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Ultra-rapid modulation of neurite outgrowth in a gigahertz acoustic streaming system
Abstract
The development of rapid and efficient tools to modulate neurons is vital for the treatment of nervous system diseases. Here, a novel non-invasive neurite outgrowth modulation method based on a controllable acoustic streaming effect induced by an electromechanical gigahertz resonator microchip is reported. The results demonstrate that the gigahertz acoustic streaming can induce cell structure changes within a 10 min period of stimulation, which promotes a high proportion of neurite bearing cells and encourages longer neurite outgrowth. Specifically, the resonator stimulation not only promotes outgrowth of neurites, but also can be combined with chemical mediated methods to accelerate the direct entry of nerve growth factor (NGF) into cells, resulting in higher modulation efficacy. Owing to shear stress caused by the acoustic streaming effect, the resonator microchip mediates stress fiber formation and induces the neuron-like phenotype of PC12 cells. We suggest that this method may potentially be applied to precise single-cell modulation, as well as in the development of non-invasive and rapid disease treatment strategies.
... This kind of AS is superior in solution mixing and pumping, which has been successfully implemented in microfluidic and applied for sample separation, concentration and manipulation [26][27][28]. However, besides these biological and chemical analysis applications, few studies have been devoted to on chip surface modification and analysis applications [29,30]. ...
... In this study, a microfluidic sonoelectrochemical platform was proposed by integrating the electrochemistry and gigahertz (GHz) resonator on a chip. The AS generated by the resonator can be flexibly and accurately controlled by simply adjusting the power and distance of the resonator, which has been proved by our present work [30][31][32][33]. Benefiting from the AS, sonoelectrochemical deposition of AuNPs with smaller size and even distributing on the working electrode was achieved, which showed excellent enzyme -mimic activity. ...
Developing effective strategies for the flexible control of fluid is vital for microfluidic electrochemical biosensing. In this study, a gigahertz (GHz) acoustic streaming (AS) based sonoelectrochemical system was developed to realize an on-chip surface modification and sensitive hydrogen peroxide (H2O2) detection from living cells. The flexible and controlled fluid surrounding the electrochemical chip was optimized theoretically and applied in the sonoelectrochemical deposition of Au nanoparticles (AuNPs) first. Under the steady and fast flow stimulus of AS, AuNPs could be synthesized with a smaller and evener size distribution than the normal condition, allowing AuNPs to show an excellent peroxidase-like activity. Moreover, the AS also accelerated the mass transport of target molecules and improved the catalytic rate, leading to the enhancement of H2O2 detection, with an extremely low detection limit of 32 nM and a high sensitivity of 4.34 μA/ (mM·mm²). Finally, this system was successfully applied in tracking H2O2 release from different cell lines to distinguish the cancer cells from normal cells. This study innovatively integrated the surface modification and molecules detection process on a chip, and also proposed a simple but sensitive platform for microfluidic biosensing application.
... And the gigahertz acoustic wave can be an excellent candidate to generate localized AS with high liquid speed and intense shear force. 25 Most importantly, with the previous studies from our group, this AS effect is able to realize controllable cell deformation 25,26 and promote the intracellular delivery of exogenous payloads. 27−29 In this work, an AuNRs-AS system is proposed, which realizes the rapid intracellular delivery of AuNRs together with controllable cell mechanical motion regulation by utilizing the fluid shear force generated by gigahertz AS. ...
... The cytoskeleton is a dynamic structure that continuously assembles and disassembles into specific functional arrangements in response to extracellular stimulation, which plays an important role in cell mechanical motion. 40,41 As shown in Figure 3A and SI Figure S6, when cells were exposed to AS, Factin (labeled with TRITC-phalloidin, red color) was partly assembled into ordered microfilaments in the cytoplasm compared to the nontreatment group 26 (Figure 3B and SI Figure S6) and responded to the external fluid shear force. However, there were still some actin proteins within a diffuse distribution in both the cytoplasm and nuclear region of cells exposed to AS stimulation. ...
Cell mechanical motion is a key physiological process that relies on the dynamics of actin filaments. Herein, a localized shear-force system based on gigahertz acoustic streaming (AS) is proposed, which can simultaneously realize intracellular delivery and cellular mechanical regulation. The results demonstrate that gold nanorods (AuNRs) can be delivered into the cytoplasm and even the nuclei of cancer and normal cells within a few minutes by AS stimulation. The delivery efficiency of AS stimulation is four times higher than that of endocytosis. Moreover, AS can effectively promote cytoskeleton assembly, regulate cell stiffness and change cell morphology. Since the inhibitory effect of AuNRs on cytoskeleton assembly, this AuNRs-AS system is able to inhibit or promote cell mechanical motion in a controlled manner by regulating the mechanical properties of cells. The bidirectional regulation of cell motion is further verified via scratch experiments, in which AuNRs-treated cells recover their motion ability through AS stimulation. In particular, the results of AuNRs-AS mechanical regulation on cell are related to the intrinsic properties of cell lines, revealing to more obvious effects on the cells with higher motor capacities. In summary, this acoustic technology has shown superiorities in controllable cell-motion manipulation, indicating its potential in building a multifunctional, integrated cytomechanics regulation platform.
... It has recently been confirmed that the strong acoustic force driven by BAW can realize in-situ mixing and microparticles concentrating [36,37]. In addition, recent works from our group also found that this strong acoustic streaming could drive the membrane of mammalian cells [38] as well as inducing deformation [35] and differentiation of cells [39], to facilitate the biological applications. ...
... When such acoustic waves are coupled from solid substrates to microfluidics, the wave energy absorbed by liquid will bring flow of the fluid, thus triggering the AS. AS is the result of acoustic energy flux dissipation within the fluid, its force is strongly dependent on the applied power of resonator and the distance from resonator surface [35,39,41]. In this work, we found that by selecting the appropriate microchannel height, the system could achieve excellent functions in selectively capturing larger size of particles and deforming the cell membrane for intracellular delivery of CDs. ...
Currently, researches on nanomaterials have been restricted by slow and multistep synthesis procedures. Herein, we demonstrate an ultrafast, one step method of purification and delivery of quantum dots into living cells, actuated by the acoustic streaming (AS) produced through a gigahertz resonator. Results demonstrate that the impurities in the carbon dots (CDs) can be extracted immediately aided by the acoustic forcing, with extra high purification efficiency of 93%. The system can also efficiently deliver the CDs into cells, showing excellent nucleus and mitochondria uptake under 3 min of acoustic streaming treatment, and making the organelles of cells to be recorded more easily and simultaneously. More importantly, the AS is found to further accelerate the bioreaction inside the cells, thus realizes the enhanced biosensing of Fe³⁺ in single living cells. This work develops a novel type of multifunctional method for effective purification, intracellular delivery and biosensing of nanomaterials, inspiring the biological/medical nanotechnology researches at subcellular level.
