Nanobubble-actuated ultrasound neuromodulation for selectively shaping behavior in mice.

Ultrasound is an acoustic wave which can noninvasively penetrate the skull to deep brain regions, enabling neuromodulation. However, conventional ultrasound's spatial resolution is diffraction-limited and low-precision. Here, we report acoustic nanobubble-mediated ultrasound stimulation capable of localizing ultrasound's effects to only the desired brain region in male mice. By varying the delivery site of nanobubbles, ultrasound could activate specific regions of the mouse motor cortex, evoking EMG signaling and limb movement, and could also, separately, activate one of two nearby deep brain regions to elicit distinct behaviors (freezing or rotation). Sonicated neurons displayed reversible, low-latency calcium responses and increased c-Fos expression in the sub-millimeter-scale region with nanobubbles present. Ultrasound stimulation of the relevant region also modified depression-like behavior in a mouse model. We also provide evidence of a role for mechanosensitive ion channels. Altogether, our treatment scheme allows spatially-targetable, repeatable and temporally-precise activation of deep brain circuits for neuromodulation without needing genetic modification.

Ultra-High Frequency Self-Focusing Ultrasonic Sensors With Half-Concave Geometry for Visualization of Mouse Brain Atrophy.

Ultra-high frequency (>100 MHz) acoustic waves feature biocompatibility and high sensitivity and allow biomedical imaging and acoustic tweezers. Primarily, excellent spatial resolution and broad bandwidth at ultra-high frequency is the goal for pathological research and cell selection at the cellular level. Here, we propose an efficient approach to visualize mouse brain atrophy by self-focused ultrasonic sensors at ultra-high frequency with ultra-broad bandwidth. The numerical models of geometry and theoretically predicted acoustic parameters for half-concave piezoelectric elements are calculated by the differential method, which agrees with measured results (lateral resolution: 24 µm, and bandwidth: 115% at -6 dB). Compared with the brain slices of 2-month-old mouse, the atrophy visualization of the 6-month-old mouse brain was realized by C-mode imaging with an acoustic microscopy system, which is a potential prospect for diagnosis and treatment of Alzheimer's disease (AD) combined with neuroscience. Meanwhile, the acoustic properties of the brain slices were quantitatively measured by the acoustic microscopy. These encouraging results demonstrate the promising application for high-resolution imaging in vitro biological tissue with ultra-high frequency self-focusing ultrasonic sensors.

Modulation of deep neural circuits with sonogenetics.

Noninvasive control of neuronal activity in the deep brain can be illuminating for probing brain function and treating dysfunctions. Here, we present a sonogenetic approach for controlling distinct mouse behavior with circuit specificity and subsecond temporal resolution. Targeted neurons in subcortical regions were made to express a mutant large conductance mechanosensitive ion channel (MscL-G22S), enabling ultrasound to trigger activity in MscL-expressing neurons in the dorsal striatum and increase locomotion in freely moving mice. Ultrasound stimulation of MscL-expressing neurons in the ventral tegmental area could activate the mesolimbic pathway to trigger dopamine release in the nucleus accumbens and modulate appetitive conditioning. Moreover, sonogenetic stimulation of the subthalamic nuclei of Parkinson's disease model mice improved their motor coordination and mobile time. Neuronal responses to ultrasound pulse trains were rapid, reversible, and repeatable. We also confirmed that the MscL-G22S mutant is more effective to sensitize neurons to ultrasound compared to the wild-type MscL. Altogether, we lay out a sonogenetic approach which can selectively manipulate targeted cells to activate defined neural pathways, affect specific behaviors, and relieve symptoms of neurodegenerative disease.

The mechanosensitive ion channel Piezo1 contributes to ultrasound neuromodulation.

