A 3D neuronal network read-out interface with high recording performance using a neuronal cluster patterning on a microelectrode array.

In recent years, in vitro three-dimensional (3D) neuronal network models utilizing extracellular matrices have been advancing. To understand the network activity from these models, attempts have been made to measure activity in multiple regions simultaneously using a microelectrode array (MEA). Although there hve been many attempts to measure the activity of 3D networks using 2-dimensional (2D) MEAs, the physical coupling between the 3D network and the microelectrodes was not stable and needed to be improved. In this study, we proposed a neuronal cluster interface that improves the active channel ratio of commercial 2D MEAs, enabling reliable measurement of 3D network activity. To achieve this, neuronal clusters, which consist of a small number of neurons, were patterned on microelectrodes and used as mediators to transmit the signal between the 3D network and the microelectrodes. We confirmed that the patterned neuronal clusters enhanced the active channel ratio and SNR(signal-to-noise-ratio) about 3D network recording and stimulation for a month. Our interface was able to functionally connect with 3D networks and measure the 3D network activity without significant alternation of activity characteristics. Finally, we demonstrated that our interface can be used to analyze the differences in the dynamics of 3D and 2D networks and to construct the 3D clustered network. This method is expected to be useful for studying the functional activity of various 3D neuronal network models, offering broad applications for the use of these models.

Connectivity and network burst properties of in-vitro neuronal networks induced by a clustered structure with alginate hydrogel patterning.

Modularity is one of the important structural properties that affect information processing and other functionalities of neuronal networks. Researchers have developed in-vitro clustered network models for reproducing the modularity, but it is still challenging to control the segregation and integration of several sub-populations of them. We cultured clustered networks with alginate patterning and collected the electrophysiological signals to investigate the changes in functional properties during the development. We built inter-connected neuronal clusters using alginate micro-patterning with a circular shape on the surface of the micro-electrode array. The neuronal clusters were enabled to be connected at 3 or 10 days-in-vitro (DIV) by removing the barrier. The neuronal signals from different types of networks were collected from 16 to 34 DIV, and functional characteristics were examined. Connectivity and burst motif analysis were carried out to find out the relation between the structure and function of the networks. Neuronal networks with clustered structure showed different activity properties from the random networks along the development. The clustered networks had more short-range connections compared to the random networks. In the network burst motif analysis, the clustered networks showed more various patterns and a slower propagation of the activation patterns. In this study, we successfully cultured neuronal networks with clustered structure, and the structure affected the functional properties. The network model suggested in this study will be a good solution for observing the effect of structure on function during their development. Supplementary Information: The online version contains supplementary material available at 10.1007/s13534-023-00289-5.

Enhancement of Thermoplasmonic Neural Modulation Using a Gold Nanorod-Immobilized Polydopamine Film.

Photothermal neural activity inhibition has emerged as a minimally invasive neuromodulation technology with submillimeter precision. One of the techniques involves the utilization of plasmonic gold nanoparticles (AuNPs) to modulate neural activity by photothermal effects ("thermoplasmonics"). A surface modification technique is often required to integrate AuNPs onto the neural interface. Here, polydopamine (pDA), a multifunctional adhesive polymer with a wide light absorption spectrum, is introduced both as a primer layer for the immobilization of gold nanorods (GNRs) on the neural interface and as an additional photothermal agent by absorbing near-infrared red (NIR) lights for more efficient photothermal effects. First, the optical and photothermal properties of pDA as well as the characteristics of GNRs attached onto the pDA film are investigated for the optimized photothermal neural interface. Due to the covalent bonding between GNR surfaces and pDA, GNRs immobilized on pDA showed strong attachment onto the surface, yielding a more stable photothermal platform. Lastly, when photothermal neural stimulation was applied to the primary rat hippocampal neurons, the substrate with GNRs immobilized on the pDA film allowed more laser power-efficient photothermal neuromodulation as well as photothermal cell death. This study suggests the feasibility of using pDA as a surface modification material for developing a photothermal platform for the inhibition of neural activities.

Neurons-on-a-Chip: In Vitro NeuroTools.

Neurons-on-a-Chip technology has been developed to provide diverse in vitro neuro-tools to study neuritogenesis, synaptogensis, axon guidance, and network dynamics. The two core enabling technologies are soft-lithography and microelectrode array technology. Soft lithography technology made it possible to fabricate microstamps and microfluidic channel devices with a simple replica molding method in a biological laboratory and innovatively reduced the turn-around time from assay design to chip fabrication, facilitating various experimental designs. To control nerve cell behaviors at the single cell level via chemical cues, surface biofunctionalization methods and micropatterning techniques were developed. Microelectrode chip technology, which provides a functional readout by measuring the electrophysiological signals from individual neurons, has become a popular platform to investigate neural information processing in networks. Due to these key advances, it is possible to study the relationship between the network structure and functions, and they have opened a new era of neurobiology and will become standard tools in the near future.

