Initiation and termination of reentry-like activity in rat cardiomyocytes cultured in a microelectrode array.

Cardiac reentry is a lethal arrhythmia associated with cardiac diseases. Although arrhythmias are reported to be due to localized propagation abnormalities, little is known about the mechanisms underlying the initiation and termination of reentry. This is primarily because of a lack of an appropriate experimental system in which activity pattern switches between reentry and normal beating can be investigated. In this study, we aimed to develop a culture system for measuring the spatial dynamics of reentry-like activity during its onset and termination. Rat cardiomyocytes were seeded in microelectrode arrays and purified with a glucose-free culture medium to generate a culture with a heterogeneous cell density. Reentry-like activity was recorded in purified cardiomyocytes, but not in the controls. Reentry-like activity occurred by a unidirectional conduction block after shortening of the inter-beat interval. Furthermore, reentry-like activity was terminated after propagation with a conduction delay of less than 300 ms, irrespective of whether the propagation pattern changed or not. These results indicate that a simple purification process is sufficient to induce reentry-like activity. In the future, a more detailed evaluation of spatial dynamics will contribute to the development of effective treatment methods.

A Study of the Effects of Plasma Surface Treatment on Lipid Bilayers Self-Spreading on a Polydimethylsiloxane Substrate under Different Treatment Times.

Plasma-treated poly(dimethylsiloxane) (PDMS)-supported lipid bilayers are used as functional tools for studying cell membrane properties and as platforms for biotechnology applications. Self-spreading is a versatile method for forming lipid bilayers. However, few studies have focused on the effect of plasma treatment on self-spreading lipid bilayer formation. In this paper, we performed lipid bilayer self-spreading on a PDMS surface with different treatment times. Surface characterization of PDMS treated with different treatment times is evaluated by AFM and SEM, and the effects of plasma treatment of the PDMS surface on lipid bilayer self-spreading behavior is investigated by confocal microscopy. The front-edge velocity of lipid bilayers increases with the plasma treatment time. By theoretical analyses with the extended-DLVO modeling, we find that the most likely cause of the velocity change is the hydration repulsion energy between the PDMS surface and lipid bilayers. Moreover, the growth behavior of membrane lobes on the underlying self-spreading lipid bilayer was affected by topography changes in the PDMS surface resulting from plasma treatment. Our findings suggest that the growth of self-spreading lipid bilayers can be controlled by changing the plasma treatment time.

Synchronous firing patterns of induced pluripotent stem cell-derived cortical neurons depend on the network structure consisting of excitatory and inhibitory neurons.

The balance between glutamate-mediated excitation and GABA-mediated inhibition is critical to cortical functioning. However, the contribution of network structure consisting of the both neurons to cortical functioning has not been elucidated. We aimed to evaluate the relationship between the network structure and functional activity patterns in vitro. We used mouse induced pluripotent stem cells (iPSCs) to construct three types of neuronal populations; excitatory-rich (Exc), inhibitory-rich (Inh), and control (Cont). Then, we analyzed the activity patterns of these neuronal populations using microelectrode arrays (MEAs). Inhibitory synaptic densities differed between the three types of iPSC-derived neuronal populations, and the neurons showed spontaneously synchronized bursting activity with functional maturation for one month. Moreover, different firing patterns were observed between the three populations; Exc demonstrated the highest firing rates, including frequent, long, and dominant bursts. In contrast, Inh demonstrated the lowest firing rates and the least dominant bursts. Synchronized bursts were enhanced by disinhibition via GABAA receptor blockade. The present study, using iPSC-derived neurons and MEAs, for the first time show that synchronized bursting of cortical networks in vitro depends on the network structure consisting of excitatory and inhibitory neurons.

Functional innervation of human induced pluripotent stem cell-derived cardiomyocytes by co-culture with sympathetic neurons developed using a microtunnel technique.