... 5,6 Their fundamental sensing principles are based on the transmission of electrons from a motion target to the surface of a static microelectrode, and the speed of the target's movement in liquid is one of the most important factors affecting the speed and sensitivity of electrochemical detection. 7,8 However, in the conventional microfluidic systems, the volume of the fluid is in the range of microliters to nanoliters, and the fluid transfer rate is usually slow, which significantly limits the detection rate of electrochemical sensors. [9][10][11] Therefore, it is crucial to enhance the flow and mass transfer rates of the fluid and effectively increase the movement speed of the targeted species, thus improving the performance of the electrochemical sensor. ...
Microelectrode-based electrochemical detection methods have been extensively applied in microfluidic sensors, but there are significant challenges for achieving fast and efficient contact between analytes and the microarray electrodes and, thus, enhancing the sensing performance. In this paper, we develop a technique using asymmetrically aligned focused surface acoustic waves (FSAWs) to enhance sensitivity of microarray electrodes detection. Effects of various focusing angles of the FSAW devices on the values and distributions of acoustic wave amplitudes were analyzed using finite element simulations, and torques, which determine the acoustic streaming velocity, were calculated as a function of values and distributions of amplitude. Based on simulation results, the FSAW device with a focusing angle of 30° was used to investigate sensitivity of microarray electrochemical sensors. The maximum value of instantaneous current was increased up to 11 times, researching a current value of 4.3 μA with the applied FSAWs. This developed electrochemical sensing platform shows great potentials for highly sensitive food quality control and biochemical detections.
... The mechanical force imposed on the cells by the AS at the solid-liquid interface is caused by the sharp reduction of the velocity to zero. 31 Therefore, the closer the cells are to the SMR, the greater is the mechanical force that can be applied. Thus, the distance between the SMR and the cell can be controlled by the Z axis to produce a specific magnitude of force imposed on the cell. ...
The blood–brain barrier (BBB) is a structural and functional barrier necessary for brain homeostasis, and it plays an important role in the realization of neural function and in protecting the brain from damage by circulating toxins and pathogens. However, the extremely dense BBB also severely limits the transport of molecules across it, which is a great hindrance to the diagnosis and treatment of central nervous system (CNS) diseases. This paper reports a new method for controllable opening of the BBB, based on the gigahertz acoustic streaming (AS) generated by a bulk acoustic wave resonant device. By adjusting the input power and working distance of the device, AS with tunable flow rate can be generated to disrupt tight junction proteins (TJs) between endothelial cells. The results obtained with this method show that the gigahertz AS promotes the penetration of dextran molecules with different molecular weights across the BBB. This work provides a new platform for studying the mechanical regulation of BBB by fluid shear forces and a new method for improving the efficiency of drug delivery.
Gigahertz acoustic streaming enables the synthesis of localized microjets reaching speeds of up to meters per second, offering tremendous potential for precision micromanipulation. However, theoretical and numerical investigations of acoustic streaming at these frequencies remain so far relatively scarce due to significant challenges including: (i) the inappropriateness of classical approaches, rooted in asymptotic development, for addressing high-speed streaming with flow velocities comparable to the acoustic velocity; and (ii) the numerical cost of direct numerical simulations generally considered as prohibitive. In this paper, we investigate high-frequency bulk streaming using high-order finite difference direct numerical simulations. First, we demonstrate that high-speed micrometric jets of several meters per second can only be obtained at high frequencies, due to diffraction limits. Second, we establish that the maximum jet streaming speed at a given actuation power scales with the frequency to the power of 3/2 in the low attenuation limit and linearly with the frequency for strongly attenuated waves. Last, our analysis of transient regimes reveals a dramatic reduction in the time required to reach the maximum velocity as the frequency increases (power law in –5/2), leading to characteristic time on the order of μs at gigahertz frequencies, and hence accelerations within the Mega-g range.
Development of a micromodel that recapitulates multiple mechanical properties to mimic the complex mechanical microenvironment is crucial for cell‐based research. Herein, a microsystem combining structure of hydrogel matrix and acoustic streaming (AS) to mimic the cellular microenvironment is proposed, which can realize multiplex cellular mechanical cues, including matrix stiffness, fluid shear stress (FSS) generated by AS, and matrix roughness. The results demonstrate that the cell spreading area enlarges with the increase of matrix stiffness, and cell spreading is encouraged by integrin β1 cluster to polymerize actin when combines the hydrogel matrix with FSS. In addition, FSS has the influence on the roughness of the hydrogel, which further affects the cell morphology and mechanical properties, inducing mesenchymal stem cells (MSCs) differentiation into astrocytes rapidly. Meanwhile, cell migration is also enhanced by FSS stimulation, particularly, undifferentiated cells at 22 kPa hydrogel have the fastest migration speed, and change the movement model from contact inhibition to contact stimulation migration. Especially, matrix roughness and stiffness dominantly control of cell spreading and differentiation, whereas FSS affects cell migration. In conclusion, the microsystem in this work shows superior performance in regulating the spreading, differentiation, and migration of MSCs, which provides a new tool for cell‐microenvironment study.
Acoustic manipulation techniques have gained significant attention across various fields, particularly in medical diagnosis and biochemical research, due to their biocompatibility and non-contact operation. In this article, we review the broad range of biomedical applications of micro/nano-motors that use acoustic manipulation methods, with a specific focus on cell manipulation, targeted drug release for cancer treatment and genetic disease diagnosis. These applications are facilitated by acoustic-propelled micro/nano-motors and nanoparticles which are manipulated by acoustic tweezers. Acoustic systems enable high precision positioning and can be effectively combined with magnetic manipulation techniques. Furthermore, acoustic propulsion facilitates faster transportation speeds, making it suitable for tasks in blood flow, allowing for precise positioning and in-body manipulation of cells, microprobes, and drugs. By summarizing and understanding these acoustic manipulation methods, this review aims to provide a summary and discussion of the acoustic manipulation methods for biomedical research, diagnostic, and therapeutic applications.
Surface topography has become a powerful tool to control cell behaviors, however, it's still difficult to monitor cellular microenvironment changes during topography-induced cell responses. Here, a dual-functional platform integrating cell alignment with extracellular pH (pHe) measurement is proposed. The platform is fabricated by assembling gold nanorods (AuNRs) into micro pattern via wettability difference interface method, which provides topographical cues and surface-enhanced Raman scattering (SERS) effect for cell alignment and biochemical detection respectively. Results demonstrate that contact guidance and cell morphology changes are achieved by the AuNRs micro pattern, and pHe are also obtained by the changes of SERS spectra during cell alignment, where the pHe near cytoplasm is lower than nucleus, revealing the heterogeneity of extracellular microenvironment. Moreover, a correlation between lower extracellular pH and higher cell migration ability is revealed, and AuNRs micro pattern can differentiate cells with different migration ability, which may be an inheritable character during cell division. Furthermore, mesenchymal stem cells response dramatically to AuNRs micro pattern, showing different morphology and increased pHe level, offering the potential of impacting stem cell differentiation. This approach provides a new idea for the research of cell regulation and response mechanism.