Optical recording of neural responses to gold-nanorod mediated photothermal neural inhibition.

BACKGROUND: Photothermal stimulation is a heat-mediated neuromodulation technique. When photothermal effects are induced on neuronal membrane, it can either excite or inhibit neural spiking activities. It has been demonstrated that gold nanorod mediated photothermal stimulation could decrease the electrical activity of cultured neural network. We investigated the effect of photothermal inhibition on neural activity using calcium imaging technique. NEW METHOD: Hippocampal neurons were cultured on a gold-nanorod-coated-microelectrode array and near-infrared laser was illuminated to induce neural inhibition. The neuronal responses at a single-cell resolution were measured by an extracellular recording and calcium imaging simultaneously. RESULTS: The photothermal effect on neural spikes were confirmed by electrical recording and calcium imaging. The decrease in neural spikes in electrical recordings during NIR illumination was correlated with the neighboring neural activity quantified by calcium spikes. COMPARISON WITH EXISTING METHOD(S): Optical recording at the single cell resolution was attempted during photothermal stimulation to confirm the neural suppression effect. CONCLUSIONS: Heat mediated suppression of neural activity was optically validated in single cell level. The present study will be helpful to understand the emerging photothermal neuromodulation technology.

Development of the micro-patterned 3D neuronal-hydrogel model using soft-lithography for study a 3D neural network on a microelectrode array.

In vitro patterned neuronal models have been studied as one of the strategies to investigate the relationship between structural connectivity and functional activity of neural network. Despite the importance of three-dimensional (3D) cell models, most of these studies have been performed on two-dimensional models. In this study, we present a technique to construct the micro-pattern to 3D neuronal-hydrogel model using a micromolding in capillaries (MIMIC) technique on microelectrode array (MEA). Our technique was suitable to prevent the deformation of micro-patterned collagen model against the neuronal contracted tension during the network formation. The relationship between the growth directions of glial cells and micro-pattern direction was investigated. Lastly, we confirmed that our 3D model had synchronized activity among neurons in 3D. This model is expected to be used as a tool to study the relationship between structural connectivity and functional activity in the 3D environment.

Laser Power Determination Using Light-to-Heat Conversion Rate of Nanoplasmonic Substrates for Neural Stimulation.

Since neurons have temperature sensitive properties, gold nanorod (GNR)-mediated photothermal stimulation has been developed as a neuromodulation application. As an in vitro photothermal platform, GNR-layer was integrated with substrates to effectively apply heat stimulation to the cultured neurons. However, identifying optimal laser power for a targeted temperature on the substrate requires the consideration of thermal properties of the GNR-coated substrates. In this report, we suggest a simple numerical method to determine incident laser power on the substrates for a targeted temperature.

Label-free monitoring of 3D cortical neuronal growth in vitro using optical diffraction tomography.

The highly complex central nervous systems of mammals are often studied using three-dimensional (3D) in vitro primary neuronal cultures. A coupled confocal microscopy and immunofluorescence labeling are widely utilized for visualizing the 3D structures of neurons. However, this requires fixation of the neurons and is not suitable for monitoring an identical sample at multiple time points. Thus, we propose a label-free monitoring method for 3D neuronal growth based on refractive index tomograms obtained by optical diffraction tomography. The 3D morphology of the neurons was clearly visualized, and the developmental processes of neurite outgrowth in 3D spaces were analyzed for individual neurons.

Closed-loop control of neural spike rate of cultured neurons using a thermoplasmonics-based photothermal neural stimulation.

