Force-Based Neuromodulation.

Technologies for neuromodulation have rapidly developed in the past decade with a particular emphasis on creating noninvasive tools with high spatial and temporal precision. The existence of such tools is critical in the advancement of our understanding of neural circuitry and its influence on behavior and neurological disease. Existing technologies have employed various modalities, such as light, electrical, and magnetic fields, to interface with neural activity. While each method offers unique advantages, many struggle with modulating activity with high spatiotemporal precision without the need for invasive tools. One modality of interest for neuromodulation has been the use of mechanical force. Mechanical force encapsulates a broad range of techniques, ranging from mechanical waves delivered via focused ultrasound (FUS) to torque applied to the cell membrane.Mechanical force can be delivered to the tissue in two forms. The first form is the delivery of a mechanical force through focused ultrasound. Energy delivery facilitated by FUS has been the foundation for many neuromodulation techniques, owing to its precision and penetration depth. FUS possesses the potential to penetrate deeply (∼centimeters) into tissue while maintaining relatively precise spatial resolution, although there exists a trade-off between the penetration depth and spatial resolution. FUS may work synergistically with ultrasound-responsive nanotransducers or devices to produce a secondary energy, such as light, heat, or an electric field, in the target region. This layered technology, first enabled by noninvasive FUS, overcomes the need for bulky invasive implants and also often improves the spatiotemporal precision of light, heat, electrical fields, or other techniques alone. Conversely, the second form of mechanical force modulation is the generation of mechanical force from other modalities, such as light or magnetic fields, for neuromodulation via mechanosensitive proteins. This approach localizes the mechanical force at the cellular level, enhancing the precision of the original energy delivery. Direct interaction of mechanical force with tissue presents translational potential in its ability to interface with endogenous mechanosensitive proteins without the need for transgenes.In this Account, we categorize force-mediated neuromodulation into two categories: 1) methods where mechanical force is the primary stimulus and 2) methods where mechanical force is generated as a secondary stimulus in response to other modalities. We summarize the general design principles and current progress of each respective approach. We identify the key advantages of the limitations of each technology, particularly noting features in spatiotemporal precision, the need for transgene delivery, and the potential outlook. Finally, we highlight recent technologies that leverage mechanical force for enhanced spatiotemporal precision and advanced applications.

A Nanozyme-Based Electrode for High-Performance Neural Recording.

Implanted neural electrodes have been widely used to treat brain diseases that require high sensitivity and biocompatibility at the tissue-electrode interface. However, currently used clinical electrodes cannot meet both these requirements simultaneously, which hinders the effective recording of electronic signals. Herein, nanozyme-based neural electrodes incorporating bioinspired atomically precise clusters are developed as a general strategy with a heterogeneous design for multiscale and ultrasensitive neural recording via quantum transport and biocatalytic processes. Owing to the dual high-speed electronic and ionic currents at the electrode-tissue interface, the impedance of nanozyme electrodes is 26 times lower than that of state-of-the-art metal electrodes, and the acquisition sensitivity for the local field potential is ≈10 times higher than that of clinical PtIr electrodes, enabling a signal-to-noise ratio (SNR) of up to 14.7 dB for single-neuron recordings in rats. The electrodes provide more than 100-fold higher antioxidant and multi-enzyme-like activities, which effectively decrease 67% of the neuronal injury area by inhibiting glial proliferation and allowing sensitive and stable neural recording. Moreover, nanozyme electrodes can considerably improve the SNR of seizures in acute epileptic rats and are expected to achieve precise localization of seizure foci in clinical settings.

Activation of mechanoluminescent nanotransducers by focused ultrasound enables light delivery to deep-seated tissue in vivo.

