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[This corrects the article DOI: 10.3389/fncel.2023.1287089.].
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While there is a growing appreciation of three-dimensional (3D) neural tissues (i.e., hydrogel-based, organoids, and spheroids), shown to improve cellular health and network activity to mirror brain-like activity in vivo, functional assessment using current electrophysiology techniques (e.g., planar multi-electrode arrays or patch clamp) has been technically challenging and limited to surface measurements at the bottom or top of the 3D tissue. As next-generation MEAs, specifically 3D MEAs, are being developed to increase the spatial precision across all three dimensions (X, Y, Z), development of improved computational analytical tools to discern region-specific changes within the Z dimension of the 3D tissue is needed. In the present study, we introduce a novel computational analytical pipeline to analyze 3D neural network activity recorded from a "bottom-up" 3D MEA integrated with a 3D hydrogel-based tissue containing human iPSC-derived neurons and primary astrocytes. Over a period of ~6.5 weeks, we describe the development and maturation of 3D neural activity (i.e., features of spiking and bursting activity) within cross sections of the 3D tissue, based on the vertical position of the electrode on the 3D MEA probe, in addition to network activity (identified using synchrony analysis) within and between cross sections. Then, using the sequential addition of postsynaptic receptor antagonists, bicuculline (BIC), 2-amino-5-phosphonovaleric acid (AP-5), and 6-cyano-5-nitroquinoxaline-2,3-dione (CNQX), we demonstrate that networks within and between cross sections of the 3D hydrogel-based tissue show a preference for GABA and/or glutamate synaptic transmission, suggesting differences in the network composition throughout the neural tissue. The ability to monitor the functional dynamics of the entire 3D reconstructed neural tissue is a critical bottleneck; here we demonstrate a computational pipeline that can be implemented in studies to better interpret network activity within an engineered 3D neural tissue and have a better understanding of the modeled organ tissue.
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ABSTRACT: At the start of the COVID-19 pandemic, the US faced nationwide shortages of nasopharyngeal swabs due to both overwhelmed supply chains and an increase in demand. To address this shortfall, multiple 3D printed swabs were ultimately produced and sold for COVID-19 testing. In this work, we present a framework for mechanical and functional bench-testing of nasopharyngeal swabs using standard and widely available material testing equipment. Using this framework, we offer a comprehensive, quantitative comparison of the 3D printed swabs to benchmark their performance against traditional flocked swabs. The test protocols were designed to emulate the clinical use of the nasopharyngeal swabs and to evaluate potential failure modes. Overall, the 3D printed swabs performed comparably to, or outperformed, the traditional swabs in all mechanical tests. While traditional swabs outperformed some of the new 3D printed swabs in terms of sample uptake and retention, similar amounts of RNA were recovered from both 3D printed and traditional swabs.
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The hippocampus is diversely interconnected with other brain systems along its axis. Cycles of theta-frequency activity are believed to propagate from the septal to temporal pole, yet it is unclear how this one-way route supports the flexible cognitive capacities of this structure. We leveraged novel thin-film microgrid arrays conformed to the human hippocampal surface to track neural activity two-dimensionally in vivo. All oscillation frequencies identified between 1-15 Hz propagated across the tissue. Moreover, they dynamically shifted between two roughly opposite directions oblique to the long axis. This predominant propagation axis was mirrored across participants, hemispheres, and consciousness states. Directionality was modulated in a participant who performed a behavioral task, and it could be predicted by wave amplitude topography over the hippocampal surface. Our results show that propagation directions may thus represent distinct meso-scale network computations, operating along versatile spatiotemporal processing routes across the hippocampal body.