Objective.Photothermal neural stimulation has been developed in a variety of interfaces as an alternative technology that can perturb neural activity. The demonstrations of these techniques have heavily relied on open-loop stimulation or complete suppression of neural activity. To extend the controllability of photothermal neural stimulation, combining it with a closed-loop system is required. In this work, we investigated whether photothermal suppression mechanism can be used in a closed-loop system to reliably modulate neural spike rate to non-zero setpoints.Approach. To incorporate the photothermal inhibition mechanism into the neural feedback system, we combined a thermoplasmonic stimulation platform based on gold nanorods (GNRs) and near-infrared illuminations (808 nm, spot size: 2 mm or 200µm in diameter) with a proportional-integral (PI) controller. The closed-loop feedback control system was implemented to track predetermined target spike rates of hippocampal neuronal networks cultured on GNR-coated microelectrode arrays.Main results. The closed-loop system for neural spike rate control was successfully implemented using a PI controller and the thermoplasmonic neural suppression platform. Compared to the open-loop control, the target-channel spike rates were precisely modulated to remain constant or change in a sinusoidal form in the range below baseline spike rates. The spike rate response behaviors were affected by the choice of the controller gain. We also demonstrated that the functional connectivity of a synchronized bursting network could be altered by controlling the spike rate of one of the participating channels.Significance.The thermoplasmonic feedback controller proved that it can precisely modulate neural spike rate of neural activityin vitro. This technology can be used for studying neuronal network dynamics and might provide insights in developing new neuromodulation techniques in clinical applications.

Contribution of Extracellular Matrix Component Landscapes in the Adult Subventricular Zone to the Positioning of Neural Stem/Progenitor Cells.

Neurogenesis persists in restricted regions of the adult brain, including the subventricular zone (SVZ). Adult neural stem cells (NSCs) in the SVZ proliferate and give rise to new neurons and glial cells depending on intrinsic and environmental cues. Among the multiple factors that contribute to the chemical, physical, and mechanical components of the neurogenic niche, we focused on the composition of the extracellular matrix (ECM) of vasculature and fractones in the SVZ. The SVZ consists of ECM-rich blood vessels and fractones during development and adulthood, and adult neural stem/progenitor cells (NS/PCs) preferentially attach to the laminin-rich basal lamina. To examine the ECM preference of adult NS/PCs, we designed a competition assay using cell micropatterning. Although both laminin and collagen type IV, which are the main components of basal lamina, act as physical scaffolds, adult NS/PCs preferred to adhere to laminin over collagen type IV. Interestingly, the ECM preference of adult NS/PCs could be manipulated by chemokines such as stromal-derived factor 1 (SDF1) and α6 integrin. As SDF1 re-routes NSCs and their progenitors toward the injury site after brain damage, these results suggest that the alteration in ECM preferences may provide a molecular basis for contextdependent NS/PC positioning.

Thermoplasmonic neural chip platform for in situ manipulation of neuronal connections in vitro.

Cultured neuronal networks with a controlled structure have been widely studied as an in vitro model system to investigate the relationship between network structure and function. However, most cell culture techniques lack the ability to control network structures during cell cultivation, making it difficult to assess functional changes induced by specific structural changes. In this study, we present an in situ manipulation platform based on gold-nanorod-mediated thermoplasmonics to interrogate an in vitro network model. We find that it is possible to induce new neurite outgrowths, eliminate interconnecting neurites, and estimate functional relationships in matured neuronal networks. This method is expected to be useful for studying functional dynamics of neural networks under controlled structural changes.

Thermoplasmonic Optical Fiber for Localized Neural Stimulation.

Thermoplasmonic effect-based neural stimulation has been suggested as an alternative optical neural stimulation technology without genetic modification. Integration of near-infrared light with plasmonic gold nanoparticles has been demonstrated as a neuromodulation tool on in vitro neuronal network models. In order to further test the validity of the thermoplasmonic neural stimulation across multiple biological models (in vitro, ex vivo, and in vivo) avoiding genetic modification in optical neuromodulation, versatile engineering approaches to apply the thermoplasmonic effect would be required. In this work, we developed a gold nanorod attached optical fiber technology for the localized neural stimulation based on a thermoplasmonic effect. A simple fabrication process was developed for efficient nanoparticle coating on commercial optical fibers. The thermoplasmonic optical fiber proved that it can locally modulate the neural activity in vitro. Lastly, we simulated the spatiotemporal temperature change by the thermoplasmonic optical fiber and analyzed its applicability to in vivo animal models.

Compact 256-channel multi-well microelectrode array system for in vitro neuropharmacology test.