Microelectrode array (MEA) based-drug screening with human induced pluripotent stem cell-derived cardiomyocytes (hiPSCM) is a potent pre-clinical assay for efficiently assessing proarrhythmic risks in new candidates. Furthermore, predicting sympathetic modulation of the proarrhythmic side-effects is an important issue. Although we have previously developed an MEA-based co-culture system of rat primary cardiomyocyte and sympathetic neurons (rSNs), it is unclear if this co-culture approach is applicable to develop and investigate sympathetic innervation of hiPSCMs. In this study, we developed a co-culture of rSNs and hiPSCMs on MEA substrate, and assessed functional connections. The inter-beat interval of hiPSCM was significantly shortened by stimulation in SNs depending on frequency and pulse number, indicating functional connections between rSNs and hiPSCM and the dependency of chronotropic effects on rSN activity pattern. These results suggest that our co-culture approach can evaluate sympathetic effects on hiPSCMs and would be a useful tool for assessing sympathetic modulated-cardiotoxicity in human cardiac tissue.

Recording axonal conduction to evaluate the integration of pluripotent cell-derived neurons into a neuronal network.

Stem cell transplantation is a promising therapy to treat neurodegenerative disorders, and a number of in vitro models have been developed for studying interactions between grafted neurons and the host neuronal network to promote drug discovery. However, methods capable of evaluating the process by which stem cells integrate into the host neuronal network are lacking. In this study, we applied an axonal conduction-based analysis to a co-culture study of primary and differentiated neurons. Mouse cortical neurons and neuronal cells differentiated from P19 embryonal carcinoma cells, a model for early neural differentiation of pluripotent stem cells, were co-cultured in a microfabricated device. The somata of these cells were separated by the co-culture device, but their axons were able to elongate through microtunnels and then form synaptic contacts. Propagating action potentials were recorded from these axons by microelectrodes embedded at the bottom of the microtunnels and sorted into clusters representing individual axons. While the number of axons of cortical neurons increased until 14 days in vitro and then decreased, those of P19 neurons increased throughout the culture period. Network burst analysis showed that P19 neurons participated in approximately 80% of the bursting activity after 14 days in vitro. Interestingly, the axonal conduction delay of P19 neurons was significantly greater than that of cortical neurons, suggesting that there are some physiological differences in their axons. These results suggest that our method is feasible to evaluate the process by which stem cell-derived neurons integrate into a host neuronal network.

Synthetic DNA nanopores for direct molecular transmission between lipid vesicles.

Lipid vesicles hold potential as artificial cells in bottom-up synthetic biology, and as tools in drug delivery and biosensing. Transmitting molecular signals is a key function for vesicle-based systems. One strategy to achieve this function is by releasing molecular signals from vesicles through nanopores. Nevertheless, in this strategy, an excess of molecular signals may be required to reach the targets, due to the dispersion of the signals during diffusion. The key to achieving the efficient utilization of signals is to shorten the distance between the sender vesicle and the target. Here, we present a pair of DNA nanopores that can connect and form a direct molecular pathway between vesicles. The nanopores are self-assembled from nine single DNA strands, including six 14-nucleotide single-stranded overhangs as sticky-end segments, enabling them to bind with each other. Incorporating nanopores shortens the distance between different populations of vesicles, allowing less diffusion of molecules into bulk solution. To further reduce the loss of molecules, a DNA nanocap is added to one of the nanopore's openings. The nanocap can be removed through the toehold-mediated DNA strand displacement when the nanopore meets its counterpart. Our DNA nanopores provide a novel molecular transmission tool to lipid vesicles-based systems.

Ultracompact mirror device for forming 20-nm achromatic soft-X-ray focus toward multimodal and multicolor nanoanalyses.

Nanoscale soft-X-ray microscopy is a powerful analysis tool in biological, chemical, and physical sciences. To enhance its probe sensitivity and leverage multimodal soft-X-ray microscopy, precise achromatic focusing devices, which are challenging to fabricate, are essential. Here, we develop an ultracompact Kirkpatrick-Baez (ucKB) mirror, which is ideal for the high-performance nanofocusing of broadband-energy X-rays. We apply our advanced fabrication techniques and short-focal-length strategy to realize diffraction-limited focusing over the entire soft-X-ray range. We achieve a focus size of 20.4 nm at 2 keV, which represents a significant improvement in achromatic soft-X-ray focusing. The ucKB mirror extends soft-X-ray fluorescence microscopy by producing a bicolor nanoprobe with a 1- or 2-keV photon energy. We propose a subcellular chemical mapping method that allows a comprehensive analysis of specimen morphology and the distribution of light elements and metal elements. ucKB mirrors will improve soft-X-ray nanoanalyses by facilitating photon-hungry, multimodal, and polychromatic methods, even with table-top X-ray sources.