Light is used extensively in biological and medical research for optogenetic neuromodulation, fluorescence imaging, photoactivatable gene editing and light-based therapies. The major challenge to the in vivo implementation of light-based methods in deep-seated structures of the brain or of internal organs is the limited penetration of photons in biological tissue. The presence of light scattering and absorption has resulted in the development of invasive techniques such as the implantation of optical fibers, the insertion of endoscopes and the surgical removal of overlying tissues to overcome light attenuation and deliver it deep into the body. However, these procedures are highly invasive and make it difficult to reposition and adjust the illuminated area in each animal. Here, we detail a noninvasive approach to deliver light (termed 'deLight') in deep tissue via systemically injected mechanoluminescent nanotransducers that can be gated by using focused ultrasound. This approach achieves localized light emission with sub-millimeter resolution and millisecond response times in any vascularized organ of living mice without requiring invasive implantation of light-emitting devices. For example, deLight enables optogenetic neuromodulation in live mice without a craniotomy or brain implants. deLight provides a generalized method for applications that require a light source in deep tissues in vivo, such as deep-brain fluorescence imaging and photoactivatable genome editing. The implementation of the entire protocol for an in vivo application takes ~1-2 weeks.

Wireless deep-brain neuromodulation using photovoltaics in the second near-infrared spectrum.

Conventional electrical neuromodulation techniques are constrained by the need for invasive implants in neural tissues, whereas methods using optogenetic are subjected to genetic alterations and hampered by the poor tissue penetration of visible light. Photovoltaic neuromodulation using light from the second near-infrared (NIR-II) spectrum, which minimizes scattering and enhances tissue penetration, shows promise as an alternative to existing neuromodulation technologies. NIR-II light has been used in deep-tissue imaging and in deep-brain photothermal neuromodulation via nanotransducers. This Perspective will provide an overview for the underpinning mechanisms of photovoltaic neuromodulation and identify avenues for future research in materials science and bioengineering that can further advance NIR-II photovoltaic neuromodulation methods.

Laminin-coated electronic scaffolds with vascular topography for tracking and promoting the migration of brain cells after injury.

In the adult brain, neural stem cells are largely restricted into spatially discrete neurogenic niches, and hence areas of neuron loss during neurodegenerative disease or following a stroke or traumatic brain injury do not typically repopulate spontaneously. Moreover, understanding neural activity accompanying the neural repair process is hindered by a lack of minimally invasive devices for the chronic measurement of the electrophysiological dynamics in damaged brain tissue. Here we show that 32 individually addressable platinum microelectrodes integrated into laminin-coated branched polymer scaffolds stereotaxically injected to span a hydrogel-filled cortical lesion and deeper regions in the brains of mice promote neural regeneration while allowing for the tracking of migrating host brain cells into the lesion. Chronic measurements of single-unit activity and neural-circuit analyses revealed the establishment of spiking activity in new neurons in the lesion and their functional connections with neurons deeper in the brain. Electronic implants mimicking the topographical and surface properties of brain vasculature may aid the stimulation and tracking of neural-circuit restoration following injury.

Direct-print three-dimensional electrodes for large- scale, high-density, and customizable neural inter- faces.

Silicon-based planar microelectronics is a powerful tool for scalably recording and modulating neural activity at high spatiotemporal resolution, but it remains challenging to target neural structures in three dimensions (3D). We present a method for directly fabricating 3D arrays of tissue-penetrating microelectrodes onto silicon microelectronics. Leveraging a high-resolution 3D printing technology based on 2-photon polymerization and scalable microfabrication processes, we fabricated arrays of 6,600 microelectrodes 10-130 µm tall and at 35-µm pitch onto a planar silicon-based microelectrode array. The process enables customizable electrode shape, height and positioning for precise targeting of neuron populations distributed in 3D. As a proof of concept, we addressed the challenge of specifically targeting retinal ganglion cell (RGC) somas when interfacing with the retina. The array was customized for insertion into the retina and recording from somas while avoiding the axon layer. We verified locations of the microelectrodes with confocal microscopy and recorded high-resolution spontaneous RGC activity at cellular resolution. This revealed strong somatic and dendritic components with little axon contribution, unlike recordings with planar microelectrode arrays. The technology could be a versatile solution for interfacing silicon microelectronics with neural structures and modulating neural activity at large scale with single-cell resolution.

Nanotransducer-Enabled Deep-Brain Neuromodulation with NIR-II Light.