Over the past few decades, acoustofluidics, one of the branches of microfluidics, has rapidly developed as a multidisciplinary cutting edge research topic, covering many biomedical and bioanalytical applications. Acoustofluidics usually utilizes acoustic pressure and acoustic streaming effects to manipulate liquids and bioparticles. Acoustic manipulation using acoustic radiation force has been widely studied; however, with the recent development of new piezoelectric devices that enable faster acoustic streaming, particle manipulations using drag force induced by acoustic streaming have attracted more attention. Despite many review articles on acoustic radiation force-based acoustophoresis, acoustic streaming is less frequently covered. Here, we review the recent development of microscale acoustic streaming, especially high-frequency transducer-induced high-speed streaming, confinement and programed streaming, and acoustic streaming tweezers, which combine the acoustic radiation force and drag force to tackle the size limitations of conventional acoustic manipulations. A brief review of acoustic streaming theory and its generation is summarized. Recent progress in applying acoustic streaming for fluidic handling and bioparticle manipulations is reviewed. Representative applications of micro acoustic streaming are provided, and the key issues in these applications are analyzed. Finally, the future prospects of micro acoustic streaming in bioanalytical and biomedical applications are discussed.
The hydrodynamic method mimics the in vivo environment of the mechanical effect on cell stimulation, which not only modulates cell physiology but also shows excellent intracellular delivery ability. Herein, a hydrodynamic intracellular delivery system based on the gigahertz acoustic streaming (AS) effect is proposed, which presents powerful targeted delivery capabilities with high efficiency and universality. Results indicate that the range of cells with AuNR introduction is related to that of AS, enabling a tunable delivery range due to the adjustability of the AS radius. Moreover, with the assistance of AS, the organelle localization delivery of AuNRs with different modifications is enhanced. AuNRs@RGD is inclined to accumulate in the nucleus, while AuNRs@BSA tend to enter the mitochondria and AuNRs@PEGnK tend to accumulate in the lysosome. Finally, the photothermal effect is proved based on the large quantities of AuNRs introduced via AS. The abundant introduction of AuNRs under the action of AS can achieve rapid cell heating with the irradiation of a 785 nm laser, which has great potential in shortening the treatment cycle of photothermal therapy (PTT). Thereby, an efficient hydrodynamic technology in AuNR introduction based on AS has been demonstrated. The outstanding location delivery and organelle targeting of this method provides a new idea for precise medical treatment.
Low-intensity ultrasound is an emerging modality for neuromodulation. Yet, transcranial neuromodulation using low-frequency piezo-based transducers offers poor spatial confinement of excitation volume, often bigger than a few millimeters in diameter. In addition, the bulky size limits their implementation in a wearable setting and prevents integration with other experimental modalities. Here, we report spatially confined optoacoustic neural stimulation through a miniaturized Fiber-Optoacoustic Converter (FOC). The FOC has a diameter of 600 μm and generates omnidirectional ultrasound wave locally at the fiber tip through the optoacoustic effect. We show that the acoustic wave generated by FOC can directly activate individual cultured neurons and generate intracellular Ca2+ transients. The FOC activates neurons within a radius of 500 μm around the fiber tip, delivering superior spatial resolution over conventional piezo-based low-frequency transducers. Finally, we demonstrate direct and spatially confined neural stimulation of mouse brain and modulation of motor activity in vivo. Low-intensity ultrasound can be used for neuromodulation in vivo, but it has poor spatial confinement and can result in unwanted cochlear pathway activation. Here the authors use the optoacoustic effect to generate spatially confined ultrasound waves to activate neurons within a 500 μm radius in the mouse brain.
Background
Controllable and multiple DNA release is critical in modern gene-based therapies. Current approaches require complex assistant molecules for combined release. To overcome the restrictions on the materials and environment, a novel and versatile DNA release method using a nano-electromechanical (NEMS) hypersonic resonator of gigahertz (GHz) frequency is developed.
Results
The micro-vortexes excited by ultra-high frequency acoustic wave can generate tunable shear stress at solid–liquid interface, thereby disrupting molecular interactions in immobilized multilayered polyelectrolyte thin films and releasing embedded DNA strands in a controlled fashion. Both finite element model analysis and experiment results verify the feasibility of this method. The release rate and released amount are confirmed to be well tuned. Owing to the different forces generated at different depth of the films, release of two types of DNA molecules with different velocities is achieved, which further explores its application in combined gene therapy.
Conclusions
Our research confirmed that this novel platform based on a nano-electromechanical hypersonic resonator works well for controllable single and multi-DNA release. In addition, the unique features of this resonator such as miniaturization and batch manufacturing open its possibility to be developed into a high-throughput, implantable and site targeting DNA release and delivery system.
Tools for noninvasively modulating neural signaling in peripheral organs will advance the study of nerves and their effect on homeostasis and disease. Herein, we demonstrate a noninvasive method to modulate specific signaling pathways within organs using ultrasound (U/S). U/S is first applied to spleen to modulate the cholinergic anti-inflammatory pathway (CAP), and US stimulation is shown to reduce cytokine response to endotoxin to the same levels as implant-based vagus nerve stimulation (VNS). Next, hepatic U/S stimulation is shown to modulate pathways that regulate blood glucose and is as effective as VNS in suppressing the hyperglycemic effect of endotoxin exposure. This response to hepatic U/S is only found when targeting specific sub-organ locations known to contain glucose sensory neurons, and both molecular (i.e. neurotransmitter concentration and cFOS expression) and neuroimaging results indicate US induced signaling to metabolism-related hypothalamic sub-nuclei. These data demonstrate that U/S stimulation within organs provides a new method for site-selective neuromodulation to regulate specific physiological functions.
Controlling the outgrowth of neurites is important for enhancing the repair of injured nerves and understanding the development of nervous systems. Herein we report a simple strategy for enhancing the outgrowth of neurites through a unique integration of topographical guidance and a chemical cue. We use electrospray to easily functionalize the surface of a substrate with microparticles of natural fatty acids at a controllable density. Through a synergistic effect from the surface roughness arising from the microparticles and the chemical cue offered by the fatty acids, the outgrowth of neurites from PC12 cells is greatly enhanced. We also functionalize the surfaces of uniaxially aligned, electrospun microfibers with the microparticles and further demonstrate that the substrates can guide and enhance directional outgrowth of neurites from both PC12 multicellular spheroids and chick embryonic dorsal root ganglia bodies.
Controllable exchange of molecules between the interior and the external environment of vesicles is critical in drug delivery and micro/nano‐reactors. While many approaches exist to trigger release from vesicles, controlled loading remains a challenge. Here, we show that gigahertz acoustic streaming generated by a nanoelectromechanical resonator can control the loading and release of cargo into/from vesicles. Polymer‐shelled vesicles showed loading and release of molecules both in solution and on a solid substrate. We observed deformation of individual giant unilamellar vesicles and propose that the shear stress generated by gigahertz acoustic streaming induces the formation of transient nanopores in the vesicle membranes. The size of these pores was estimated to be on the order of 100 nm by loading nanoparticles of different sizes into the vesicles. Forming such pores with gigahertz acoustic streaming provides a non‐invasive method to control materials exchange across membranes of different types of vesicles. This method could allow site‐specific release of therapeutics and controlled loading into cells, as well as tunable microreactors.
