Design, Fabrication, and Implantation of Invasive Microelectrode Arrays as in vivo Brain Machine Interfaces: A Comprehensive Review.

Invasive Microelectrode Arrays (MEAs) have been a significant and useful tool for us to gain a fundamental understanding of how the brain works through high spatiotemporal resolution neuron-level recordings and/or stimulations. Through decades of research, various types of microwire, silicon, and flexible substrate-based MEAs have been developed using the evolving new materials, novel design concepts, and cutting-edge advanced manufacturing capabilities. Surgical implantation of the latest minimal damaging flexible MEAs through the hard-to-penetrate brain membranes introduces new challenges and thus the development of implantation strategies and instruments for the latest MEAs. In this paper, studies on the design considerations and enabling manufacturing processes of various invasive MEAs as in vivo brain-machine interfaces have been reviewed to facilitate the development as well as the state-of-art of such brain-machine interfaces from an engineering perspective. The challenges and solution strategies developed for surgically implanting such interfaces into the brain have also been evaluated and summarized. Finally, the research gaps have been identified in the design, manufacturing, and implantation perspectives, and future research prospects in invasive MEA development have been proposed.

Unveiling the impact of low-frequency electrical stimulation on network synchronization and learning behavior in cultured hippocampal neural networks.

Understanding the dynamics of neural networks and their response to external stimuli is crucial for unraveling the mechanisms associated with learning processes. In this study, we hypothesized that electrical stimulation (ES) would lead to significant alterations in the activity patterns of hippocampal neuronal networks and investigated the effects of low-frequency ES on hippocampal neuronal populations using the microelectrode arrays (MEAs). Our findings revealed significant alterations in the activity of hippocampal neuronal networks following low-frequency ES trainings. Post-stimulation, the neural activity exhibited an organized burst firing pattern characterized by increased spike and burst firings, increased synchronization, and enhanced learning behaviors. Analysis of peri-stimulus time histograms (PSTHs) further revealed that low-frequency ES (1Hz) significantly enhanced neural plasticity, thereby facilitating the learning process of cultured neurons, whereas high-frequency ES (>10Hz) impeded this process. Moreover, we observed a substantial increase in correlations and connectivity within neuronal networks following ES trainings. These alterations in network properties indicated enhanced synaptic plasticity and emphasized the positive impact of low-frequency ES on hippocampal neural activities, contributing to the brain's capacity for learning and memory.

Neuromodulatory Compensation of Cortical Neural Activity on Electrodeposited Pt/Ir Modified Microelectrode Arrays for Temperature Transients.

Temperature has a profound influence on various neuromodulation processes and has emerged as a focal point. However, the effects of acute environmental temperature fluctuations on cultured cortical networks have been inadequately elucidated. To bridge this gap, we have developed a brain-on-a-chip platform integrating cortical networks and electrodeposited Pt/Ir modified microelectrode arrays (MEAs) with 3D-printed bear-shaped triple chambers, facilitating control of temperature transients. This innovative system administers thermal stimuli while concurrently monitoring neuronal activity, including spikes and local field potentials, from 60 microelectrodes (diameter: 30 µm; impedance: 9.34 ± 1.37 kΩ; and phase delay: -45.26 ± 2.85°). Temperature transitions of approximately ±10 °C/s were applied to cortical networks on MEAs via in situ perfusion within the triple chambers. Subsequently, we examined the spatiotemporal dynamics of the brain-on-a-chip under temperature regulation at both the group level (neuronal population) and their interactions (network dynamics) and the individual level (cellular activity). Specifically, we found that after the temperature reduction neurons enhanced the overall information transmission efficiency of the network through synchronous firing to compensate for the decreased efficiency of single-cell level information transmission, in contrast to temperature elevation. By leveraging the integration of high-performance MEAs with perfusion chambers, this investigation provides a comprehensive understanding of the impact of temperature on the spatiotemporal dynamics of neural networks, thereby facilitating future exploration of the intricate interplay between temperature and brain function.