Transcranial low-intensity ultrasound is a promising neuromodulation modality, with the advantages of noninvasiveness, deep penetration, and high spatiotemporal accuracy. However, the underlying biological mechanism of ultrasonic neuromodulation remains unclear, hindering the development of efficacious treatments. Here, the well-known Piezo1 was studied through a conditional knockout mouse model as a major mediator for ultrasound neuromodulation ex vivo and in vivo. We showed that Piezo1 knockout (P1KO) in the right motor cortex of mice significantly reduced ultrasound-induced neuronal calcium responses, limb movement, and muscle electromyogram (EMG) responses. We also detected higher Piezo1 expression in the central amygdala (CEA), which was found to be more sensitive to ultrasound stimulation than the cortex was. Knocking out the Piezo1 in CEA neurons showed a significant reduction of response under ultrasound stimulation, while knocking out astrocytic Piezo1 showed no-obvious changes in neuronal responses. Additionally, we excluded an auditory confound by monitoring auditory cortical activation and using smooth waveform ultrasound with randomized parameters to stimulate P1KO ipsilateral and contralateral regions of the same brain and recording evoked movement in the corresponding limb. Thus, we demonstrate that Piezo1 is functionally expressed in different brain regions and that it is an important mediator of ultrasound neuromodulation in the brain, laying the ground for further mechanistic studies of ultrasound.

Measuring sound velocity based on acoustic resonance using multiple narrow band transducers.

The sound velocity in a medium is closely related to its material properties, including its composition, structure, density, pressure, and temperature. Various methods have been developed to determine the sound velocity through materials. Among them, a strategy based on ultrasound resonance frequency has been most widely used due to the simplicity. However, it requires a transducer with a wide bandwidth to cover enough resonance frequencies to perform the consequent calculations. In this paper, we develop a resonance method for measuring sound velocity, using multi-frequency narrow-band transducers breaking through the limitation of transducer bandwidth on the utilization of the resonance method. We use different transducers at different center frequencies and with different bandwidth to measure the sound velocity in 100-µm and 400-µm thick steel pieces. The measurement results of different combinations are in good agreement, verifying that the use of multi-frequency narrow-band transducer combinations. Given that most therapeutic transducers have a narrow bandwidth, this method can be used during intracranial ultrasound stimulation to optimize targeting by non-invasively measuring the sound velocity in the skull, especially at thinner locations.

A tool for monitoring cell type-specific focused ultrasound neuromodulation and control of chronic epilepsy.

Focused ultrasound (FUS) is a powerful tool for noninvasive modulation of deep brain activity with promising therapeutic potential for refractory epilepsy; however, tools for examining FUS effects on specific cell types within the deep brain do not yet exist. Consequently, how cell types within heterogeneous networks can be modulated and whether parameters can be identified to bias these networks in the context of complex behaviors remains unknown. To address this, we developed a fiber Photometry Coupled focused Ultrasound System (PhoCUS) for simultaneously monitoring FUS effects on neural activity of subcortical genetically targeted cell types in freely behaving animals. We identified a parameter set that selectively increases activity of parvalbumin interneurons while suppressing excitatory neurons in the hippocampus. A net inhibitory effect localized to the hippocampus was further confirmed through whole brain metabolic imaging. Finally, these inhibitory selective parameters achieved significant spike suppression in the kainate model of chronic temporal lobe epilepsy, opening the door for future noninvasive therapies.

Sonogenetics: Recent advances and future directions.

Sonogenetics refers to the use of genetically encoded, ultrasound-responsive mediators for noninvasive and selective control of neural activity. It is a promising tool for studying neural circuits. However, due to its infancy, basic studies and developments are still underway, including gauging key in vivo performance metrics such as spatiotemporal resolution, selectivity, specificity, and safety. In this paper, we summarize recent findings on sonogenetics to highlight technical hurdles that have been cleared, challenges that remain, and future directions for optimization.

Associations of Parenting Style and Resilience With Depression and Anxiety Symptoms in Chinese Middle School Students.

Background: Parenting style and resilience are independently associated with symptoms of depression and anxiety. However, no study has tested the interaction effects between the patterns of parenting style and resilience on mental health in adolescent populations. Therefore, this study aimed to explore the interaction effects between the patterns of parenting style and resilience on depression/anxiety symptoms among middle school students in China. Methods: A sample of 2,179 Chinese middle school students were included in this study. Latent profile analysis (LPA) was used to examine parenting style patterns. Multivariable logistic regression was used to analyze the associations of different parenting patterns and resilience with depression/anxiety symptoms, as well as the interaction effect. Results: Latent profile analysis results showed that the most suitable model included three-profile solution, which were labeled as positive parenting, negative parenting, and moderate parenting. Subsequent analyses indicated that students across profiles exhibited significant differences in their depression/anxiety symptoms. Specifically, compared to moderate parenting, negative parenting was positively associated with depression/anxiety symptoms, while positive parenting was negatively associated with these symptoms. Moreover, low levels of resilience were positively associated with depression/anxiety symptoms compared to a high level of resilience. Although the interaction effect was not significant, there were differences in the associations between different parenting patterns and symptoms of depression and anxiety when stratifying resilience. Conclusion: The present study identified three-profile solution of parenting styles among Chinese middle school students using LPA as a person-centered approach. Future interventions targeting depression/anxiety symptoms in adolescents may consider the potential influence of patterns of parenting styles, or improved resilience, to achieve better intervention outcomes.