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Hipocampo/fisiología , Ritmo Teta/fisiología , Conducta/fisiología , Electrocorticografía , Humanos , Modelos Neurológicos , Tabique del Cerebro/fisiología , Lóbulo Temporal/fisiologíaRESUMEN
Three-dimensional (3D) in vitro models have become increasingly popular as systems to study cell-cell and cell-ECM interactions dependent on the spatial, mechanical, and chemical cues within the environment of the tissue, which is limited in traditional two-dimensional (2D) models. Although electrophysiological recordings of neuronal action potentials through 2D microelectrode arrays (MEAs) are a common and trusted method of evaluating neuronal function, network communication, and response to chemicals and biologicals, there are currently limited options for measuring electrophysiological activity from many locations simultaneously throughout a 3D network of neurons in vitro. Here, we have developed a thin-film, 3D flexible microelectrode array (3DMEA) that non-invasively interrogates a 3D culture of neurons and can accommodate 256 channels of recording or stimulation. Importantly, the 3DMEA is straightforward to fabricate and integrates with standard commercially available electrophysiology hardware. Polyimide probe arrays were microfabricated on glass substrates and mechanically actuated to collectively lift the arrays into a vertical position, relying solely on plastic deformation of their base hinge regions to maintain vertical alignment. Human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes were entrapped in a collagen-based hydrogel and seeded onto the 3DMEA, enabling growth of suspended cells in the matrix and the formation and maturation of a neural network around the 3DMEA probes. The 3DMEA supported the growth of functional neurons in 3D with action potential spike and burst activity recorded over 45 days in vitro. This platform is an important step in facilitating noninvasive electrophysiological characterization of 3D networks of electroactive cells in vitro.
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Células Madre Pluripotentes Inducidas , Potenciales de Acción , Encéfalo , Humanos , Microelectrodos , NeuronasRESUMEN
From animal experiments by Cohen and Suzuki et al. in the 1960s to the first-in-human clinical trials now in progress, prosthetic electrical stimulation targeting semicircular canal branches of the vestibular nerve has proven effective at driving directionally appropriate vestibulo-ocular reflex eye movements, postural responses, and perception. That work was considerably facilitated by the fact that all hair cells and primary afferent neurons in each canal have the same directional sensitivity to head rotation, the three canals' ampullary nerves are geometrically distinct from one another, and electrically evoked three-dimensional (3D) canal-ocular reflex responses approximate a simple vector sum of linearly independent components representing relative excitation of each of the three canals. In contrast, selective prosthetic stimulation of the utricle and saccule has been difficult to achieve, because hair cells and afferents with many different directional sensitivities are densely packed in those endorgans and the relationship between 3D otolith-ocular reflex responses and the natural and/or prosthetic stimuli that elicit them is more complex. As a result, controversy exists regarding whether selective, controllable stimulation of electrically evoked otolith-ocular reflexes (eeOOR) is possible. Using micromachined, planar arrays of electrodes implanted in the labyrinth, we quantified 3D, binocular eeOOR responses to prosthetic electrical stimulation targeting the utricle, saccule, and semicircular canals of alert chinchillas. Stimuli delivered via near-bipolar electrode pairs near the maculae elicited sustained ocular countertilt responses that grew reliably with pulse rate and pulse amplitude, varied in direction according to which stimulating electrode was employed, and exhibited temporal dynamics consistent with responses expected for isolated macular stimulation.NEW & NOTEWORTHY As the second in a pair of papers on Binocular 3D Otolith-Ocular Reflexes, this paper describes new planar electrode arrays and vestibular prosthesis architecture designed to target the three semicircular canals and the utricle and saccule. With this technological advancement, electrically evoked otolith-ocular reflexes due to stimulation via utricle- and saccule-targeted electrodes were recorded in chinchillas. Results demonstrate advances toward achieving selective stimulation of the utricle and saccule.
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Chinchilla/fisiología , Movimientos Oculares/fisiología , Prótesis Neurales , Membrana Otolítica/fisiología , Reflejo Vestibuloocular/fisiología , Sáculo y Utrículo/fisiología , Canales Semicirculares/fisiología , Animales , Estimulación Eléctrica , Tecnología de Seguimiento OcularRESUMEN
Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas. The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days. This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.