Investigations over half a century have indicated that mechanical forces induce neurite growth - with neurites elongating at a rate of 0.1-0.3{\mu}mh^{-1} per pico-Newton (pN) of applied force - when mechanical tension exceeds a threshold, with this being identified as 400-1000 pN for neurites of PC12 cells. Here we demonstrate that there is no threshold for neurite elongation of PC12 cells in response to applied mechanical forces. Instead, this proceeds at the same previously identified rate, on the application of tensions with intensity below 1pN. This supports the idea of mechanical tension as an endogenous signal used by neurons for promoting neurite elongation.
On-demand droplet dispensing systems are indispensable tools in bioanalytical fields, such as microarray
fabrication. Biomaterial solutions can be very limited and expensive, so minimizing the use of solution per
spot produced is highly desirable. Here, we proposed a novel droplet dispensing method which utilizes a
gigahertz (GHz) acoustic resonator to deposit well-defined droplets on-demand. This ultra-high frequency
acoustic resonator induces a highly localized and strong body force at the solid–liquid interface, which
pushes the liquid to generate a stable and sharp “liquid needle” and further delivers droplets to the target
substrate surface by transient contact. This approach is between contact and non-contact methods, thus
avoiding some issues of traditional methods (such as nozzle clogging or satellite spots). We demonstrated
the feasibility of this approach by fabricating high quality DNA and protein microarrays on glass and flexible
substrates. Notably, the spot size can be delicately controlled down to a few microns (femtoliter in volume). Because of the CMOS compatibility, we expect this technique to be readily applied to advanced biofabrication processes.
Transcranial electric stimulation is a non-invasive tool that can influence brain activity; however, the parameters necessary to affect local circuits in vivo remain to be explored. Here, we report that in rodents and human cadaver brains, ~75% of scalp-applied currents are attenuated by soft tissue and skull. Using intracellular and extracellular recordings in rats, we find that at least 1 mV/mm voltage gradient is necessary to affect neuronal spiking and subthreshold currents. We designed an 'intersectional short pulse' stimulation method to inject sufficiently high current intensities into the brain, while keeping the charge density and sensation on the scalp surface relatively low. We verify the regional specificity of this novel method in rodents; in humans, we demonstrate how it affects the amplitude of simultaneously recorded EEG alpha waves. Our combined results establish that neuronal circuits are instantaneously affected by intensity currents that are higher than those used in conventional protocols.
Neurological and psychiatric diseases often involve the dysfunction of specific neural circuits in particular regions of the brain. Existing treatments, including drugs and implantable brain stimulators, aim to modulate the activity of these circuits, but are typically not cell type-specific, lack spatial targeting or require invasive procedures. Here, we introduce an approach to modulating neural circuits noninvasively with spatial, cell-type and temporal specificity. This approach, called acoustically targeted chemogenetics, or ATAC, uses transient ultrasonic opening of the blood brain barrier to transduce neurons at specific locations in the brain with virally-encoded engineered G-protein-coupled receptors, which subsequently respond to systemically administered bio-inert compounds to activate or inhibit the activity of these neurons. We demonstrate this concept in mice by using ATAC to noninvasively modify and subsequently activate or inhibit excitatory neurons within the hippocampus, showing that this enables pharmacological control of memory formation. This technology allows a brief, noninvasive procedure to make one or more specific brain regions capable of being selectively modulated using orally bioavailable compounds, thereby overcoming some of the key limitations of conventional brain therapies.
Ultrasound neuro-modulation has gained increasing attention as a non-invasive method. In this paper, we present an ultrasound neuro-modulation chip, capable of initiating reversal behaviour and activating neurons of C. elegans under the stimulation of a single-shot, short-pulsed ultrasound. About 85.29% ± 6.17% of worms respond to the ultrasound stimulation exhibiting reversal behaviour. Furthermore, the worms can adapt to the ultrasound stimulation with a lower acoustic pulse duration of stimulation. In vivo calcium imaging shows that the activity of ASH, a polymodal sensory neuron in C. elegans, can be directly evoked by the ultrasound stimulation. On the other hand, AFD, a thermal sensitive neuron, cannot be activated by the ultrasound stimulation using the same parameter and the temperature elevation during the stimulation process is relatively small. Consistent with the calcium imaging results, the tax-4 mutants, which are insensitive to temperature increase, do not show a significant difference in avoidance probability compared to the wild type. Therefore, the mechanical effects induced by ultrasound are the main reason for neural and behavioural modulation of C. elegans. With the advantages of confined acoustic energy on the surface, compatible with standard calcium imaging, this neuro-modulation chip could be a powerful tool for revealing the molecular mechanisms of ultrasound neuro-modulation.
Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties.
Despite increasing demand in the manipulation of nanoscale objects for next generation biological and industrial processes, there is a lack of methods for reliable separation, concentration and purification of nanoscale objects. Acoustic methods have proven their utility in contactless manipulation of microscale objects mainly relying on the acoustic radiation effect, though the influence of acoustic streaming has typically prevented manipulation at smaller length scales. In this work, however, we explicitly take advantage of the strong acoustic streaming in the vicinity of a highly focused, high frequency surface acoustic wave (SAW) beam emanating from a series of focused 6 μm substrate wavelength interdigital transducers patterned on a piezoelectric lithium niobate substrate and actuated with a 633 MHz sinusoidal signal. This streaming field serves to focus fluid streamlines such that incoming particles interact with the acoustic field similarly regardless of their initial starting positions, and results in particle displacements that would not be possible with a travelling acoustic wave force alone. This streaming-induced manipulation of nanoscale particles is maximized with the formation of micro-vortices that extend the width of the microfluidic channel even with the imposition of a lateral flow, occurring when the streaming-induced flow velocities are an order of magnitude larger than the lateral one. We make use of this acoustic streaming to demonstrate the continuous and differential focusing of 100 nm, 300 nm and 500 nm particles.
A major challenge in neuroscience is to reliably activate individual neurons, particularly those in deeper brain regions. Current optogenetic approaches require invasive surgical procedures to deliver light of specific wavelengths to target cells to activate or silence them. Here, we demonstrate the use of low-pressure ultrasound as a non-invasive trigger to activate specific ultrasonically sensitized neurons in the nematode, Caenorhabditis elegans. We first show that wild-type animals are insensitive to low-pressure ultrasound and require gas-filled microbubbles to transduce the ultrasound wave. We find that neuron-specific misexpression of TRP-4, the pore-forming subunit of a mechanotransduction channel, sensitizes neurons to ultrasound stimulus, resulting in behavioural outputs. Furthermore, we use this approach to manipulate the function of sensory neurons and interneurons and identify a role for PVD sensory neurons in modifying locomotory behaviours. We suggest that this method can be broadly applied to manipulate cellular functions in vivo.
Mechanotransduction - how cells sense physical forces
and translate them into biochemical and biological
responses - is a vibrant and rapidly-progressing field,
and is important for a broad range of biological
phenomena. This forum explores the role of
mechanotransduction in a variety of cellular activities
and highlights intriguing questions that deserve further
attention.
