibiting astroglial integration of futile swimming. In sum, our work provides evidence that ketamine exerts its antidepressant effects by inhibiting an astroglial population that integrates futility and changes behavioral state. Astroglia play central roles in modulating circuit dynamics, and our work argues that targeting astroglial signaling may be a fruitful strategy for designing new rapid-acting antidepressants.

The field of ultrasound neuromodulation has rapidly developed over the past decade, a consequence of the discovery of strain-sensitive structures in the membrane and organelles of cells extending into the brain, heart, and other organs. Notably, clinical trials are underway for treating epilepsy using focused ultrasound to elicit an organized local electrical response. A key limitation to this approach is the formation of standing waves within the skull. In standing acoustic waves, the maximum ultrasound intensity spatially varies from near zero to double the mean in one half a wavelength, and can lead to localized tissue damage and disruption of normal brain function while attempting to evoke a broader response. This phenomenon also produces a large spatial variation in the actual ultrasound exposure in tissue, leading to heterogeneous results and challenges with interpreting these effects. One approach to overcome this limitation is presented herein: transducer-mounted diffusers that result in spatiotemporally incoherent ultrasound. The signal is numerically and experimentally quantified in an enclosed domain with and without the diffuser. Specifically, we show that adding the diffuser leads to a two-fold increase in ultrasound responsiveness of hsTRPA1 transfected HEK cells. Furthermore, we demonstrate the diffuser allow us to produce an uniform spatial distribution of pressure in the rodent skull. Collectively, we propose that our approach leads to a means to deliver uniform ultrasound into irregular cavities for sonogenetics.