The second near-infrared window (NIR-II window), which ranges from 1000 to 1700 nm in wavelength, exhibits distinctive advantages of reduced light scattering and thus deep penetration in biological tissues in comparison to the visible spectrum. The NIR-II window has been widely employed for deep-tissue fluorescence imaging in the past decade. More recently, deep-brain neuromodulation has been demonstrated in the NIR-II window by leveraging nanotransducers that can efficiently convert brain-penetrant NIR-II light into heat. In this Perspective, we discuss the principles and potential applications of this NIR-II deep-brain neuromodulation technique, together with its advantages and limitations compared with other existing optical methods for deep-brain neuromodulation. We also point out a few future directions where the advances in materials science and bioengineering can expand the capability and utility of NIR-II neuromodulation methods.

An optimized bioluminescent substrate for non-invasive imaging in the brain.

Bioluminescence imaging (BLI) allows non-invasive visualization of cells and biochemical events in vivo and thus has become an indispensable technique in biomedical research. However, BLI in the central nervous system remains challenging because luciferases show relatively poor performance in the brain with existing substrates. Here, we report the discovery of a NanoLuc substrate with improved brain performance, cephalofurimazine (CFz). CFz paired with Antares luciferase produces greater than 20-fold more signal from the brain than the standard combination of D-luciferin with firefly luciferase. At standard doses, Antares-CFz matches AkaLuc-AkaLumine/TokeOni in brightness, while occasional higher dosing of CFz can be performed to obtain threefold more signal. CFz should allow the growing number of NanoLuc-based indicators to be applied to the brain with high sensitivity. Using CFz, we achieve video-rate non-invasive imaging of Antares in brains of freely moving mice and demonstrate non-invasive calcium imaging of sensory-evoked activity in genetically defined neurons.

Ultrasound-activated luminescence with color tunability enabled by mechanoluminescent colloids and perovskite quantum dots.

Ultrasound represents a wireless and non-contact route for energy delivery and device control, owing to its ability to propagate and focus in various mediums including biological tissue. Specifically, ultrasound-activated mechanoluminescence from a colloidal suspension of mechanoluminescent (ML) nanocrystals offers a wireless means to remotely control a light source, such as wirelessly addressing a multicolor display. However, the limited color purity and tunability, as well as the large sizes of conventional ML materials prevent their use in an ultrasound-mediated flexible color display. Here, we apply a biomineral-inspired suppressed dissolution approach to synthesize ML colloids with bright blue emission under ultrasound and small sizes down to 20 nm. In addition, we leverage the bandgap engineering strategy of all-inorganic perovskite quantum dots (PQDs) to achieve wavelength tunability of the mechanoluminescence of ML colloid/PQD composites. Remarkably, the ultrasound-activated emission of the ML colloid/PQD composites exhibits a highly saturated color gamut covering the entire visible spectrum. Based on these advantages, we assembled a pixel array composed of different ML colloid/PQD composites in a silicone elastomer and demonstrated the proof-of-concept of a flexible and wireless multicolor display with each pixel individually addressed by scanning focused ultrasound.

Ultrasound-Triggered In Situ Photon Emission for Noninvasive Optogenetics.

Optogenetics has revolutionized neuroscience understanding by allowing spatiotemporal control over cell-type specific neurons in neural circuits. However, the sluggish development of noninvasive photon delivery in the brain has limited the clinical application of optogenetics. Focused ultrasound (FUS)-derived mechanoluminescence has emerged as a promising tool for in situ photon emission, but there is not yet a biocompatible liquid-phase mechanoluminescence system for spatiotemporal optogenetics. To achieve noninvasive optogenetics with a high temporal resolution and desirable biocompatibility, we have developed liposome (Lipo@IR780/L012) nanoparticles for FUS-triggered mechanoluminescence in brain photon delivery. Synchronized and stable blue light emission was generated in solution under FUS irradiation due to the cascade reactions in liposomes. In vitro tests revealed that Lipo@IR780/L012 could be triggered by FUS for light emission at different stimulation frequencies, resulting in activation of opsin-expressing spiking HEK cells under the FUS irradiation. In vivo optogenetic stimulation further demonstrated that motor cortex neurons could be noninvasively and reversibly activated under the repetitive FUS irradiation after intravenous injection of lipid nanoparticles to achieve limb movements.

Principles and applications of sono-optogenetics.