We previously developed an easy and sensitive method for real-time monitoring of dopamine release from PC12 cells using a combination of enzymatic oxidation of dopamine and luminal reaction. Here, we applied our enzyme-luminescence method for the evaluation of neuronal differentiation in PC12 cells with NGF, via real-time monitoring of dopamine release response. ATP and muscarine-stimulated dopamine release from PC12 cells was compared before and after differentiation by the luminescence peak intensity responding to agonists' injection. A clear enhancement of dopamine release ability in PC12 cells after differentiation was demonstrated. Therefore, this enhancement in luminescence peak intensity by dopamine release upon agonist stimulation may be a good marker of differentiation in PC12 cells. These results imply that the enzyme-luminescence method might be used as a promising tool for evaluation of the effects of various growth factors and chemicals on neuronal differentiation in neuronal progenitor cells.
Few But Powerful
Drug activation of the different types of acetylcholine receptors in cholinergic neurons often generates opposing or conflicting effects. Using optogenetic techniques in transgenic mice, Witten et al. (p. 1677 ) investigated the function of a rather enigmatic subpopulation of cholinergic neurons, the giant interneurons of the nucleus accumbens. Their excitation paradoxically reduced neighboring medium spiny neuron firing, while their inhibition increased medium spiny neuron firing. Furthermore, the giant interneurons were directly activated by cocaine, and silencing their drug-induced activity during cocaine exposure in freely behaving animals disrupted cocaine reward.
This mini-symposium aims to provide an integrated perspective on recent developments in optogenetics. Research in this emerging field combines optical methods with targeted expression of genetically encoded, protein-based probes to achieve experimental manipulation and measurement of neural systems with superior temporal and spatial resolution. The essential components of the optogenetic toolbox consist of two kinds of molecular devices: actuators and reporters, which respectively enable light-mediated control or monitoring of molecular processes. The first generation of genetically encoded calcium reporters, fluorescent proteins, and neural activators has already had a great impact on neuroscience. Now, a second generation of voltage reporters, neural silencers, and functionally extended fluorescent proteins hold great promise for continuing this revolution. In this review, we will evaluate and highlight the limitations of presently available optogenic tools and discuss where these technologies and their applications are headed in the future.
The regulation of neuronal differentiation and neurite outgrowth is essential during development of the nervous system and is crucial in developing therapies to promote axon regeneration after nerve injury or in neurodegenerative diseases. The serine/threonine kinase Akt has been well documented to promote neuronal survival. More recently Akt has also been revealed as key mediator of several aspects of neurite outgrowth, including elongation, branching and calibre. Downstream of Akt, several substrates have been identified that are likely to play key roles in Akt-mediated neurite outgrowth, such as glycogen synthase kinase 3beta, peripherin, mammalian target of rapamycin and delta-catenin. The physical interaction between Akt and Hsp27, another protein that has been linked with neurite outgrowth, may also be significant in the process of neurite outgrowth. This review will unite and discuss the research to date that has examined the functionality of Akt in neuronal differentiation during development and neurite outgrowth.
Deep brain stimulation (DBS) is a therapeutic option for intractable neurological and psychiatric disorders, including Parkinson's disease and major depression. Because of the heterogeneity of brain tissues where electrodes are placed, it has been challenging to elucidate the relevant target cell types or underlying mechanisms of DBS. We used optogenetics and solid-state optics to systematically drive or inhibit an array of distinct circuit elements in freely moving parkinsonian rodents and found that therapeutic effects within the subthalamic nucleus can be accounted for by direct selective stimulation of afferent axons projecting to this region. In addition to providing insight into DBS mechanisms, these results demonstrate an optical approach for dissection of disease circuitry and define the technological toolbox needed for systematic deconstruction of disease circuits by selectively controlling individual components.
A single cell clonal line which responds reversibly to nerve growth factor (NGF) has been established from a transplantable rat adrenal pheochromocytoma. This line, designated PC12, has a homogeneous and near-diploid chromosome number of 40. By 1 week's exposure to NGF, PC12 cells cease to multiply and begin to extend branching varicose processes similar to those produced by sympathetic neurons in primary cell culture. By several weeks of exposure to NGF, the PC12 processes reach 500-1000 mum in length. Removal of NGF is followed by degeneration of processes within 24 hr and by resumption of cell multiplication within 72 hr. PC12 cells grown with or without NGF contain dense core chromaffin-like granules up to 350 nm in diameter. The NGF-treated cells also contain small vesicles which accumulate in process varicosities and endings. PC12 cells synthesize and store the catecholamine neurotransmitters dopamine and norepinephrine. The levels (per mg of protein) of catecholamines and of the their synthetic enzymes in PC12 cells are comparable to or higher than those found in rat adrenals. NGF-treatment of PC12 cells results in no change in the levels of catecholamines or of their synthetic enzymes when expressed on a per cell basis, but does result in a 4- to 6-fold decrease in levels when expressed on a per mg of protein basis. PC12 cells do not synthesize epinephrine and cannot be induced to do so by treatment with dexamethasone. The PC12 cell line should be a useful model system for neurobiological and neurochemical studies.
Treatment of cells with a variety of growth factors triggers a phosphorylation cascade that leads to activation of mitogen-activated protein kinases (MAPKs, also called extracellular signal-regulated kinases, or ERKs). We have identified a synthetic inhibitor of the MAPK pathway. PD 098059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one] selectively inhibited the MAPK-activating enzyme, MAPK/ERK kinase (MEK), without significant inhibitory activity of MAPK itself. Inhibition of MEK by PD 098059 prevented activation of MAPK and subsequent phosphorylation of MAPK substrates both in vitro and in intact cells. Moreover, PD 098059 inhibited stimulation of cell growth and reversed the phenotype of ras-transformed BALB 3T3 mouse fibroblasts and rat kidney cells. These results indicate that the MAPK pathway is essential for growth and maintenance of the ras-transformed phenotype. Further, PD 098059 is an invaluable tool that will help elucidate the role of the MAPK cascade in a variety of biological settings.
The mitogen-activated protein kinase (MAP kinase) pathway is thought to play an important role in the actions of neurotrophins. A small molecule inhibitor of the upstream kinase activator of MAP kinase, MAP kinase kinase (MEK) was examined for its effect on the cellular action of nerve growth factor (NGF) in PC-12 pheochromocytoma cells. PD98059 selectively blocks the activity of MEK, inhibiting both the phosphorylation and activation of MAP kinases in vitro. Pretreatment of PC-12 cells with the compound completely blocked the 4-fold increase in MAP kinase activity produced by NGF. Half-maximal inhibition was observed at 2 microM PD98059, with maximal effects at 10-100 microM. The tyrosine phosphorylation of immunoprecipitated MAP kinase was also completely blocked by the compound. In contrast, the compound was without effect on NGF-dependent tyrosine phosphorylation of the pp140trk receptor or its substrate Shc and did not block NGF-dependent activation of phosphatidylinositol 3'-kinase. However, PD98059 completely blocked NGF-induced neurite formation in these cells without altering cell viability. These data indicate that the MAP kinase pathway is absolutely required for NGF-induced neuronal differentiation in PC-12 cells.