The Profile of Network Spontaneous Activity and Functional Organization Interplay in Hierarchically Connected Modular Neural Networks In Vitro.

Modern microtechnology methods are widely used to create neural networks on a chip with a connection architecture demonstrating properties of modularity and hierarchy similar to brain networks. Such in vitro networks serve as a valuable model for studying the interplay of functional architecture within modules, their activity, and the effectiveness of inter-module interaction. In this study, we use a two-chamber microfluidic platform to investigate functional connectivity and global activity in hierarchically connected modular neural networks. We found that the strength of functional connections within the module and the profile of network spontaneous activity determine the effectiveness of inter-modular interaction and integration activity in the network. The direction of intermodular activity propagation configures the different densities of inhibitory synapses in the network. The developed microfluidic platform holds the potential to explore function-structure relationships and efficient information processing in two- or multilayer neural networks, in both healthy and pathological states.

Novel Multiparametric Bioelectronic Measurement System for Monitoring Virus-Induced Alterations in Functional Neuronal Networks.

Development and optimisation of bioelectronic monitoring techniques like microelectrode array-based field potential measurement and impedance spectroscopy for the functional, label-free and non-invasive monitoring of in vitro neuronal networks is widely investigated in the field of biosensors. Thus, these techniques were individually used to demonstrate the capabilities of, e.g., detecting compound-induced toxicity in neuronal culture models. In contrast, extended application for investigating the effects of central nervous system infecting viruses are rarely described. In this context, we wanted to analyse the effect of herpesviruses on functional neuronal networks. Therefore, we developed a unique hybrid bioelectronic monitoring platform that allows for performing field potential monitoring and impedance spectroscopy on the same microelectrode. In the first step, a neuronal culture model based on primary hippocampal cells from neonatal rats was established with reproducible and stable synchronised electrophysiological network activity after 21 days of cultivation on microelectrode arrays. For a proof of concept, the pseudorabies model virus PrV Kaplan-ΔgG-GFP was applied and the effect on the neuronal networks was monitored by impedance spectroscopy and field potential measurement for 72 h in a multiparametric mode. Analysis of several bioelectronic parameters revealed a virus concentration-dependent degeneration of the neuronal network within 24-48 h, with a significant early change in electrophysiological activity, subsequently leading to a loss of activity and network synchronicity. In conclusion, we successfully developed a microelectrode array-based hybrid bioelectronic measurement platform for quantitative monitoring of pathologic effects of a herpesvirus on electrophysiological active neuronal networks.

Tentacle Microelectrode Arrays Uncover Soft Boundary Neurons in Hippocampal CA1.

Hippocampal CA1 neurons show intense firing at specific spatial locations, modulated by isolated landmarks. However, the impact of real-world scene transitions on neuronal activity remains unclear. Moreover, long-term neural recording during movement challenges device stability. Conventional rigid-based electrodes cause inflammatory responses, restricting recording durations. Inspired by the jellyfish tentacles, the multi-conductive layer ultra-flexible microelectrode arrays (MEAs) are developed. The tentacle MEAs ensure stable recordings during movement, thereby enabling the discovery of soft boundary neurons. The soft boundary neurons demonstrate high-frequency firing that aligns with the boundaries of scene transitions. Furthermore, the localization ability of soft boundary neurons improves with more scene transition boundaries, and their activity decreases when these boundaries are removed. The innovation of ultra-flexible, high-biocompatible tentacle MEAs improves the understanding of neural encoding in spatial cognition. They offer the potential for long-term in vivo recording of neural information, facilitating breakthroughs in the understanding and application of brain spatial navigation mehanisms.

Engineering an in vitro retinothalamic nerve model.