Improving the accuracy of decoding monkey brain-machine interface data by estimating the state of unobserved cell assemblies.

BACKGROUND: The brain-machine interface is a technology that has been used for improving the quality of life of individuals with physical disabilities and also healthy individuals. It is important to improve the methods used for decoding the brain-machine interface data as the accuracy and speed of movements achieved using the existing technology are not comparable to the normal body. COMPARISON WITH THE EXISTING METHOD: Decoding of brain-machine interface data using the proposed method resulted in improved decoding accuracy compared to the existing method. CONCLUSIONS: The results demonstrated the usefulness of cell assembly state estimation method for decoding the brain-machine interface data. NEW METHOD: We incorporated a novel method of estimating cell assembly states using spike trains with the existing decoding method that used only firing rate data. Synaptic connectivity pattern was used as feature values in addition to firing rate. Publicly available monkey brain-machine interface datasets were used in the study. RESULTS: As long as the decoding was successful, the root mean square error of the proposed method was significantly smaller than the existing method. Artificial neural netowork-based decoding method resulted in more stable decoding, and also improved the decoding accuracy due to incorporation of synaptic connectivity pattern.

Development of a Hypersensitivity Evaluation Method for Cultured Sensory Neurons Using Electrical Activity Recording.

Investigation of hypersensitivity caused by peripheral sensitization progression is important for developing novel pain treatments. Existing methods cannot record plastic changes in neuronal activity because they occur over a few days. We aimed to establish an efficient method to evaluate neuronal activity alterations caused by peripheral sensitization on high-density microelectrode arrays (HD-MEAs) which can record neuronal activity for a long time. Rat dorsal root ganglion (DRG) neurons were dissected from rat embryos and cultured on HD-MEAs. DRG neurons were labeled with NeuO, live staining dye. Neurons were detected with the fluorescence signal and electrodes were selected with the fluorescence images. The number of DRG neurons, whose activity were recorded, detected based on fluorescence observation was five times greater than that based on neuronal activity. Analysis of changes in neuronal activity observed in pharmacological stimulation experiments suggested that substance P induced peripheral sensitization and enhanced capsaicin sensitivity. In addition, results of immunofluorescence staining suggested that peripheral sensitization occurred mostly in neurons that co-expressed transient receptor potential vanilloid 1 (TRPV1) and neurokinin 1 receptor (NK1R). In conclusion, we established an efficient method for assessing the effects of peripheral sensitization on DRG neurons cultured on HD-MEAs.

Observing Cell Assemblies From Spike Train Recordings Based on the Biological Basis of Synaptic Connectivity.

Cell assemblies are difficult to observe because they consist of many neurons. We aimed to observe cell assemblies based on biological statistics, such as synaptic connectivity. We developed an estimation method to estimate the activity and synaptic connectivity of cell assemblies from spike trains using mathematical models of individual neurons and cell assemblies. Synaptic transmissions were averaged to generate postsynaptic currents with the same timing and waveform but different amplitudes, as the number of presynaptic neurons was large. We estimated the average synaptic transmission and synaptic connectivity from active cell assemblies based on the stochastic prediction of membrane potentials and verified the estimation ability of the average synaptic transmission and synaptic connectivity using the proposed method on simulated neural activity. Different cell assembly activities evoked by electrical stimuli were correctly sorted into various clusters in experiments using rat cortical neurons cultured on microelectrode arrays. We observed multiple cell assemblies from the spontaneous activity of rat cortical networks on microelectrode arrays, based on the synaptic connectivity patterns estimated by the proposed method. The proposed method was superior to the conventional method for detecting the activity of multiple cell assemblies. Using the proposed method, it is possible to observe multiple cell assemblies based on the biological basis of synaptic connectivity. In summary, we report a novel method to observe cell assemblies from spike train recordings based on the biological basis of synaptic connectivity, rather than merely relying on a statistical method.