Mechanical signals transmitted through the cytoplasmic actin cytoskeleton must be relayed to the nucleus to control gene expression. LIM domains are protein-protein interaction modules found in cytoskeletal proteins and transcriptional regulators. Here, we identify three LIM protein families (zyxin, paxillin, and FHL) whose members preferentially localize to the actin cytoskeleton in mechanically stimulated cells through their tandem LIM domains. A minimal actin-myosin reconstitution system reveals that representatives of all three families directly bind F-actin only in the presence of mechanical force. Point mutations at a site conserved in each LIM domain of these proteins disrupt tensed F-actin binding in vitro and cytoskeletal localization in cells, demonstrating a common, avidity-based mechanism. Finally, we find that binding to tensed F-actin in the cytoplasm excludes the cancer-associated transcriptional co-activator FHL2 from the nucleus in stiff microenvironments. This establishes direct force-activated F-actin binding as a mechanosensing mechanism by which cytoskeletal tension can govern nuclear localization.
Visualizing and perturbing neural activity on a brain-wide scale in model animals and humans is a major goal of neuroscience technology development. Established electrical and optical techniques typically break down at this scale due to inherent physical limitations. In contrast, ultrasound readily permeates the brain, and in some cases the skull, and interacts with tissue with a fundamental resolution on the order of 100 μm and 1 ms. This basic ability has motivated major efforts to harness ultrasound as a modality for large-scale brain imaging and modulation. These efforts have resulted in already-useful neuroscience tools, including high-resolution hemodynamic functional imaging, focused ultrasound neuromodulation, and local drug delivery. Furthermore, recent breakthroughs promise to connect ultrasound to neurons at the genetic level for biomolecular imaging and sonogenetic control. In this article, we review the state of the art and ongoing developments in ultrasonic neurotechnology, building from fundamental principles to current utility, open questions, and future potential.
Electrical stimulation (ES) as an easy and effective inducing method has been widely used in induction differentiation of stem cells, e.g. osteogenic differentiation of mesenchymal stem cells (MSCs) for bone healing and bone tissue therapies. However, micro-effect of inhomogeneous electric field has rarely been investigated for ES in induction differentiation, and conventionally used ex-situ assay may preclude accurate assessment due to variation from cell inoculations and treatments. Here, a novel electrical stimulation method with microelectrode array chip is proposed for osteogenic differentiation of MSCs. Electric field applied onto the MSCs by the microelectrode array is designed similarly with natural aggregation distribution of differentiated MSCs. The proposed ES method accelerates the osteoblast proliferation and differentiation in the electrode array region and generates more amount of mineralized deposits, which are assayed via in-situ alizarin red staining and morphology observation as well as immunocytochemistry. In addition, this method allows a directly in-situ assessment to compare the osteogenic differentiations of MSCs with and without ES on single chip to avoid culture environment difference. The method provides a fundamental platform for investigating induced differentiation of stem cells and allows integration with multifunctional cell assays to achieve in-situ tracking for differentiation process of stem cells.
Microbubbles (MBs) are known to serve as an amplifier of the mechanical effects of ultrasound, which combined with ultrasound are widely used in brain. The goal of this study is to investigate the effect of oscillating MBs on the neuronal activity in the central nervous system (CNS) of mammals. The motor cortex of mice brain was subjected to ultrasound stimulation with and without MBs, and evoked electromyogram signals were recorded. A c-fos immunofluorescence assay was performed to evaluate the neuronal activation in the region of ultrasound stimulation. BBB integrity during ultrasound stimulation with MBs was assessed in this study. Moreover, the safety of ultrasound stimulation with MBs was examined. Using ultrasound at 620 kHz, the injection of MBs significantly increased the success rate of motor response from 0.065 ± 0.06 to 0.28 ± 0.10 when stimulation was applied at 0.12 MPa and from 0.38 ± 0.09 to 0.77 ± 0.18 at 0.25 MPa (p < 0.001). The results of the c-fos immunofluorescence assay showed that the mean densities of c-fos+ cells were significantly increased from 15.67 ± 3.51 to 53.01 ± 9.54 at 0.12 MPa acoustic pressure. At 0.25 MPa, the mean density of c-fos + cells was 81 ± 10.97 without MBs and it significantly increased to 124.12 ± 25.71 with MBs (p < 0.05). Enhanced neuronal activities were observed with 0.12 MPa ultrasound stimulation with MBs, while the integrity of BBB was not compromised, but 0.25 MPa ultrasound stimulation with MBs resulted in BBB disruption. These findings reveal that the oscillations of MBs can enhance neuronal activity in the CNS of mammals, and may provide an insight into the application of MBs combined with ultrasound in brain.
Electrical stimulation (ES) is widely applied to promote nerve regeneration. Currently, metal needles are used to exert external ES, which may cause pain and risk of infection. In this work, a multiblock conductive nerve scaffold with self‐powered ES by the consumption of glucose and oxygen is prepared. The conductive substrate is prepared by in situ polymerization of polypyrrole (PPy) on the nanofibers of bacterial cellulose (BC). Platinum nanoparticles are electrodeposited on the anode side for glucose oxidation, while nitrogen‐doped carbon nanotubes (N‐CNTs) are loaded on the cathode side for oxygen reduction. The scaffold shows good mechanical property, flexibility and conductivity. The scaffold can form a potential difference of above 300 mV between the anode and the cathode in PBS with 5 × 10⁻³m glucose. Dorsal root ganglions cultured on the Pt‐BC/PPy‐N‐CNTs scaffold are 55% longer in mean neurite length than those cultured on BC/PPy. In addition, in vivo study indicates that the Pt‐BC/PPy‐N‐CNTs scaffold promotes nerve regeneration compared with the BC/PPy group. This paper presents a novel design of a nerve scaffold with self‐powered ES. In the future, it can be combined with other features to promote nerve regeneration.
For the investigation of cell-cell interaction in general and for neural communication and future applications of neural networks, a controllable and well-defined network structure is crucial. We here propose the implementation of an acoustically driven system for tunable and deliberate stimulation and manipulation of cell growth on a chip. This piezoelectric chip allows us to generate a checkerboard-like standing surface acoustic wave pattern coupled to a fluid layer in a microfluidic chamber on top. Such a dynamically induced patterning lattice is shown to allow for the active positioning of the neurons and subsequent guided neurite outgrowth, thus finally overcoming the limitations of static approaches. After thorough characterization of the resulting tunable potential landscape, we successfully demonstrate cell adhesion and even growth of the such positioned cells within the well-defined pressure nodes. We demonstrate neuron growth at predetermined positions and observe a subsequent neurite outgrowth, even being correlated with the artificial potential landscape. For the very delicate and sensitive primary neural cells, this is a change of paradigm! Our experimental findings give us confidence that our hybrid lab-on-a-chip system in the near future will allow researchers to study cell-cell interaction of primary neurons. If scaled to a true network level, it will enable us to control and study how neural networks connect, interact, and communicate.