Understanding the retinogeniculate pathway in vitro can offer insights into its development and potential for future therapeutic applications. This study presents a Polydimethylsiloxane-based two-chamber system with axon guidance channels, designed to replicate unidirectional retinogeniculate signal transmission in vitro. Using embryonic rat retinas, we developed a model where retinal spheroids innervate thalamic targets through up to 6 mm long microfluidic channels. Using a combination of electrical stimulation and functional calcium imaging we assessed how channel length and electrical stimulation frequency affects thalamic target response. In the presented model we integrated up to 20 identical functional retinothalamic neural networks aligned on a single transparent microelectrode array, enhancing the robustness and quality of recorded functional data. We found that network integrity depends on channel length, with 0.5-2 mm channels maintaining over 90% morphological and 50% functional integrity. A reduced network integrity was recorded in longer channels. The results indicate a notable reduction in forward spike propagation in channels longer than 4 mm. Additionally, spike conduction fidelity decreased with increasing channel length. Yet, stimulation-induced thalamic target activity remained unaffected by channel length. Finally, the study found that a sustained thalamic calcium response could be elicited with stimulation frequencies up to 31 Hz, with higher frequencies leading to transient responses. In conclusion, this study presents a high-throughput platform that demonstrates how channel length affects retina to brain network formation and signal transmission in vitro.

Investigating Communication Dynamics in Neuronal Network using 3D Gold Microelectrode Arrays.

Although in vitro neuronal network models hold great potential for advancing neuroscience research, with the capacity to provide fundamental insights into mechanisms underlying neuronal functions, the dynamics of cell communication within such networks remain poorly understood. Here, we develop a customizable, polymer modified three-dimensional gold microelectrode array with sufficient stability for high signal-to-noise, long-term, neuronal recording of cultured networks. By using directed spatial and temporal patterns of electrical stimulation of cells to explore synaptic-based communication, we monitored cell network dynamics over 3 weeks, quantifying communication capability using correlation heatmaps and mutual information networks. Analysis of synaptic delay and signal speed between cells enabled us to establish a communication connectivity model. We anticipate that our discoveries of the dynamic changes in communication across the neuronal network will provide a valuable tool for future studies in understanding health and disease as well as in developing effective platforms for evaluating therapies.

High-Performance Bidirectional Microelectrode Array for Assessing Sevoflurane Anesthesia Effects and In Situ Electrical Stimulation in Deep Brain Regions.

Precise assessment of wakefulness states during sevoflurane anesthesia and timely arousal are of paramount importance to refine the control of anesthesia. To tackle this issue, a bidirectional implantable microelectrode array (MEA) is designed with the capability to detect electrophysiological signal and perform in situ deep brain stimulation (DBS) within the dorsomedial hypothalamus (DMH) of mice. The MEA, modified with platinum nanoparticles/IrOx nanocomposites, exhibits exceptional characteristics, featuring low impedance, minimal phase delay, substantial charge storage capacity, high double-layer capacitance, and longer in vivo lifetime, thereby enhancing the sensitivity of spike firing detection and electrical stimulation (ES) effectiveness. Using this MEA, sevoflurane-inhibited neurons and sevoflurane-excited neurons, together with changes in the oscillation characteristics of the local field potential within the DMH, are revealed as indicative markers of arousal states. During the arousal period, varying-frequency ESs are applied to the DMH, eliciting distinct arousal effects. Through in situ detection and stimulation, the disparity between these outcomes can be attributed to the influence of DBS on different neurons. These advancements may further our understanding of neural circuits and their potential applications in clinical contexts.

Inhibition of mTOR prevents glucotoxicity-mediated increase of SA-beta-gal, p16INK4a, and insulin hypersecretion, without restoring electrical features of mouse pancreatic islets.