An Optimized Miniaturized Ultrasound Transducer for Transcranial Neuromodulation.

Transcranial ultrasound stimulation (TUS) is a young neuromodulation technology, which uses ultrasound to achieve non-invasive stimulation or inhibition of deep intracranial brain regions, with the advantages of non-invasive, deep penetration, and high resolution. It is widely considered to be one of the most promising techniques for probing brain function and treating brain diseases. In preclinical studies, developing miniaturized transducers to facilitate neuromodulation in freely moving small animals is critical for understanding the mechanism and exploring potential applications. In this article, a miniaturized transducer with a half-concave structure is proposed. Based on the finite element simulation models established by PZFlex software, several ultrasound transducers with different concave curvatures were designed and analyzed. Based on the simulation results, half-concave focused ultrasonic transducers with curvature radii of 5 mm and 7.5 mm were fabricated. Additionally, the emission acoustic fields of the ultrasonic transducers with different structures were characterized at their thickness resonance frequencies of 1 MHz using a multifunctional ultrasonic test platform built in the laboratory. To verify the practical ability for neuromodulation, different ultrasound transducers were used to induce muscle activity in mice. As a result, the stimulation success rates were (32 ± 10)%, (65 ± 8)%, and (84 ± 7)%, respectively, by using flat, #7, and #5 transducers, which shows the simulation and experimental results have a good agreement and that the miniaturized half-concave transducer could effectively converge the acoustic energy and achieve precise and effective ultrasonic neuromodulation.

Photonic Nanojet-Mediated Optogenetics.

Optogenetics has become a widely used technique in neuroscience research, capable of controlling neuronal activity with high spatiotemporal precision and cell-type specificity. Expressing exogenous opsins in the selected cells can induce neuronal activation upon light irradiation, and the activation depends on the power of incident light. However, high optical power can also lead to off-target neuronal activation or even cell damage. Limiting the incident power, but enhancing power distribution to the targeted neurons, can improve optogenetic efficiency and reduce off-target effects. Here, the use of optical lenses made of polystyrene microspheres is demonstrated to achieve effective focusing of the incident light of relatively low power to neighboring neurons via photonic jets. The presence of microspheres significantly localizes and enhances the power density to the target neurons both in vitro and ex vivo, resulting in increased inward current and evoked action potentials. In vivo results show optogenetic stimulation with microspheres that can evoke significantly more motor behavior and neuronal activation at lowered power density. In all, a proof-of-concept of a strategy is demonstrated to increase the efficacy of optogenetic neuromodulation using pulses of reduced optical power.

Evolvable Acoustic Field Generated by a Transducer with 3D-Printed Fresnel Lens.

Evolvable acoustic fields are considered an effective method for solving technical problems related to fields such as biological imaging, particle manipulation, drug therapy and intervention. However, because of technical difficulties and the limited technology available for realizing flexible adjustments of sound fields, few studies have reported on this aspect in recent years. Herein, we propose a novel solution, using a Fresnel lens-focused ultrasonic transducer for generating excited-signal-dependent acoustic pressure patterns. Finite element analysis (FEA) is used to predict the performance of a transducer with a Fresnel lens. The Fresnel lens is printed using 3D additive manufacturing. Normalized intensity maps of the acoustic pressure fields are characterized from the Fresnel lens-focused transducer under various numbers of excited-signal cycles. The results demonstrate that under different cycle excitations, a temporal evolution acoustic intensity can be generated and regulated by an ultrasound transducer with a 3D Fresnel lens. This acoustical pattern control method is not only simple to realize but also has considerable application prospects.

Gas-filled protein nanostructures as cavitation nuclei for molecule-specific sonodynamic therapy.