Microelectrode arrays (MEAs) have been extensively used to measure extracellular spike activity from cultured neurons using multiple electrodes embedded in a planar glass substrate. This system has been implemented to investigate drug effects by detecting pharmacological perturbation reflected in spontaneous network activity. By configuring multiple wells in an MEA, a high-throughput electrophysiological assay has become available, speeding up drug tests. Despite its merits in acquiring massive amounts of electrophysiological data, the high cost and the bulky size of commercial multi-well MEA systems and most importantly its lack of customizability prevent potential users from fully implementing the system in drug experiments. In this work, we have developed a microelectrode array based drug testing platform by incorporating a custom-made compact 256-channel multi-well MEA in a standard microscope slide and commercial application-specific integrated circuit (ASIC) chip based recording system. We arranged 256 electrodes in 16 wells to maximize data collection from a single chip. The multi-well MEA in this work has a more compact design with reduced chip size compared to previously reported multi-well MEAs. Four synaptic modulators (NMDA, AMPA, bicuculline (BIC) and ATP) were applied to a multi-well MEA and neural spike activity was analyzed to study their neurophysiological effects on cultured neurons. Analyzing various neuropharmacological compounds has become much more accessible by utilizing commercially available digital amplifier chips and customizing a user-preferred analog-front-end interface design with additional benefits in reduced platform size and cost.

Correction to: SHP2 mutations induce precocious gliogenesis of Noonan syndrome-derived iPSCs during neural development in vitro.

An amendment to this paper has been published and can be accessed via the original article.

SHP2 mutations induce precocious gliogenesis of Noonan syndrome-derived iPSCs during neural development in vitro.

BACKGROUND: Noonan syndrome (NS) is a developmental disorder caused by mutations of Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2). Although NS patients have diverse neurological manifestations, the mechanisms underlying the involvement of SHP2 mutations in neurological dysfunction remain elusive. METHODS: Induced pluripotent stem cells generated from dermal fibroblasts of three NS-patients (NS-iPSCs) differentiated to the neural cells by using two different culture systems, 2D- and 3D-cultured systems in vitro. RESULTS: Here we represent that SHP2 mutations cause aberrant neural development. The NS-iPSCs exhibited impaired development of EBs in which BMP and TGF-ß signalings were activated. Defective early neuroectodermal development of NS-iPSCs recovered by inhibition of both signalings and further differentiated into NPCs. Intriguingly, neural cells developed from NS-NPCs exhibited abundancy of the glial cells, neurites of neuronal cells, and low electrophysiological property. Those aberrant phenotypes were also detected in NS-cerebral organoids. SHP2 inhibition in the NS-NPCs and NS-cerebral organoids ameliorated those anomalies such as biased glial differentiation and low neural activity. CONCLUSION: Our findings demonstrate that SHP2 mutations contribute to precocious gliogenesis in NS-iPSCs during neural development in vitro.

Polydopamine-doped conductive polymer microelectrodes for neural recording and stimulation.

BACKGROUND: Microelectrodes have been widely used to detect and modulate the activities of neuronal networks. Various materials have been applied to microelectrode fabrication, and the conductive polymer is one of the most intensively explored material. The properties of conductive polymer highly depend on the incorporated material, so selecting it is essential. The mussel-inspired biomolecule, polydopamine (pDA), is known to provide unique chemical and mechanical properties to biological interfaces. NEW METHOD: pDA was incorporated into poly(3,4-ethylenedioxythiophene) (PEDOT) resulting in polydopamine PEDOT hybrid (PEDOT/pDA) microelectrode by an electrochemical deposition method. The electrical properties, such as impedance, charge storage capacity (CSC), and charge injection limit (CIL), of PEDOT/pDA microelectrodes, were characterized. RESULTS: PEDOT/pDA microelectrodes had low impedance, high CSC, and high CIL, which are prerequisite for neuronal signal recording and stimulation. Then, neuronal recordings and electrical stimulations were conducted to verify the functionality of the PEDOT/pDA microelectrodes. Spontaneous and evoked extracellular neuronal signals were successfully measured from cultured rat hippocampal neuronal networks, and the recorded signals showed excellent signal-to-noise ratio for the detection of extracellular spikes. COMPARISON WITH EXISTING METHODS: Compared with existing conductive polymer based neural electrodes, the PEDOT/pDA microelectrode had chemically functional material, pDA, embedded in the electrode, while it had comparable level of impedance and CSC and CIL for neural stimulation and recordings. CONCLUSIONS: We have shown that it is possible to fabricate a microelectrode array of pDA doped PEDOT microelectrodes and validated its performance for neuronal signal recording and electrical stimulation. The PEDOT/pDA microelectrode with excellent electrical performance and biocompatibility will be a promising tool for studying neuronal networks.

Slow-Wave Recordings From Micro-Sized Neural Clusters Using Multiwell Type Microelectrode Arrays.