An over-activation of the mechanistic target of rapamycin (mTOR) pathway promotes senescence and age-related diseases like type 2 diabetes. Besides, the regenerative potential of pancreatic islets deteriorates with aging. Nevertheless, the role of mTOR on senescence promoted by metabolic stress in islet cells as well as its relevance for electrophysiological aspects is not yet known. Here, we investigated whether parameters suggested to be indicative for senescence are induced in vitro in mouse islet cells by glucotoxicity and if mTOR inhibition plays a protective role against this. Islet cells exhibit a significant increase (~ 76%) in senescence-associated beta-galactosidase (SA-beta-gal) activity after exposure to glucotoxicity for 72 h. Glucotoxicity does not markedly influence p16INK4a protein within 72 h, but p16INK4a levels increase significantly after a 7-days incubation period. mTOR inhibition with a low rapamycin concentration (1 nM) entirely prevents the glucotoxicity-mediated increase of SA-beta-gal and p16INK4a. At the functional level, reactive oxygen species, calcium homeostasis, and electrical activity are disturbed by glucotoxicity, and rapamycin fails to prevent this. In contrast, rapamycin significantly attenuates the insulin hypersecretion promoted by glucotoxicity by modifying the mRNA levels of Vamp2 and Snap25 genes, related to insulin exocytosis. Our data indicate an influence of glucotoxicity on pancreatic islet-cell senescence and a reduction of the senescence markers by mTOR inhibition, which is relevant to preserve the regenerative potential of the islets. Decreasing the influence of mTOR on islet cells exposed to glucotoxicity attenuates insulin hypersecretion, but is not sufficient to prevent electrophysiological disturbances, indicating the involvement of mTOR-independent mechanisms.

Utilizing GO/PEDOT:PSS/PtNPs-enhanced high-stability microelectrode arrays for investigating epilepsy-induced striatal electrophysiology alterations.

The striatum plays a crucial role in studying epilepsy, as it is involved in seizure generation and modulation of brain activity. To explore the complex interplay between the striatum and epilepsy, we engineered advanced microelectrode arrays (MEAs) specifically designed for precise monitoring of striatal electrophysiological activities in rats. These observations were made during and following seizure induction, particularly three and 7 days post-initial modeling. The modification of graphene oxide (GO)/poly (3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS)/platinu-m nanoparticles (PtNPs) demonstrated a marked reduction in impedance (10.5 ± 1.1 kΩ), and maintained exceptional stability, with impedance levels remaining consistently low (23 kΩ) even 14 days post-implantation. As seizure intensity escalated, we observed a corresponding increase in neuronal firing rates and local field potential power, with a notable shift towards higher frequency peaks and augmented inter-channel correlation. Significantly, during the grand mal seizures, theta and alpha bands became the dominant frequencies in the local field potential. Compared to the normal group, the spike firing rates on day 3 and 7 post-modeling were significantly higher, accompanied by a decreased firing interval. Power in both delta and theta bands exhibited an increasing trend, correlating with the duration of epilepsy. These findings offer valuable insights into the dynamic processes of striatal neural activity during the initial and latent phases of temporal lobe epilepsy and contribute to our understanding of the neural mechanisms underpinning epilepsy.

Purinergic Signalling Mediates Aberrant Excitability of Developing Neuronal Circuits in the Fmr1 Knockout Mouse Model.

Neuronal hyperexcitability within developing cortical circuits is a common characteristic of several heritable neurodevelopmental disorders, including Fragile X Syndrome (FXS), intellectual disability and autism spectrum disorders (ASD). While this aberrant circuitry is typically studied from a neuron-centric perspective, glial cells secrete soluble factors that regulate both neurite extension and synaptogenesis during development. The nucleotide-mediated purinergic signalling system is particularly instrumental in facilitating these effects. We recently reported that within a FXS animal model, the Fmr1 KO mouse, the purinergic signalling system is upregulated in cortical astrocytes leading to altered secretion of synaptogenic and plasticity-related proteins. In this study, we examined whether elevated astrocyte purinergic signalling also impacts neuronal morphology and connectivity of Fmr1 KO cortical neurons. Here, we found that conditioned media from primary Fmr1 KO astrocytes was sufficient to enhance neurite extension and complexity of both wildtype and Fmr1 KO neurons to a similar degree as UTP-mediated outgrowth. Significantly enhanced firing was also observed in Fmr1 KO neuron-astrocyte co-cultures grown on microelectrode arrays but was associated with large deficits in firing synchrony. The selective P2Y2 purinergic receptor antagonist AR-C 118925XX effectively normalized much of the aberrant Fmr1 KO activity, designating P2Y2 as a potential therapeutic target in FXS. These results not only demonstrate the importance of astrocyte soluble factors in the development of neural circuitry, but also show that P2Y purinergic receptors play a distinct role in pathological FXS neuronal activity.