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Electrodos Implantados/normas , Fenómenos Electrofisiológicos/fisiología , Polímeros/normas , Animales , RatasRESUMEN
This protocol demonstrates a method for electrochemical roughening of thin-film platinum electrodes without preferential dissolution at grain boundaries of the metal. Using this method, a crack free, thin-film macroelectrode surface with up to 40 times increase in active surface area was obtained. The roughening is easy to do in a standard electrochemical characterization laboratory and incudes the application of voltage pulses followed by extended application of a reductive voltage in a perchloric acid solution. The protocol includes the chemical and electrochemical preparation of both a macroscale (1.2 mm diameter) and microscale (20 µm diameter) platinum disc electrode surface, roughening the electrode surface and characterizing the effects of surface roughening on electrode active surface area. This electrochemical characterization includes cyclic voltammetry and impedance spectroscopy and is demonstrated for both the macroelectrodes and the microelectrodes. Roughening increases electrode active surface area, decreases electrode impedance, increases platinum charge injection limits to those of titanium nitride electrodes of same geometry and improves substrates for adhesion of electrochemically deposited films.
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Técnicas Electroquímicas , Platino (Metal) , Impedancia Eléctrica , Electrodos , Propiedades de SuperficieRESUMEN
OBJECTIVE: Electrode arrays for chronic implantation in the brain are a critical technology in both neuroscience and medicine. Recently, flexible, thin-film polymer electrode arrays have shown promise in facilitating stable, single-unit recordings spanning months in rats. While array flexibility enhances integration with neural tissue, it also requires removal of the dura mater, the tough membrane surrounding the brain, and temporary bracing to penetrate the brain parenchyma. Durotomy increases brain swelling, vascular damage, and surgical time. Insertion using a bracing shuttle results in additional vascular damage and brain compression, which increase with device diameter; while a higher-diameter shuttle will have a higher critical load and more likely penetrate dura, it will damage more brain parenchyma and vasculature. One way to penetrate the intact dura and limit tissue compression without increasing shuttle diameter is to reduce the force required for insertion by sharpening the shuttle tip. APPROACH: We describe a novel design and fabrication process to create silicon insertion shuttles that are sharp in three dimensions and can penetrate rat dura, for faster, easier, and less damaging implantation of polymer arrays. Sharpened profiles are obtained by reflowing patterned photoresist, then transferring its sloped profile to silicon with dry etches. MAIN RESULTS: We demonstrate that sharpened shuttles can reliably implant polymer probes through dura to yield high quality single unit and local field potential recordings for at least 95 days. On insertion directly through dura, tissue compression is minimal. SIGNIFICANCE: This is the first demonstration of a rat dural-penetrating array for chronic recording. This device obviates the need for a durotomy, reducing surgical time and risk of damage to the blood-brain barrier. This is an improvement to state-of-the-art flexible polymer electrode arrays that facilitates their implantation, particularly in multi-site recording experiments. This sharpening process can also be integrated into silicon electrode array fabrication.
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Encéfalo/fisiología , Duramadre/fisiología , Electrodos Implantados , Diseño de Equipo/métodos , Microtecnología/métodos , Silicio , Animales , Materiales Biocompatibles , Diseño de Equipo/instrumentación , Masculino , Microelectrodos , Microtecnología/instrumentación , Ratas , Ratas Long-EvansRESUMEN
The brain is a massive neuronal network, organized into anatomically distributed sub-circuits, with functionally relevant activity occurring at timescales ranging from milliseconds to years. Current methods to monitor neural activity, however, lack the necessary conjunction of anatomical spatial coverage, temporal resolution, and long-term stability to measure this distributed activity. Here we introduce a large-scale, multi-site, extracellular recording platform that integrates polymer electrodes with a modular stacking headstage design supporting up to 1,024 recording channels in freely behaving rats. This system can support months-long recordings from hundreds of well-isolated units across multiple brain regions. Moreover, these recordings are stable enough to track large numbers of single units for over a week. This platform enables large-scale electrophysiological interrogation of the fast dynamics and long-timescale evolution of anatomically distributed circuits, and thereby provides a new tool for understanding brain activity.