OBJECTIVE: The use of microelectrode array (MEA) recordings is a very effective neurophysiological method because it is able to continuously and noninvasively obtain the spatiotemporal information of electrical activity from many neurons constituting a neural network. Very recently, studies have been published that used MEAs for the measurement of a low-frequency component of electrical activity as an indicator of diverse activity of cultured neurons. The occurrence of low-frequency activities has electrophysiological information that does not include the information from fast spikes. However, there is no in vitro experimental model suitable for measuring the low-frequency activities (slow-waves) for further study. METHODS: Neural clusters consisting of dozens of neurons were placed directly onto each electrode of an MEA from which fast spikes and slow-waves were measured. RESULTS: We obtained sufficient data on the early development patterns of the slow-waves and the spikes measured from many independent neural clusters confirming that the slow-waves occurred first before the emergence of the spikes in the neural clusters. We also showed that changes in the occurrence frequency of the slow-waves for synaptic blockers were measured from a large number of independent cultures. CONCLUSION: Microsized neural cluster arrays, which can be combined with conventional MEAs, are suitable for multiple simultaneous recordings of slow-waves. SIGNIFICANCE: Our technology provides a simple but useful method to study the generation of a low-frequency component of the electrical activity in cultured neural networks that are not yet well known as well as to expand the use of conventional MEAs.

A monitoring system for axonal growth dynamics using micropatterns of permissive and Semaphorin 3F chemorepulsive signals.

Neurons reach their correct targets by directional outgrowth of axons, which is mediated by attractive or repulsive cues. Growing axons occasionally cross a field of repulsive cues and stop at intermediate targets on the journey to their final destination. However, it is not well-understood how individual growth cones make decisions, and pass through repulsive territory to reach their permissive target regions. We developed a microcontact printing culture system that could trap individual axonal tips in a permissive dot area surrounded by the repulsive signal, semaphorin 3F (Sema3F). Axons of rat hippocampal neurons on the Sema3F/PLL dot array extended in the checkboard pattern with a significantly slow growth rate. The detailed analysis of the behaviors of axonal growth cones revealed the saccadic dynamics in the dot array system. The trapped axonal tips in the permissive area underwent growth cone enlargement with remarkably spiky filopodia, promoting their escape from the Sema3F constraints with straight extension of axons. This structured axonal growth on the dot pattern was disrupted by increased inter-dot distance, or perturbing intracellular signaling machineries. These data indicate that axons grow against repulsive signals by jumping over the repulsive cues, depending on contact signals and intracellular milieu. Our study suggests that our dot array culture system can be used as a screening system to easily and efficiently evaluate ECM or small molecule inhibitors interfering growth cone dynamics leading to controlling axonal growth.

Single-Cell Photothermal Neuromodulation for Functional Mapping of Neural Networks.

Photothermal neuromodulation is one of the emerging technologies being developed for neuroscience studies because it can provide minimally invasive control of neural activity in the deep brain with submillimeter precision. However, single-cell modulation without genetic modification still remains a challenge, hindering its path to broad applications. Here, we introduce a nanoplasmonic approach to inhibit single-neural activity with high temporal resolution. Low-intensity near-infrared light was focused at the single cell size on a gold-nanorod-integrated microelectrode array platform, generating a photothermal effect underneath a target neuron for photothermal stimulation. We found that the photothermal stimulation modulates the spontaneous activity of a target neuron in an inhibitory manner. Single neuron inhibition was fast and highly reliable without thermal damage, and it can induce changes in network firing patterns, potentially suggesting their application for in vivo circuit modulation and functional connectomes.

Thermo-plasmonic gold nanofilms for simple and mass-producible photothermal neural interfaces.

In recent years, photothermal stimulation methods using plasmonic metal nanoparticles have emerged as non-genetic optical techniques in neuromodulation. Although nanoparticle-based photothermal stimulation shows great potential in the excitation and the inhibition of neural activity, the complex synthesis processes of the nanoparticles and the lack of large-area deposition methods can be limiting factors for the development of photothermal neural devices. In this paper, we propose a plasmonic gold nanofilm, fabricated by a standard thermal evaporation process, as a simple and mass-producible photothermal neural interface layer for microelectrode array (MEA) chips. The absorption of the gold nanofilm at near infrared wavelengths is optimized to maximize the photothermal effect by varying the thickness and microstructure of the gold nanofilm. With the optimized conditions, a significantly strong photothermal effect is applied on MEAs without affecting the neural signal recording capability. Finally, primary rat hippocampal neuronal cultures are used to show that the photothermal neural inhibition using the gold nanofilm is as effective as that using the plasmonic nanoparticles. Due to the greater simplicity and versatility of the fabrication process, the plasmonic gold nanofilm can provide a promising solution for the mass production of photothermal platforms.