Recording Saltatory Conduction Along Sensory Axons Using a High-Density Microelectrode Array.

Myelinated fibers are specialized neurological structures used for conducting action potentials quickly and reliably, thus assisting neural functions. Although demyelination leads to serious functional impairments, little is known the relationship between myelin structural change and increase in conduction velocity during myelination and demyelination processes. There are no appropriate methods for the long-term evaluation of spatial characteristics of saltatory conduction along myelinated axons. Herein, we aimed to detect saltatory conduction from the peripheral nervous system neurons using a high-density microelectrode array. Rat sensory neurons and intrinsic Schwann cells were cultured. Immunofluorescence and ultrastructure examination showed that the myelinating Schwann cells appeared at 1 month, and compact myelin was formed by 10 weeks in vitro. Activity of rat sensory neurons was evoked with optogenetic stimulation, and axon conduction was detected with high-density microelectrode arrays. Some conductions included high-speed segments with low signal amplitude. The same segment could be detected with electrical recording and immunofluorescent imaging for a myelin-related protein. The spatiotemporal analysis showed that some segments show a velocity of more than 2 m/s and that ends of the segments show a higher electrical sink, suggesting that saltatory conduction occurred in myelinated axons. Moreover, mathematical modeling supported that the recorded signal was in the appropriate range for axon and electrode sizes. Overall, our method could be a feasible tool for evaluating spatial characteristics of axon conduction including saltatory conduction, which is applicable for studying demyelination and remyelination.

Coupling of in vitro Neocortical-Hippocampal Coculture Bursts Induces Different Spike Rhythms in Individual Networks.

Brain-state alternation is important for long-term memory formation. Each brain state can be identified with a specific process in memory formation, e.g., encoding during wakefulness or consolidation during sleeping. The hippocampal-neocortical dialogue was proposed as a hypothetical framework for systems consolidation, which features different cross-frequency couplings between the hippocampus and distributed neocortical regions in different brain states. Despite evidence supporting this hypothesis, little has been reported about how information is processed with shifts in brain states. To address this gap, we developed an in vitro neocortical-hippocampal coculture model to study how activity coupling can affect connections between coupled networks. Neocortical and hippocampal neurons were cultured in two different compartments connected by a micro-tunnel structure. The network activity of the coculture model was recorded by microelectrode arrays underlying the substrate. Rhythmic bursting was observed in the spontaneous activity and electrical evoked responses. Rhythmic bursting activity in one compartment could couple to that in the other via axons passing through the micro-tunnels. Two types of coupling patterns were observed: slow-burst coupling (neocortex at 0.1-0.5 Hz and hippocampus at 1 Hz) and fast burst coupling (neocortex at 20-40 Hz and hippocampus at 4-10 Hz). The network activity showed greater synchronicity in the slow-burst coupling, as indicated by changes in the burstiness index. Network synchronicity analysis suggests the presence of different information processing states under different burst activity coupling patterns. Our results suggest that the hippocampal-neocortical coculture model possesses multiple modes of burst activity coupling between the cortical and hippocampal parts. With the addition of external stimulation, the neocortical-hippocampal network model we developed can elucidate the influence of state shifts on information processing.

Change in network dynamics over time by administering Notch response inhibitor DAPT to hippocampal culture.

Although previous researches have investigated the relationship between learning and memory function in the hippocampus and continuously produced newborn neurons, the detailed role of newly generated neurons remains unclear. Here, we investigated the correlation between immature neurons and the electrical activity of the hippocampus at the network level in vitro. We showed that administrating the Notch response inhibitor DAPT to the hippocampal network enhances the neuronal differentiation of newborn cells and decreases the ratio of immature neurons in hippocampal culture. Unlike the hippocampal network without DAPT, the network with DAPT decreased the burst duration and the coefficient of variation of interburst intervals over culturing time and showed a higher synchronization level of the network over time. Moreover, the number of neurons playing a receiver or sender neuron was lower in the network with DAPT than without DAPT. Our results indicate that immature neurons may contribute to assigning neurons specific nodes as the receiver of the sender and to the diversity of the network activity while altering connections among neurons in the network.Clinical Relevance- Our research demonstrated the effect of DAPT on the ratio of immature neurons. Furthermore, our study showed the role of immature neurons in the hippocampus at the network level.