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Encéfalo/fisiología , Electrodos Implantados/normas , Fenómenos Electrofisiológicos/fisiología , Red Nerviosa/fisiología , Polímeros/normas , Animales , Electrodos Implantados/tendencias , Masculino , Ratas , Ratas Long-EvansRESUMEN
Understanding the detailed dynamics of neuronal networks will require the simultaneous measurement of spike trains from hundreds of neurons (or more). Currently, approaches to extracting spike times and labels from raw data are time consuming, lack standardization, and involve manual intervention, making it difficult to maintain data provenance and assess the quality of scientific results. Here, we describe an automated clustering approach and associated software package that addresses these problems and provides novel cluster quality metrics. We show that our approach has accuracy comparable to or exceeding that achieved using manual or semi-manual techniques with desktop central processing unit (CPU) runtimes faster than acquisition time for up to hundreds of electrodes. Moreover, a single choice of parameters in the algorithm is effective for a variety of electrode geometries and across multiple brain regions. This algorithm has the potential to enable reproducible and automated spike sorting of larger scale recordings than is currently possible.
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Potenciales de Acción/fisiología , Algoritmos , Neuronas/fisiología , Procesamiento de Señales Asistido por Computador , Programas Informáticos , Animales , Automatización , Encéfalo/fisiología , Masculino , RatasRESUMEN
BACKGROUND: Current source density analysis of recordings from penetrating electrode arrays has traditionally been used to examine the layer- specific cortical activation and plastic changes associated with changed afferent input. We report on a related analysis, the second spatial derivative (SSD) of surface local field potentials (LFPs) recorded using custom designed thin-film polyimide substrate arrays. RESULTS: SSD analysis of tone- evoked LFPs generated from the auditory cortex under the recording array demonstrated a stereotypical single local minimum, often flanked by maxima on both the caudal and rostral sides. In contrast, tone-pips at frequencies not represented in the region under the array, but known (on the basis of normal tonotopic organization) to be represented caudal to the recording array, had a more complex pattern of many sources and sinks. COMPARISON WITH EXISTING METHODS: Compared to traditional analysis of LFPs, SSD analysis produced a tonotopic map that was more similar to that obtained with multi-unit recordings in a normal-hearing animal. Additionally, the statistically significant decrease in the number of acoustically responsive cortical locations in partially deafened cats following 6 months of cochlear implant use compared to unstimulated cases observed with multi-unit data (p=0.04) was also observed with SSD analysis (p=0.02), but was not apparent using traditional analysis of LFPs (p=0.6). CONCLUSIONS: SSD analysis of surface LFPs from the thin-film array provides a rapid and robust method for examining the spatial distribution of cortical activity with improved spatial resolution compared to more traditional LFP recordings.
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Corteza Auditiva/fisiología , Percepción Auditiva/fisiología , Electrodos Implantados , Procesamiento de Señales Asistido por Computador , Estimulación Acústica , Animales , Corteza Auditiva/fisiopatología , Gatos , Implantes Cocleares , Sordera/fisiopatología , Sordera/rehabilitación , Modelos Animales de Enfermedad , Diseño de Equipo , Potenciales Evocados Auditivos , KanamicinaRESUMEN
The brain is a massively interconnected network of specialized circuits. Even primary sensory areas, once thought to support relatively simple, feed-forward processing, are now known to be parts of complex feedback circuits. All brain functions depend on millisecond timescale interactions across these brain networks. Current approaches cannot measure or manipulate such large-scale interactions. Here we demonstrate that polymer-based, penetrating, micro-electrode arrays can provide high quality neural recordings from awake, behaving animals over periods of months. Our results indicate that polymer electrodes are a viable substrate for the development of systems that can record from thousands of channels across months to years. This is our first step towards developing a 1000+ electrode system capable of providing high-quality, long-term neural recordings.