Sonodynamic therapy (SDT) is a promising alternative for cancer therapy, understood to exert cytotoxicity through cavitation and subsequent production of large amounts of reactive oxygen species (ROS). Gas-filled protein nanostructures (gas vesicles or GVs) produced by cyanobacteria have a hollow structure similar to microbubbles and have demonstrated comparable enhancement of ultrasound imaging contrast. We thus hypothesized that GVs may act as stable nuclei for inertial cavitation to enhance SDT with improved enhanced permeability and retention (EPR) effects due to their nanometer scale. The function of GVs to mediate cavitation, ROS production, and cell-targeted toxicity under SDT was determined. In solution, we found that GVs successfully increased cavitation and enhanced ROS production in a dose- and time-dependent manner. Then, GV surfaces were modified (FGVs) to specifically target CD44+ cells and accumulate preferentially at the tumor site. In vitro sonodynamic therapy (SDT) showed ROS production and tumor cell toxicity substantially elevated in the presence of FGVs, and the addition of FGVs was found to enhance cavitation and subsequently inhibit tumor growth and exert greater damage to tumors under SDT in vivo. Our results thus demonstrate that FGVs can function as stable, nanosized, nuclei for spatially accurate and cell-targeted SDT. STATEMENT OF SIGNIFICANCE: The initiation of inertial cavitation is critical for ROS generation and subsequent cellular toxicity in SDT. Thus, precise control of the occurrence of cavitation is a key factor in increasing SDT's therapeutic efficacy. We explored nanometer-sized gas vesicles (GVs) as a new class of cavitation nuclei for molecule-specific sonodynamic therapy. Our results showed that GV-mediated SDT treatment enabled targeted disruption of specific cells expressing a known surface marker within the area of insonation, providing a spatially specific and targeted SDT treatment.

Precise Ultrasound Neuromodulation in a Deep Brain Region Using Nano Gas Vesicles as Actuators.

Ultrasound is a promising new modality for non-invasive neuromodulation. Applied transcranially, it can be focused down to the millimeter or centimeter range. The ability to improve the treatment's spatial resolution to a targeted brain region could help to improve its effectiveness, depending upon the application. The present paper details a neurostimulation scheme using gas-filled nanostructures, gas vesicles (GVs), as actuators for improving the efficacy and precision of ultrasound stimuli. Sonicated primary neurons display dose-dependent, repeatable Ca2+ responses, closely synced to stimuli, and increased nuclear expression of the activation marker c-Fos in the presence of GVs. GV-mediated ultrasound triggered rapid and reversible Ca2+ responses in vivo and could selectively evoke neuronal activation in a deep-seated brain region. Further investigation indicate that mechanosensitive ion channels are important mediators of this effect. GVs themselves and the treatment scheme are also found not to induce significant cytotoxicity, apoptosis, or membrane poration in treated cells. Altogether, this study demonstrates a simple and effective method to achieve enhanced and better-targeted neurostimulation with non-invasive low-intensity ultrasound.

Protocol for the sonogenetic stimulation of mouse brain by non-invasive ultrasound.

Manipulating specific neural activity by targeted ultrasound intervention is a powerful method to gain causal insight into brain functions and treat brain disorders. The technique of sonogenetics enables controlling of cells that are genetically modulated with ultrasound-sensitive ion channels. Here, we detail the preparations, surgical procedures, ultrasound stimulation process, and simultaneous electromyogram (EMG) measurement necessary for successful sonogenetic stimulation in mice. For complete details on the use and execution of this protocol, please refer to Qiu et al. (2020).

Behavioral and Functional Assessment of Ultrasound Neuromodulation on Caenorhabditis Elegans.

Ultrasound brain stimulation is a promising modality for probing brain function and treating brain diseases. However, its mechanism is as yet unclear, and in vivo effects are not well-understood. Here, we present a top-down strategy for assessing ultrasound bioeffects in vivo, using Caenorhabditis elegans. Behavioral and functional changes of single worms and of large populations upon ultrasound stimulation were studied. Worms were observed to significantly increase their average speed upon ultrasound stimulation, adapting to it upon continued treatment. Worms also generated more reversal turns when ultrasound was ON, and within a minute post-stimulation, they performed significantly more reversal and omega turns than prior to ultrasound. In addition, in vivo calcium imaging showed that the neural activity in the worms' heads and tails was increased significantly by ultrasound stimulation. In all, we conclude that ultrasound can directly activate the neurons of worms in vivo, in both of their major neuronal ganglia, and modify their behavior.