Modulation of dynamics in a pre-existing hippocampal network by neural stem cells on a microelectrode array.

Objective.Neural stem cells (NSCs) are continuously produced throughout life in the hippocampus, which is a vital structure for learning and memory. NSCs in the brain incorporate into the functional hippocampal circuits and contribute to processing information. However, little is known about the mechanisms of NSCs' activity in a pre-existing neuronal network. Here, we investigate the role of NSCs in the neuronal activity of a pre-existing hippocampalin vitronetwork grown on microelectrode arrays.Approach.We assessed the change in internal dynamics of the network by additional NSCs based on spontaneous activity. We also evaluated the networks' ability to discriminate between different input patterns by measuring evoked activity in response to external inputs.Main results.Analysis of spontaneous activity revealed that additional NSCs prolonged network bursts with longer intervals, generated a lower number of initiating patterns, and decreased synchronization among neurons. Moreover, the network with NSCs showed higher synchronicity in close connections among neurons responding to external inputs and a larger difference in spike counts and cross-correlations during evoked response between two different inputs. Taken together, our results suggested that NSCs alter the internal dynamics of the pre-existing hippocampal network and produce more specific responses to external inputs, thus enhancing the ability of the network to differentiate two different inputs.Significance.We demonstrated that NSCs improve the ability to distinguish external inputs by modulating the internal dynamics of a pre-existing network in a hippocampal culture. Our results provide novel insights into the relationship between NSCs and learning and memory.

Microfabricated Device to Record Axonal Conduction Under Pharmacological Treatment for Functional Evaluation of Axon Ion Channel.

OBJECTIVE: Neuronal networks are fundamental structures for information processing in the central nervous system. This processing function is severely impaired by abnormal axonal conduction from changes in functional ion channel expression. The evaluation of axonal conduction properties can be effective in the early diagnosis of information-processing abnormalities. However, little is known about functional ion channel expression in axons owing to lack of an appropriate method. In this study, we developed a device to measure changes in axonal conduction properties by selective pharmacological stimulation for the functional evaluation of Na channels expressed in axons. METHODS: Axons of rat cortical neurons were guided across a pair of electrodes through microtunnel structures by employing surface patterning. RESULTS: The developed device detected more than 50 axons while recording for 10 min. The conduction delay along the axons decreased by 22.5% with neuron maturation. Tetrodotoxin and lidocaine (Na channel blockers) increased the conduction delay in a concentration-dependent manner depending on their working concentrations, indicating the effectiveness of the device. Finally, selective Na channel blockers for various Na channel subtypes were used. Phrixotoxin, a Nav1.2 blocker, markedly increased the conduction delay, suggesting that Nav1.2 is functionally expressed in the unmyelinated axons of the cerebral cortex. CONCLUSION: These results show that our device is feasible for the high-throughput functional evaluation of Na channel subtypes in axons. SIGNIFICANCE: The results obtained can contribute to the understanding of the pathogenic mechanisms of neurological diseases that involve changes in the functional expression states of ion channels in axons.

Preparation of Size-controlled Giant Vesicles Under Physiological Conditions.

Giant vesicles (GVs) are model cell membranes that function as tools for the study of cell membrane properties. Recently, researchers have been calling for GVs of specific sizes for use in studies with precise needs. In this paper, we report a method of forming GVs of specific sizes by using an agarose-swelling approach. The resulting GVs had a narrow size distribution and were successfully formed under physiological conditions.

Microdevice for Evaluating Ion Channel Expression by Axon-Targeted Recording to Cultured Neurons.