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Mapeo Encefálico/métodos , Electrodos Implantados , Neuronas/patología , Polímeros/química , Procesamiento de Señales Asistido por Computador , Animales , Animales Modificados Genéticamente , Encéfalo/fisiología , Electrofisiología/métodos , Diseño de Equipo , Ratas , Ratas Long-Evans , Interfaz Usuario-ComputadorRESUMEN
We report a novel method to fabricate chronic neural interfaces with the intent to combine the reliability and lifetime of bulk metal electrodes, with the miniaturization and mechanical flexibility of thin-film polymer microelectrode arrays. 10 µm thick platinum discs were laser cut from a foil into the shape of individual electrodes, and coated with gold on the backside. The discs were bonded to a microelectrode array with gold bond pads via gold-gold inter-diffusion using a flipchip bonder. Electrode bonding and adhesion was characterized using mechanical shear testing and electrical testing. Electrode performance was characterized in vitro using electrochemical impedance spectroscopy and cyclic voltammetry. Biphasic electrical pulsing experiments were conducted on the bonded electrodes to study degradation of the electrode; the preliminary results show that the electrodes can withstand at least 4,900 million pulses with no adverse electrochemical or visual degradation. Overall, this is a promising new method for fabricating chronic neural electrodes for stimulation or recording that combines the reliability of commercial bulk electrodes with the miniaturization and versatility of microfabricated technologies.
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Estimulación Encefálica Profunda/instrumentación , Microelectrodos , Diseño de EquipoRESUMEN
The translation of advances in neural stimulation and recording research into clinical practice hinges on the ability to perform chronic experiments in awake and behaving animal models. Advances in microelectrode array technology, most notably flexible polymer arrays, have significantly improved reliability of the neural interface. However, electrical connector technology has lagged and is prone to failure from non-biocompatibility, large size, contamination, corrosion, and difficulty of use. We present a novel chronic, percutaneous electrical connector system that is suitable for neural stimulation and recording. This system features biocompatible materials, low connect and disconnect forces, passive alignment, and a protective cap during non-use. We have successfully designed, assembled, and tested in vitro both a 16-channel system and a high density 64-channel system. Custom, polyimide, 16-channel, microelectrode arrays were electrically assembled with the connector system and tested using cyclic voltammetry and electrochemical impedance spectroscopy. This connector system is versatile and can be used with a variety of microelectrode array technologies for chronic studies.
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Microelectrodos , Animales , Estimulación Eléctrica , Electricidad , ElectroquímicaRESUMEN
We report a novel nano-cluster platinum (NCPt) film that exhibits enhanced performance as an electrode material for neural stimulation applications. Nano-cluster films were deposited using a custom physical vapor deposition process and patterned on a flexible polyimide microelectrode array using semiconductor processing technology. Electrode performance was characterized in vitro using electrochemical impedance spectroscopy and compared with sputtered thinfilm platinum (TFPt) electrodes. We characterized electrode impedance, charge storage capacity, voltage transient properties, and relative surface area enhancement in vitro. Preliminary lifetime testing of the electrode reveals that the NCPt electrodes degrade more slowly than TFPt electrodes. The combination of material biocompatibility, electrochemical performance, and preliminary lifetime results point to a promising new electrode material for neural interface devices.
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Estimulación Eléctrica/métodos , Platino (Metal)/química , Impedancia Eléctrica , Técnicas Electroquímicas , Electrodos , Electrones , Microscopía Electrónica de Rastreo , Microtecnología , Nanopartículas/química , Imagen ÓpticaRESUMEN
Microelectrode arrays for neural interface devices that are made of biocompatible thin-film polymer are expected to have extended functional lifetime because the flexible material may minimize adverse tissue response caused by micromotion. However, their flexibility prevents them from being accurately inserted into neural tissue. This article demonstrates a method to temporarily attach a flexible microelectrode probe to a rigid stiffener using biodissolvable polyethylene glycol (PEG) to facilitate precise, surgical insertion of the probe. A unique stiffener design allows for uniform distribution of the PEG adhesive along the length of the probe. Flip-chip bonding, a common tool used in microelectronics packaging, enables accurate and repeatable alignment and attachment of the probe to the stiffener. The probe and stiffener are surgically implanted together, then the PEG is allowed to dissolve so that the stiffener can be extracted leaving the probe in place. Finally, an in vitro test method is used to evaluate stiffener extraction in an agarose gel model of brain tissue. This approach to implantation has proven particularly advantageous for longer flexible probes (>3 mm). It also provides a feasible method to implant dual-sided flexible probes. To date, the technique has been used to obtain various in vivo recording data from the rat cortex.