Hyperexcitability and translational phenotypes in a preclinical mouse model of SYNGAP1-Related Intellectual Disability.

Disruption of SYNGAP1 directly causes a genetically identifiable neurodevelopmental disorder (NDD) called SYNGAP1-related intellectual disability (SRID). Without functional SynGAP1 protein, individuals are developmentally delayed and have prominent features of intellectual disability, motor impairments, and epilepsy. Over the past two decades, there have been numerous discoveries indicting the critical role of Syngap1. Several rodent models with a loss of Syngap1 have been engineered identifying precise roles in neuronal structure and function, as well as key biochemical pathways key for synapse integrity. Homozygous loss of SYNGAP1/Syngap1 is lethal. Heterozygous mutations of Syngap1 result in a broad range of behavioral phenotypes. Our in vivo functional data, using the original mouse model from the Huganir laboratory, corroborated behaviors including robust hyperactivity and deficits in learning and memory in young adults. Furthermore, we described impairments in the domain of sleep, characterized using neurophysiological data collected with wireless, telemetric electroencephalography (EEG). Syngap1+/- mice exhibited elevated spiking events and spike trains, in addition to elevated power, most notably in the delta power frequency. For the first time, we illustrated primary neurons from Syngap1+/- mice displayed increased network firing activity, greater bursts, and shorter inter-burst intervals between peaks by employing high density microelectrode arrays (HD-MEA). Our work bridges in-vitro electrophysiological neuronal activity and function with in vivo neurophysiological brain activity and function. These data elucidate quantitative, translational biomarkers in vivo and in vitro that can be utilized for the development and efficacy assessment of targeted treatments for SRID.

Printable devices for neurotechnology.

Printable electronics for neurotechnology is a rapidly emerging field that leverages various printing techniques to fabricate electronic devices, offering advantages in rapid prototyping, scalability, and cost-effectiveness. These devices have promising applications in neurobiology, enabling the recording of neuronal signals and controlled drug delivery. This review provides an overview of printing techniques, materials used in neural device fabrication, and their applications. The printing techniques discussed include inkjet, screen printing, flexographic printing, 3D printing, and more. Each method has its unique advantages and challenges, ranging from precise printing and high resolution to material compatibility and scalability. Selecting the right materials for printable devices is crucial, considering factors like biocompatibility, flexibility, electrical properties, and durability. Conductive materials such as metallic nanoparticles and conducting polymers are commonly used in neurotechnology. Dielectric materials, like polyimide and polycaprolactone, play a vital role in device fabrication. Applications of printable devices in neurotechnology encompass various neuroprobes, electrocorticography arrays, and microelectrode arrays. These devices offer flexibility, biocompatibility, and scalability, making them cost-effective and suitable for preclinical research. However, several challenges need to be addressed, including biocompatibility, precision, electrical performance, long-term stability, and regulatory hurdles. This review highlights the potential of printable electronics in advancing our understanding of the brain and treating neurological disorders while emphasizing the importance of overcoming these challenges.

Beyond a Transmission Cable-New Technologies to Reveal the Richness in Axonal Electrophysiology.