Recording axonal conduction will be a strong tool to continuously evaluate Na channel expression of cultured neurons. However, little is known about the relationship between ion channel expression and axonal conduction velocity. In this study, we aim to develop a method to evaluate the relationship. A microdevice was developed with photo- and soft-lithography. Cortical neurons were cultured, and activity propagating along axons was recorded. After spike sorting, mixed signal from multiple axons were sorted into clusters of individual axons. Axons were treated with non-selective and subtype specific Na channel blockers, and changes in conduction delay were evaluated. TTX and lidocaine increased conduction delay with a different manner, suggesting that the different affinity and binding kinetics can be detected with the device. Moreover, although Nav 1.2 blocker increased the conduction delay and eventually clocked the conduction at around IC50, the other blockers did not. This result suggests that Nav 1.2 is dominant for the conduction along unmyelinated cortical axons. Overall, our device should be a feasible tool for elucidating Na channel properties by axon-targeted recording.

Change in Evoked Response of Mature Neuronal Network to Spatial Pattern Stimulation by Immature Neurons.

Adult neurogenesis in the hippocampus is known to enhance pattern separation. However, the effect of adult neurogenesis on spatial pattern separation at the cellular assembly level is unclear. In order to elucidate how newborn and immature neurons change learning of spatial pattern of mature neuronal network, we evaluated evoked response to two types of spatial patterns of the cultured hippocampal network with or without added neural stem cells by using electrical stimulation on microelectrode array. Results show that the existence of newborn and immature neurons changed evoked response of mature neuronal network to both trained and untrained patterns, suggesting that the presence of immature neurons may contribute to production of the change that mature neuronal network enhances LTP and excitation to stimuli.

Long-Term Developmental Process of the Human Cortex Revealed In Vitro by Axon-Targeted Recording Using a Microtunnel-Augmented Microelectrode Array.

OBJECTIVE: We aimed to develop a method for evaluating developmental changes in the synchronized activity of human induced pluripotent stem cell (hiPSC)-derived neurons without extrinsic signals from feeder astrocytes. METHODS: Microelectrode arrays (MEAs) and microtunnels were fabricated with photolithography and soft lithography. hiPSCs were induced to differentiate into cortical neurons, and seeded to conventional and microtunnel MEAs. Spontaneous activity was recorded every ten days, and spiking and bursting activities were elucidated. RESULTS: First, hiPSC-derived neurons were cultured on conventional MEAs. They formed aggregates and subsequently detached from the culture substrate. Hence, no MEAs showed spontaneous synchronized activity beyond 300 days post-induction. Next, we applied a microtunnel structure designed to keep the axons on the array. Synchronized activity was then recorded from all microtunnel MEAs by 450 days post-induction. The proportion of electrodes showing neural activity was greater than that in conventional MEAs. The activity pattern reached a steady state after approximately 330 days, which may be the maturation time of the human neuronal network. CONCLUSION: The use of a microtunnel MEA enables the monitoring of the long-term development of human neuronal networks of cell populations that are relatively natural given their lack of astrocyte feeders. SIGNIFICANCE: We report a more accurate method for culturing cortical neurons differentiated from hiPSCs, validating their use in elucidating cortical development and pathogenic mechanisms in humans.

Functional Scaffolding for Brain Implants: Engineered Neuronal Network by Microfabrication and iPSC Technology.

Neuroengineering methods can be effectively used in the design of new approaches to treat central nervous system and brain injury caused by neurotrauma, ischemia, or neurodegenerative disorders. During the last decade, significant results were achieved in the field of implant (scaffold) development using various biocompatible and biodegradable materials carrying neuronal cells for implantation into the injury site of the brain to repair its function. Neurons derived from animal or human induced pluripotent stem (iPS) cells are expected to be an ideal cell source, and induction methods for specific cell types have been actively studied to improve efficacy and specificity. A critical goal of neuro-regeneration is structural and functional restoration of the injury site. The target treatment area has heterogeneous and complex network topology with various types of cells that need to be restored with similar neuronal network structure to recover correct functionality. However, current scaffold-based technology for brain implants operates with homogeneous neuronal cell distribution, which limits recovery in the damaged area of the brain and prevents a return to fully functional biological tissue. In this study, we present a neuroengineering concept for designing a neural circuit with a pre-defined unidirectional network architecture that provides a balance of excitation/inhibition in the scaffold to form tissue similar to that in the injured area using various types of iPS cells. Such tissue will mimic the surrounding niche in the injured site and will morphologically and topologically integrate into the brain, recovering lost function.