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.

Activation of N-Methyl-D-aspartate receptor contributed to the ultrasonic modulation of neurons in vitro.

Ultrasound stimulation is increasingly used to investigate brain function and treat brain diseases due to its high level of safety and precise spatiotemporal resolution. Therefore, it is crucial to understand the underlying mechanisms involved in ultrasound brain stimulation. In this study, we investigate the role of NMDA receptors in mediating the effects of ultrasound on primary hippocampal neurons in mice. Our results show that ultrasound alone can activate heterologous NMDA receptor subunits, including NR1A, NR2A, and NR2B, in 293T cells, as well as endogenous NMDA receptors in primary neurons. This activation leads to an influx of calcium and an increase in nuclear c-Fos expression in primary neurons that have not been pre-treated with an NMDA receptor inhibitor. In conclusion, our findings demonstrate that NMDA receptors contribute to neuronal activation by ultrasound stimulation in vitro, providing insight into the molecular mechanisms of ultrasound neuromodulation and a new mediator for the sonogenetics technique.

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.

The mechanosensitive ion channel Piezo1 modulates the migration and immune response of microglia.

Microglia are the brain's resident immune cells, performing surveillance to promote homeostasis and healthy functioning. While microglial chemical signaling is well-studied, mechanical cues regulating their function are less well-understood. Here, we investigate the role of the mechanosensitive ion channel Piezo1 in microglia migration, pro-inflammatory cytokine production, and stiffness sensing. In Piezo1 knockout transgenic mice, we demonstrated the functional expression of Piezo1 in microglia and identified genes whose expression was consequently affected. Functional assays revealed that Piezo1 deficiency in microglia enhanced migration toward amyloid ß-protein, and decreased levels of pro-inflammatory cytokines produced upon stimulation by lipopolysaccharide, both in vitro and in vivo. The phenomenon could be mimicked or reversed chemically using a Piezo1-specific agonist or antagonist. Finally, we also showed that Piezo1 mediated the effect of substrate stiffness-induced migration and cytokine expression. Altogether, we show that Piezo1 is an important molecular mediator for microglia, its activation modulating microglial migration and immune responses.

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.

Metformin accelerates zebrafish heart regeneration by inducing autophagy.

Metformin is one of the most widely used drugs for type 2 diabetes and it also exhibits cardiovascular protective activity. However, the underlying mechanism of its action is not well understood. Here, we used an adult zebrafish model of heart cryoinjury, which mimics myocardial infarction in humans, and demonstrated that autophagy was significantly induced in the injured area. Through a systematic evaluation of the multiple cell types related to cardiac regeneration, we found that metformin enhanced the autophagic flux and improved epicardial, endocardial and vascular endothelial regeneration, accelerated transient collagen deposition and resolution, and induced cardiomyocyte proliferation. Whereas, when the autophagic flux was blocked, then all these processes were delayed. We also showed that metformin transiently enhanced the systolic function of the heart. Taken together, our results indicate that autophagy is positively involved in the metformin-induced acceleration of heart regeneration in zebrafish and suggest that this well-known diabetic drug has clinical value for the prevention and amelioration of myocardial infarction.

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.

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.

Surface-modified GVs as nanosized contrast agents for molecular ultrasound imaging of tumor.

Nanobubbles, as a kind of new ultrasound contrast agent (UCAs), have shown promise to penetrate tumor vasculature to allow for targeted imaging. However, their inherent physical instability is an ongoing concern that could weaken their imaging ability with ultrasound. Gas vesicles (GVs), which are genetically encoded, naturally stable nanostructures, have been developed as the first ultrasonic biomolecular reporters which showed strong contrast enhancement. However, further development of tumor imaging with GVs is limited by the quick clearance of GVs by the reticuloendothelial system (RES). Here, we developed PEGylated HA-GVs (PH-GVs) for in-tumor molecular ultrasound imaging by integrating polyethylene glycol (PEG) and hyaluronic acid (HA) in GV shells. PH-GVs were observed to accumulate around CD44-positive cells (SCC7) but not be internalized by macrophage cell line RAW 264.7. Green fluorescence from PH-GVs was found around cell nuclei in the tumor site after 6 h and the signal was sustained over 48 h following tail injection, demonstrating PH-GVs' ability to escape the clearance from the RES and to penetrate tumor vasculature through enhanced permeability and retention (EPR) effects. Further, PH-GVs produced strong ultrasound contrast in the tumor site in vivo, with no obvious side-effects detected following intravenous injection. Thus, we demonstrate the potential of PH-GVs as novel, nanosized and targeted UCAs for efficient and specific molecular tumor imaging, paving the way for the application of GVs in precise and personalized medicine.

Estrogen accelerates heart regeneration by promoting the inflammatory response in zebrafish.

Sexual differences have been observed in the onset and prognosis of human cardiovascular diseases, but the underlying mechanisms are not clear. Here, we found that zebrafish heart regeneration is faster in females, can be accelerated by estrogen and is suppressed by the estrogen-antagonist tamoxifen. Injuries to the zebrafish heart, but not other tissues, increased plasma estrogen levels and the expression of estrogen receptors, especially esr2a. The resulting endocrine disruption induces the expression of the female-specific protein vitellogenin in male zebrafish. Transcriptomic analyses suggested heart injuries triggered pronounced immune and inflammatory responses in females. These responses, previously shown to elicit heart regeneration, could be enhanced by estrogen treatment in males and reduced by tamoxifen in females. Furthermore, a prior exposure to estrogen preconditioned the zebrafish heart for an accelerated regeneration. Altogether, this study reveals that heart regeneration is modulated by an estrogen-inducible inflammatory response to cardiac injury. These findings elucidate a previously unknown layer of control in zebrafish heart regeneration and provide a new model system for the study of sexual differences in human cardiac repair.