Externally controlling the excitation of a neuronal subset through ion channels activation can modulate the firing pattern of an entire neural circuit in vivo. As nanovalves in the cell membrane, ion channels can be opened by light (optogenetics) or ultrasonic (sonogenetics) means. A thoroughly analyzed force sensor is the Escherichia coli mechanosensitive channel of large conductance (MscL). Here we expressed MscL in rat hippocampal neurons in a primary culture and showed that it could be activated by low-pressure ultrasound pulses. The gain-of-function mutation, I92L, sensitized MscL’s sonic response, triggering action potentials at a peak negative pressure as low as 0.25 MPa. Further, the I92L MscL reliably elicited individual spikes by timed brief pulses, making excitation programmable. Because MscL opens to tension in the lipid bilayer, requiring no other proteins or ligands, it could be developed into a general noninvasive sonogenetic tool to manipulate the activities of neurons or other cells and potential nanodevices.
Nonspecific binding (NSB) is a general issue for surface based biosensors. Various approaches have been developed to prevent or remove the NSBs. However, these approaches either increased the background signals of the sensors or limited to specific transducers interface. In this work, we developed a hydrodynamic approach to selectively remove the NSBs using a microfabricated hypersonic resonator with 2.5 gigahertz (GHz) resonant frequency. The high frequency device facilitates to generate multiple controlled micro-vortices which then create cleaning forces at the solid-liquid interfaces. The competitive adhesive and cleaning forces have been investigated using the finite element method (FEM) simulation, identifying the feasibility of the vortices induced NSB removal. NSB proteins have been selectively removed experimentally both on the surface of the resonator and on other substrates which contact the vortices. Thus, the developed hydrodynamic approach is believed to be a simple and versatile tool for NSB removal and compatible to many sensor systems. The unique feature of the hypersonic resonator is that it can be used as a gravimetric sensor as well, thus a combined NSB removal and protein detection dual functional biosensor system is developed.
Efficient delivery of genes and therapeutic agents to the interior of the cell is critical for
modern biotechnology. Herein, a new type of chemical-free cell poration method—
hypersonic poration—is developed to improve the cellular uptake, especially the
nucleus uptake. The hypersound (≈GHz) is generated by a designed piezoelectric
nano-electromechanical resonator, which directly induces normal/shear stress and
“molecular bombardment” effects on the bilayer membranes, and creates reversible
temporal nanopores improving the membrane permeability. Both theory analysis
and cellular uptake experiments of exogenous compounds prove the high delivery
efficiency of hypersonic poration. Since target molecules in cells are accumulated with
the treatment, the delivered amount can be controlled by tuning the treatment time.
Furthermore, owing to the intrinsic miniature of the resonator, localized drug delivery
at a confined spatial location and tunable arrays of the resonators that are compatible
with multiwell plate can be achieved. The hypersonic poration method shows great
delivery efficacy combined with advantage of scalability, tunable throughput, and
simplification in operation and provides a potentially powerful strategy in the field of
molecule delivery, cell transfection, and gene therapy.
In the mammalian nervous system, billions of neurons connected by quadrillions of synapses exchange electrical, chemical and mechanical signals. Disruptions to this network manifest as neurological or psychiatric conditions. Despite decades of neuroscience research, our ability to treat or even to understand these conditions is limited by the capability of tools to probe the signalling complexity of the nervous system. Although orders of magnitude smaller and computationally faster than neurons, conventional substrate-bound electronics do not recapitulate the chemical and mechanical properties of neural tissue. This mismatch results in a foreign-body response and the encapsulation of devices by glial scars, suggesting that the design of an interface between the nervous system and a synthetic sensor requires additional materials innovation. Advances in genetic tools for manipulating neural activity have fuelled the demand for devices that are capable of simultaneously recording and controlling individual neurons at unprecedented scales. Recently, flexible organic electronics and bio- and nanomaterials have been developed for multifunctional and minimally invasive probes for long-term interaction with the nervous system. In this Review, we discuss the design lessons from the quarter-century-old field of neural engineering, highlight recent materials-driven progress in neural probes and look at emergent directions inspired by the principles of neural transduction. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Current bioelectronic medicines for neurological therapies generally involve treatment with a bioelectronic system comprising a power supply unit and a bioelectrode device. Further integration of wireless and self-powered units is of practical importance for implantable bioelectronics. In this study we developed biocompatible organic photovoltaics (OPVs) for serving as wireless electrical power supply units that can be operated under illumination with near-infrared (NIR) light, and organic bioelectronic interface (OBEI) electrode devices as neural stimulation electrodes. The OPV/OBEI integrated system is capable to provide electrical stimulation (ES) as a means of enhancing neuron-like PC12 cell differentiation and neurite outgrowth. For the OPV design, we prepared devices incorporating two photoactive material systems-β-carotene/N,N´-dioctyl-3,4,9,10-perylenedicarboximide (β-carotene/PTCDI-C8) and poly(3-hexylthiophene)/phenyl-C61-butyric acid methyl ester (P3HT/PCBM)-that exhibited open circuit voltages of 0.11 and 0.49 V, respectively, under NLED illumination. Then, we connected OBEI devices with different electrode gaps, incorporating biocompatible poly(hydroxymethylated-3,4-ethylenedioxythiophene), to OPVs to precisely tailor the direct current electric field conditions during the culturing of PC12 cells. This NIR light-driven OPV/OBEI system could be engineered to provide tunable control over the electric field (from 220 to 980 mV mm-1) to promote 64% enhancement in the neurite length, and/or to direct the neurite orientation on chips. The OPV/OBEI integrated systems under NIR illumination appear to function as effective power delivery platforms that should meet the requirements for wirelessly offering medical ES to a portion of the nervous system; they might also be a key technology for the development of next-generation implantable bioelectronics.
Neurons, like most cells, exhibit strong morphological responses to the physical features of their environment, and topographical structures are often utilized to elicit unique neuronal behavior. On page 1148, I. S. Choi and co-workers demonstrate directional control over the neurites of primary hippocampal neurons by using anisotropic pillar topographies as a culture platform. The relationship between inter-pillar distances and the fidelity of unidirectional neurite alignment is explored, and it is shown that neurites preferentially elongate along the closest available pillars. This work features a purely physical means of controlling the orientation of neurite outgrowth, and highlights a valuable platform for studies regarding neuroregeneration or neuronal network formation.
Acoustic streaming underpins an exciting range of fluid manipulation phenomena of rapidly growing significance in microfluidics, where the streaming often assumes the form of a steady, laminar jet emanating from the device surface, driven by the attenuation of acoustic energy within the beam of sound propagating through the liquid. The frequencies used to drive such phenomena are often chosen ad hoc to accommodate fabrication and material issues. In this work, we seek a better understanding of the effects of sound frequency and power on acoustic streaming. We present and, using surface acoustic waves, experimentally verify a laminar jet model that is based on the turbulent jet model of Lighthill, which is appropriate for acoustic streaming seen at micro- to nanoscales, between 20 and 936 MHz and over a broad range of input power. Our model eliminates the critically problematic acoustic source singularity present in Lighthill's model, replacing it with a finite emission area and enabling determination of the streaming velocity close to the source. At high acoustic power P (and hence high jet Reynolds numbers ReJ associated with fast streaming), the laminar jet model predicts a one-half power dependence (U∼P1/2∼ ReJ) similar to the turbulent jet model. However, the laminar model may also be applied to jets produced at low powers-and hence low jet Reynolds numbers ReJ-where a linear relationship between the beam power and streaming velocity exists: U∼P∼ReJ2. The ability of the laminar jet model to predict the acoustic streaming behavior across a broad range of frequencies and power provides a useful tool in the analysis of microfluidics devices, explaining peculiar observations made by several researchers in the literature. In particular, by elucidating the effects of frequency on the scale of acoustically driven flows, we show that the choice of frequency is a vitally important consideration in the design of small-scale devices employing acoustic streaming for microfluidics.