The forbidden band and size selectivity of acoustic radiation force trapping.

Acoustic micro-beams produced by highly focused ultrasound transducer have been investigated for micro-particle and cell manipulation. Here we report the selective trapping of microspheres via the acoustic force using the single acoustical beam. The forbidden band theory of acoustic radiation force trapping is proposed, which indicates that the trapping of particles via the acoustic beam is directly related to the particle diameter-to-beam wavelength ratio as well as excitation frequency of the ultrasonic acoustic tweezers. Three tightly focused LiNbO3 transducers with different center frequencies were fabricated for use as selective single beam acoustic tweezers (SBATs). These SBATs were capable of selectively manipulating microspheres of sizes 5-45 µm by adjusting the wavelength of acoustic beam. Our observations could introduce new avenues for research in biology and biophysics by promoting the development of a tool for selectively manipulating microspheres or cells of certain selected sizes, by carefully setting the acoustic beam shape and wavelength.

Targeted Neurostimulation in Mouse Brains with Non-invasive Ultrasound.

Recently developed brain stimulation techniques have significantly advanced our ability to manipulate the brain's function. However, stimulating specific neurons in a desired region without significant surgical invasion remains a challenge. Here, we demonstrate a neuron-specific and region-targeted neural excitation strategy using non-invasive ultrasound through activation of heterologously expressed mechanosensitive ion channels (MscL-G22S). Low-intensity ultrasound is significantly better at inducing Ca2+ influx and neuron activation in vitro and at evoking electromyogram (EMG) responses in vivo in targeted cells expressing MscL-G22S. Neurons in the cerebral cortex or dorsomedial striatum of mice are made to express MscL-G22S and stimulated ultrasonically. We find significant upregulation of c-Fos in neuron nuclei only in the regions expressing MscL-G22S compared with the non-MscL controls, as well as in various other regions in the same brain. Thus, we detail an effective approach for activating specific regions and cell types in intact mouse brains by sensitizing them to ultrasound using a mechanosensitive ion channel.

Biogenic nanobubbles for effective oxygen delivery and enhanced photodynamic therapy of cancer.

Tumor hypoxia is believed to be a factor limiting successful outcomes of oxygen-consuming cancer therapy, thereby reducing patient survival. A key strategy to overcome tumor hypoxia is to increase the prevalence of oxygen at the tumor site. Oxygen-containing microbubbles/nanobubbles have been developed to supply oxygen and enhance the effects of therapies such as radiotherapy and photodynamic therapy. However, the application of these bubbles is constrained by their poor stability, requiring major workarounds to increase their half-lives. In this study, we explore the potential of biogenic gas vesicles (GVs) as a new kind of oxygen carrier to alleviate tumor hypoxia. GVs, which are naturally formed, gas-filled, protein-shelled compartments, were modified on the surface of their protein shells by a layer of liposome. A substantial improvement of oxygen concentration was observed in hypoxic solution, in hypoxic cells, as well as in subcutaneous tumors when lipid-GVs(O2) were added/tail-injected. Significant enhancement of tumor cell apoptosis and necrosis was also observed during photodynamic therapy (PDT) in the presence of lipid-GVs(O2) both in vitro and in vivo. Lipid-GVs(O2) alone induced no obvious change in cell viability in vitro or any apparent pathological abnormalities after mice were tail-injected with them. In all, lipid-GVs exhibited promising performance for intravenous gas delivery, enhanced PDT efficacy and low toxicity, a quality that may be applied to alleviate hypoxia in cancers, as well as hypoxia-related clinical treatments. STATEMENT OF SIGNIFICANCE: The development of stable oxygen-filled micro/nanobubbles capable of delivering oxygen to tumor sites is a major hurdle to enhancing the efficacy of cancer therapy. Currently, micro/nanobubbles are limited by their instability when oxygen is encapsulated, creating a large pressure gradient and surface tension. To improve stability, we modified the surfaces of GVs, a biogenic stable nanoscale hollow structure, as a new class of oxygen carriers. Lipid-coated GVs were found to be stable in solution and effective O2 carriers. This will overcome the limitations of coalescence, short circulation time of synthetic bubbles during application. Our surface-modified GVs demonstrated low toxicity in vitro cell in vivo, while also being able to overcome hypoxia-associated therapy resistance when combined with photodynamic therapy.