Optogenetics has revolutionized neuroscience research through its spatiotemporally precise activation of specific neurons by illuminating light on opsin-expressing neurons. A long-standing challenge of in vivo optogenetics arises from the limited penetration depth of visible light in the neural tissue due to scattering and absorption of photons. To address this challenge, sono-optogenetics has been developed to enable spatiotemporally precise light production in a three-dimensional volume of neural tissue by leveraging the deep tissue penetration and focusing ability of ultrasound as well as circulation-delivered mechanoluminescent nanotransducers. Here, we present a comprehensive review of the sono-optogenetics method from the physical principles of ultrasound and mechanoluminescence to its emerging applications for unique neuroscience studies. We also discuss a few promising directions in which sono-optogenetics can make a lasting transformative impact on neuroscience research from the perspectives of mechanoluminescent materials, ultrasound-tissue interaction, to the unique neuroscience opportunities of "scanning optogenetics".

Protocol for wireless deep brain stimulation in freely behaving mice with infrared light.

Here, we present a protocol for deep brain stimulation in freely behaving mice using through-scalp wide-field illumination in the second near-infrared window (NIR-II). We first describe the injection of the TRPV1 (transient receptor potential cation channel subfamily V member 1)-expressing viruses and macromolecular infrared nanotransducers for deep brain stimulation (MINDS). We then detail NIR-II neuromodulation in a conditioned place preference test, followed by immunohistochemical studies. This approach is especially useful for tether-free deep brain stimulation in social interacting experiments involving multiple subjects. For complete details on the use and execution of this protocol, please refer to Wu et al. (2022).

Shedding light on neurons: optical approaches for neuromodulation.

Today's optical neuromodulation techniques are rapidly evolving, benefiting from advances in photonics, genetics and materials science. In this review, we provide an up-to-date overview of the latest optical approaches for neuromodulation. We begin with the physical principles and constraints underlying the interaction between light and neural tissue. We then present advances in optical neurotechnologies in seven modules: conventional optical fibers, multifunctional fibers, optical waveguides, light-emitting diodes, upconversion nanoparticles, optical neuromodulation based on the secondary effects of light, and unconventional light sources facilitated by ultrasound and magnetic fields. We conclude our review with an outlook on new methods and mechanisms that afford optical neuromodulation with minimal invasiveness and footprint.

Palette of Rechargeable Mechanoluminescent Fluids Produced by a Biomineral-Inspired Suppressed Dissolution Approach.

Mechanoluminescent materials, which emit light in response to mechanical stimuli, have recently been explored as promising candidates for photonic skins, remote optogenetics, and stress sensing. All mechanoluminescent materials reported thus far are bulk solids with micron-sized grains, and their light emission is only produced when fractured or deformed in bulk form. In contrast, mechanoluminescence has never been observed in liquids and colloidal solutions, thus limiting its biological application in living organisms. Here, we report the synthesis of mechanoluminescent fluids via a suppressed dissolution approach. We demonstrate that this approach yields stable colloidal solutions comprising mechanoluminescent nanocrystals with bright emissions in the range of 470-610 nm and diameters down to 20 nm. These colloidal solutions can be recharged and discharged repeatedly under photoexcitation and hydrodynamically focused ultrasound, respectively, thus yielding rechargeable mechanoluminescent fluids that can store photon energy in a reversible manner. This rechargeable fluid can facilitate a systemically delivered light source gated by tissue-penetrant ultrasound for biological applications that require light in the tissue, such as optogenetic stimulation in the brain.

Controlled afterglow luminescent particles for photochemical tissue bonding.

Upconversion materials (UCMs) have been developed to convert tissue-penetrating near-infrared (NIR) light into visible light. However, the low energy conversion efficiency of UCMs has limited their further biophotonic applications. Here, we developed controlled afterglow luminescent particles (ALPs) of ZnS:Ag,Co with strong and persistent green luminescence for photochemical tissue bonding (PTB). The co-doping of Ag+ and Co2+ ions into ZnS:Ag,Co particles with the proper vacancy formation of host ions resulted in high luminescence intensity and long-term afterglow. In addition, the ALPs of ZnS:Ag,Co could be recharged rapidly under short ultraviolet (UV) irradiation, which effectively activated rose bengal (RB) in hyaluronate-RB (HA-RB) conjugates for the crosslinking of dissected collagen layers without additional light irradiation. The remarkable PTB of ZnS:Ag,Co particles with HA-RB conjugates was confirmed by in vitro collagen fibrillogenesis assay, in vivo animal wound closure rate analysis, and in vivo tensile strength evaluation of incised skin tissues. Taken together, we could confirm the feasibility of controlled ALPs for various biophotonic applications.