In nerve-smooth muscle preparations beta-nicotinamide adenine dinucleotide (beta-NAD) has emerged as a novel extracellular substance with putative neurotransmitter and neuromodulator functions. beta-NAD is released, along with noradrenaline and adenosine 5'-triphosphate (ATP), upon firing of action potentials in blood vessels, urinary bladder and large intestine. At present it is unclear whether noradrenaline, ATP and beta-NAD are stored in and released from common populations of synaptic vesicles. The answer is unattainable in complex systems such as nerve-smooth muscle preparations. Adrenal chromaffin cells are thus used here as a single-cell model to examine mechanisms of concomitant neurosecretion. Using high-performance liquid chromatography techniques with electrochemical and fluorescence detection we simultaneously evaluated secretion of dopamine (DA), ATP, adenosine 5'-diphosphate, adenosine 5'-monophosphate, adenosine, beta-NAD and its immediate metabolites ADP-ribose and cyclic ADP-ribose in superfused nerve growth factor-differentiated rat pheochromocytoma PC12 cells. beta-NAD, DA and ATP were released constitutively and upon stimulation with high-K(+) solution or nicotine. Botulinum neurotoxin A tended to increase the spontaneous secretion of all substances and abolished the high-K(+)-evoked release of beta-NAD and DA but not of ATP. Subcellular fractionation by continuous glycerol and sucrose gradients along with immunoblot analysis of the vesicular marker proteins synaptophysin and secretogranin II revealed that beta-NAD, ATP and DA are stored in both small synaptic-like vesicles and large dense-core-like vesicles. However, the three substances appear to have different preferential sites of release upon membrane depolarization including sites associated with SNAP-25 and sites not associated with SNAP-25.
A key issue in signal transduction is how signaling pathways common to many systems—so-called canonical signaling cassettes—integrate
signals from molecules having a wide spectrum of activities, such as hormones and neurotrophins, to deliver distinct biological
outcomes. The neuroendocrine cell line PC12, derived from rat pheochromocytoma, provides an example of how one canonical signaling
cassette—the Raf → mitogen-activated protein kinase kinase (MEK) → extracellular signal-regulated kinase (ERK) pathway—can
promote distinct outcomes, which in this case include neuritogenesis, gene induction, and proliferation. Two growth hormones,
epidermal growth factor (EGF) and nerve growth factor (NGF), use the same pathway to cause PC12 proliferation and differentiation,
respectively. In addition, pituitary adenylate cyclase–activating polypeptide (PACAP), a neurotransmitter that also causes
differentiation, uses the same canonical cassette as NGF but in a different way. The Connections Map for PC12 Cell Differentiation
brings into focus the complex array of specific cellular responses that rely on canonical signal transduction systems.
Interest in brain stimulation therapies has been rejuvenated over the last decade and brain stimulation therapy has become an alternative treatment for many neurological and psychiatric disorders, including Parkinson's disease (PD), dystonia, pain, epilepsy, depression, and schizophrenia. The effects of brain stimulation on PD are well described, and this treatment has been widely used for such conditions worldwide. Treatments for other conditions are still in experimental stages and large-scale, well controlled studies are needed to refine the treatment procedures. In the treatment of intractable brain disorders, brain stimulation, especially transcranial magnetic stimulation (TMS), is an attractive alternative to surgical lesioning as it is relatively safe, reversible, and flexible. Brain stimulation, delivered either via deeply implanted electrodes or from a surface-mounted transcranial magnetic device, can alter abnormal neural circuits underlying brain disorders. The neural mechanisms mediating the beneficial effects of brain stimulation, however, are poorly understood. Conflicting theories and experimental data have been presented. It seems that the action of stimulation on brain circuitry is not limited to simple excitation or inhibition. Alterations of neural firing patterns and long-term effects on neurotransmitter and receptor systems may also play important roles in the therapeutic effects of brain stimulation. Future research on both the basic and clinical fronts will deepen our understanding of how brain stimulation works. Real-time computation of neural activity allows for integration of brain stimulation signals into ongoing neural processing. In this way abnormal circuit activity can be adjusted by optimal therapeutic brain stimulation paradigms.
In vitro techniques are used increasingly to screen for and characterize neurotoxicants. In many cases, chemical-induced injury to developing neurons has been examined in vitro by assessing morphological changes in differentiation and neurite growth. This research evaluated the use of proteins associated with axonal growth and synaptogenesis as surrogates for morphological measurement of neuronal differentiation. PC12 cells, which differentiate upon nerve growth factor (NGF) stimulation, were used as the in vitro model. NGF-induced (50 ng/ml) differentiation (cells with at least one neurite with a length equal to the cell body diameter) and neurite growth (length of longest neurite) were determined using light microscopy and computer-based quantitative image analysis. PC12 cell differentiation and neurite growth reached a plateau after 6 days in culture. Expression of the axonal growth associated protein 43 (GAP-43) and the synaptic protein synapsin I were assessed simultaneously by Western blot during cell differentiation. Expression of GAP-43 was low on Culture Day 0 and increased progressively to maximum levels on Culture Day 5. Likewise, synapsin I expression increased slowly on Days 0-4, and then rapidly on Days 5-7 of culture. Pharmacologic inhibitors of NGF-induced signaling were used to test the sensitivity of the proteins to chemical disruption of differentiation. The MAP kinase inhibitor, U0126 (5-30 microM) and the PKC inhibitor, bisindolylmaleimide I (Bis I; 1.25-5 microM) inhibited differentiation and neurite outgrowth in a concentration-dependent manner. U0126 and Bis I significantly decreased GAP-43, but not synapsin I expression. Interestingly, the PI-PLC inhibitor edelfosine (ET-18; 5-30 microM) stimulated differentiation at early times of exposure followed by a significant decrease in neurite length at later time points. However, ET-18 did not alter the expression of GAP-43 or synapsin I. These data suggest that GAP-43 may be a useful indicator of the status of PC12 cell differentiation.
Microenvironments appear important in stem cell lineage specification but can be difficult to adequately characterize or control with soft tissues. Naive mesenchymal stem cells (MSCs) are shown here to specify lineage and commit to phenotypes with extreme sensitivity to tissue-level elasticity. Soft matrices that mimic brain are neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone prove osteogenic. During the initial week in culture, reprogramming of these lineages is possible with addition of soluble induction factors, but after several weeks in culture, the cells commit to the lineage specified by matrix elasticity, consistent with the elasticity-insensitive commitment of differentiated cell types. Inhibition of nonmuscle myosin II blocks all elasticity-directed lineage specification-without strongly perturbing many other aspects of cell function and shape. The results have significant implications for understanding physical effects of the in vivo microenvironment and also for therapeutic uses of stem cells.
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