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Implantes Absorbibles , Adhesivos/química , Encéfalo/fisiología , Microelectrodos , Polietilenglicoles/química , Animales , Encéfalo/cirugía , RatonesRESUMEN
We present here a microfabricated, multi-functional neural interface with the ability to selectively apply electrical and chemical stimuli, while simultaneously monitoring both electrical and chemical activity in the brain. Such a comprehensive approach is required to understand and treat neuropsychiatric disorders, such as major depressive disorder (MDD), and to understand the mechanisms underlying treatments, such as pharmaceutical therapies and deep brain stimulation (DBS). The polymer-based, multi-functional neural interface is capable of electrical stimulation and recording, targeted drug delivery, and electrochemical sensing. A variety of different electrode and fluidic channel arrangements are possible with this fabrication process. Preliminary testing has shown the suitability of these neural interfaces for in vivo electrical stimulation and recording, as well as in vitro chemical sensing. Testing of the in vitro drug delivery and combined in vivo functionalities this neural interface are currently underway.
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Sistemas de Liberación de Medicamentos/instrumentación , Electrodos Implantados , Polímeros/química , Encéfalo/fisiología , Estimulación Encefálica Profunda/instrumentación , Trastorno Depresivo Mayor , Estimulación Eléctrica/instrumentación , Electroquímica/instrumentación , Electroquímica/métodos , Diseño de Equipo , Humanos , Sistemas Hombre-Máquina , Microtecnología , Neuronas/metabolismoRESUMEN
Flexible polymer probes are expected to enable extended interaction with neural tissue by minimizing damage from micromotion and reducing inflammatory tissue response. However, their flexibility prevents them from being easily inserted into the tissue. This paper describes an approach for temporarily attaching a silicon stiffener with biodissolvable polyethylene glycol (PEG) so that the stiffener can be released from the probe and extracted shortly after probe placement. A novel stiffener design with wicking channels, along with flip-chip technology, enable accurate alignment of the probe to the stiffener, as well as uniform distribution of the PEG adhesive. Insertion, extraction, and electrode function were tested in both agarose gel and a rat brain. Several geometric and material parameters were tested to minimize probe displacement during stiffener extraction. We demonstrated average probe displacement of 28 ± 9 µm.
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Materiales Biocompatibles/farmacología , Encéfalo , Polietilenglicoles/farmacología , Silicio/farmacología , Adhesivos Tisulares/farmacología , Animales , Materiales Biocompatibles/química , Electrodos , Polietilenglicoles/química , Ratas , Sefarosa/química , Sefarosa/farmacología , Silicio/química , Adhesivos Tisulares/químicaRESUMEN
We present here a demonstration of a dual-sided, 4-layer metal, polyimide-based electrode array suitable for neural stimulation and recording. The fabrication process outlined here utilizes simple polymer and metal deposition and etching steps, with no potentially harmful backside etches or long exposures to extremely toxic chemicals. These polyimide-based electrode arrays have been tested to ensure they are fully biocompatible and suitable for long-term implantation; their flexibility minimizes the injury and glial scarring that can occur at the implantation site. The creation of dual-side electrode arrays with more than two layers of trace metal enables the fabrication of neural probes with more electrodes without a significant increase in probe size. This allows for more stimulation/recording sites without inducing additional injury and glial scarring.