A biomineral-inspired approach of synthesizing colloidal persistent phosphors as a multicolor, intravital light source.

Many in vivo biological techniques, such as fluorescence imaging, photodynamic therapy, and optogenetics, require light delivery into biological tissues. The limited tissue penetration of visible light discourages the use of external light sources and calls for the development of light sources that can be delivered in vivo. A promising material for internal light delivery is persistent phosphors; however, there is a scarcity of materials with strong persistent luminescence of visible light in a stable colloid to facilitate systemic delivery in vivo. Here, we used a bioinspired demineralization (BID) strategy to synthesize stable colloidal solutions of solid-state phosphors in the range of 470 to 650 nm and diameters down to 20 nm. The exceptional brightness of BID-produced colloids enables their utility as multicolor luminescent tags in vivo with favorable biocompatibility. Because of their stable dispersion in water, BID-produced nanophosphors can be delivered systemically, acting as an intravascular colloidal light source to internally excite genetically encoded fluorescent reporters within the mouse brain.

Cooling the pain.

A miniaturized, flexible cooling device can be used for precise analgesia.

Scalable Three-Dimensional Recording Electrodes for Probing Biological Tissues.

Electrophysiological recording technologies can provide critical insight into the function of the nervous system and other biological tissues. Standard silicon-based probes have limitations, including single-sided recording sites and intrinsic instabilities due to the probe stiffness. Here, we demonstrate high-performance neural recording using double-sided three-dimensional (3D) electrodes integrated in an ultraflexible bioinspired open mesh structure, allowing electrodes to sample fully the 3D interconnected tissue of the brain. In vivo electrophysiological recording using 3D electrodes shows statistically significant increases in the number of neurons per electrode, average spike amplitudes, and signal to noise ratios in comparison to standard two-dimensional electrodes, while achieving stable detection of single-neuron activity over months. The capability of these 3D electrodes is further shown for chronic recording from retinal ganglion cells in mice. This approach opens new opportunities for a comprehensive 3D interrogation, stimulation, and understanding of the complex circuitry of the brain and other electrogenic tissues in live animals over extended time periods.

Pristine carbon nanotubes are efficient absorbers at radio frequencies.

Radio frequency ablation and microwave hyperthermia are powerful tools for destroying dysfunctional biological tissues. However, wireless application of these techniques is hindered by their inability to focus the electromagnetic energy to small targets. The use of locally injected radio frequency- or microwave-absorbing nanomaterials can help to overcome this challenge by confining heat production to the injected region. Previous theoretical work suggests that high-aspect-ratio conducting nanomaterials, such as carbon nanotubes, offer powerful radio frequency and microwave absorption. While carbon nanotubes have been demonstrated as radiothermal agents, common solubilization methods may reduce their absorption efficiency, yielding undesirable nonspecific heating in the biological tissue background. In this manuscript, we hypothesize that pristine carbon nanotubes can act as efficient absorbers at radio frequencies, thus providing differential heating over the tissue background. Specifically, we use a sonication-free preparation technique to preserve both the high aspect ratio and local concentration of pristine carbon nanotubes. We validate the differential heating of these samples by 4.5-fold at 2 GHz compared to the heating of saline at a physiological concentration using infrared thermography. In addition, we successfully achieved local heating of pristine carbon nanotubes within a three-dimensional biological tissue phantom. Numerical simulations further aid in producing a temperature map within the phantom and confirming localized heating. Due to their significant differential and local heating, we believe that pristine carbon nanotubes may facilitate region-specific radio frequency ablation and microwave hyperthermia while keeping nonspecific heating to a low level in the normal tissue background.