The axon is a neuronal structure capable of processing, encoding, and transmitting information. This assessment contrasts with a limiting, but deeply rooted, perspective where the axon functions solely as a transmission cable of somatodendritic activity, sending signals in the form of stereotypical action potentials. This perspective arose, at least partially, because of the technical difficulties in probing axons: their extreme length-to-diameter ratio and intricate growth paths preclude the study of their dynamics through traditional techniques. Recent findings are challenging this view and revealing a much larger repertoire of axonal computations. Axons display complex signaling processes and structure-function relationships, which can be modulated via diverse activity-dependent mechanisms. Additionally, axons can exhibit patterns of activity that are dramatically different from those of their corresponding soma. Not surprisingly, many of these recent discoveries have been driven by novel technology developments, which allow for in vitro axon electrophysiology with unprecedented spatiotemporal resolution and signal-to-noise ratio. In this review, we outline the state-of-the-art in vitro toolset for axonal electrophysiology and summarize the recent discoveries in axon function it has enabled. We also review the increasing repertoire of microtechnologies for controlling axon guidance which, in combination with the available cutting-edge electrophysiology and imaging approaches, have the potential for more controlled and high-throughput in vitro studies. We anticipate that a larger adoption of these new technologies by the neuroscience community will drive a new era of experimental opportunities in the study of axon physiology and consequently, neuronal function.

Disrupting abnormal neuronal oscillations with adaptive delayed feedback control.

Closed-loop neuronal stimulation has a strong therapeutic potential for neurological disorders such as Parkinson's disease. However, at the moment, standard stimulation protocols rely on continuous open-loop stimulation and the design of adaptive controllers is an active field of research. Delayed feedback control (DFC), a popular method used to control chaotic systems, has been proposed as a closed-loop technique for desynchronisation of neuronal populations but, so far, was only tested in computational studies. We implement DFC for the first time in neuronal populations and access its efficacy in disrupting unwanted neuronal oscillations. To analyse in detail the performance of this activity control algorithm, we used specialised in vitro platforms with high spatiotemporal monitoring/stimulating capabilities. We show that the conventional DFC in fact worsens the neuronal population oscillatory behaviour, which was never reported before. Conversely, we present an improved control algorithm, adaptive DFC (aDFC), which monitors the ongoing oscillation periodicity and self-tunes accordingly. aDFC effectively disrupts collective neuronal oscillations restoring a more physiological state. Overall, these results support aDFC as a better candidate for therapeutic closed-loop brain stimulation.

Batch Fabrication of Microelectrode Arrays with Glassy Carbon Microelectrodes and Interconnections for Neurochemical Sensing: Promises and Challenges.

Flexible multielectrode arrays with glassy carbon (GC) electrodes and metal interconnection (hybrid MEAs) have shown promising performance in multi-channel neurochemical sensing. A primary challenge faced by hybrid MEAs fabrication is the adhesion of the metal traces with the GC electrodes, as prolonged electrical and mechanical stimulation can lead to adhesion failure. Previous devices with GC electrodes and interconnects made of a homogeneous material (all GC) demonstrated exceptional electrochemical stability but required miniaturization for enhanced tissue integration and chronic electrochemical sensing. In this study, we used two different methods for the fabrication of all GC-MEAs on thin flexible substrates with miniaturized features. The first method, like that previously reported, involves a double pattern-transfer photolithographic process, including transfer-bonding on temporary polymeric support. The second method requires a double-etching process, which uses a 2 µm-thick low stress silicon nitride coating of the Si wafer as the bottom insulator layer for the MEAs, bypassing the pattern-transfer and demonstrating a novel technique with potential advantages. We confirmed the feasibility of the two fabrication processes by verifying the practical conductivity of 3 µm-wide 2 µm-thick GC traces, the GC microelectrode functionality, and their sensing capability for the detection of serotonin using fast scan cyclic voltammetry. Through the exchange and discussion of insights regarding the strengths and limitations of these microfabrication methods, our goal is to propel the advancement of GC-based MEAs for the next generation of neural interface devices.

Improvements for recording retinal function with Microelectrode Arrays.

A microelectrode array (MEA) is a configuration of multiple electrodes that enables the concurrent targeting of multiple sites for extracellular recording and stimulation. By utilizing light pulses or electrical stimulations, MEA recordings unveil the complex patterns of electrical activities that arise from the signaling processes within the retinal network. Here, we present a stepwise approach for using microelectrode arrays (MEAs) for recording action potentials from the mouse retina in response to electrical and light stimuli. We provide detailed techniques accompanied by description of a custom optical system, example recordings, troubleshooting guidelines, and data processing methods including spike sorting and code resources for analyzing light and electrical responses. The comprehensive nature of this paper aims to guide researchers in utilizing MEAs effectively for investigating retinal functionality. In particular, it can be easy to have a MEA experiment fail, but hard to identify the source of the failure. This paper is meant to demystify that process. It includes:•A description of MEA setup, recording, and spike data validation.•A troubleshooting guide for common failure modes in MEA recordings from mouse retina.•Spike detection and sorting to precisely extract distinctive action potential.

Reactive Amine Functionalized Microelectrode Arrays Provide Short-Term Benefit but Long-Term Detriment to In Vivo Recording Performance.

Intracortical microelectrode arrays (MEAs) are used for recording neural signals. However, indwelling devices result in chronic neuroinflammation, which leads to decreased recording performance through degradation of the device and surrounding tissue. Coating the MEAs with bioactive molecules is being explored to mitigate neuroinflammation. Such approaches often require an intermediate functionalization step such as (3-aminopropyl)triethoxysilane (APTES), which serves as a linker. However, the standalone effect of this intermediate step has not been previously characterized. Here, we investigated the effect of coating MEAs with APTES by comparing APTES-coated to uncoated controls in vivo and ex vivo. First, we measured water contact angles between silicon uncoated and APTES-coated substrates to verify the hydrophilic characteristics of the APTES coating. Next, we implanted MEAs in the motor cortex (M1) of Sprague-Dawley rats with uncoated or APTES-coated devices. We assessed changes in the electrochemical impedance and neural recording performance over a chronic implantation period of 16 weeks. Additionally, histology and bulk gene expression were analyzed to understand further the reactive tissue changes arising from the coating. Results showed that APTES increased the hydrophilicity of the devices and decreased electrochemical impedance at 1 kHz. APTES coatings proved detrimental to the recording performance, as shown by a constant decay up to 16 weeks postimplantation. Bulk gene analysis showed differential changes in gene expression between groups that were inconclusive with regard to the long-term effect on neuronal tissue. Together, these results suggest that APTES coatings are ultimately detrimental to chronic neural recordings. Furthermore, interpretations of studies using APTES as a functionalization step should consider the potential consequences if the final functionalization step is incomplete.

Improving morphological and functional properties of enteric neuronal networks in vitro using a novel upside-down culture approach.

The enteric nervous system (ENS) comprises millions of neurons and glia embedded in the wall of the gastrointestinal tract. It not only controls important functions of the gut but also interacts with the immune system, gut microbiota, and the gut-brain axis, thereby playing a key role in the health and disease of the whole organism. Any disturbance of this intricate system is mirrored in an alteration of electrical functionality, making electrophysiological methods important tools for investigating ENS-related disorders. Microelectrode arrays (MEAs) provide an appropriate noninvasive approach to recording signals from multiple neurons or whole networks simultaneously. However, studying isolated cells of the ENS can be challenging, considering the limited time that these cells can be kept vital in vitro. Therefore, we developed an alternative approach cultivating cells on glass samples with spacers (fabricated by photolithography methods). The spacers allow the cells to grow upside down in a spatially confined environment while enabling acute consecutive recordings of multiple ENS cultures on the same MEA. Upside-down culture also shows beneficial effects on the growth and behavior of enteric neural cultures. The number of dead cells was significantly decreased, and neural networks showed a higher resemblance to the myenteric plexus ex vivo while producing more stable signals than cultures grown in the conventional way. Overall, our results indicate that the upside-down approach not only allows to investigate the impact of neurological diseases in vitro but could also offer insights into the growth and development of the ENS under conditions much closer to the in vivo environment.NEW & NOTEWORTHY In this study, we devised a novel approach for culturing and electrophysiological recording of the enteric nervous system using custom-made glass substrates with spacers. This allows to turn cultures of isolated myenteric plexus upside down, enhancing the use of the microelectrode array technique by allowing recording of multiple cultures consecutively using only one chip. In addition, upside-down culture led to significant improvements in the culture conditions, resulting in a more in vivo-like growth.