Search

The synchronous activity of neuronal networks is considered crucial for brain function. However, the interaction between single-neuron activity and network-wide activity remains poorly understood. This study explored this interaction within cultured networks of rat cortical neurons. Employing a combination of high-density microelectrode array recording and optogenetic stimulation, we established an experimental setup enabling simultaneous recording and stimulation at a precise single-neuron level that can be scaled to the level of the whole network. Leveraging our system, we identified a network burst-dependent response change in single neurons, providing a possible mechanism for the network-burst-dependent loss of information within the network and consequent cognitive impairment during epileptic seizures. Additionally, we directly recorded a leader neuron initiating a spontaneous network burst and characterized its firing properties, indicating that the bursting activity of hub neurons in the brain can initiate network-wide activity. Our study offers valuable insights into brain networks characterized by a combination of bottom-up self-organization and top-down regulation., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Empirical applications of the free-energy principle are not straightforward because they entail a commitment to a particular process theory, especially at the cellular and synaptic levels. Using a recently established reverse engineering technique, we confirm the quantitative predictions of the free-energy principle using in vitro networks of rat cortical neurons that perform causal inference. Upon receiving electrical stimuli-generated by mixing two hidden sources-neurons self-organised to selectively encode the two sources. Pharmacological up- and downregulation of network excitability disrupted the ensuing inference, consistent with changes in prior beliefs about hidden sources. As predicted, changes in effective synaptic connectivity reduced variational free energy, where the connection strengths encoded parameters of the generative model. In short, we show that variational free energy minimisation can quantitatively predict the self-organisation of neuronal networks, in terms of their responses and plasticity. These results demonstrate the applicability of the free-energy principle to in vitro neural networks and establish its predictive validity in this setting., (© 2023. The Author(s).)

Gamma oscillations are thought to play a role in information processing in the brain. Bursting neurons, which exhibit periodic clusters of spiking activity, are a type of neuron that are thought to contribute largely to gamma oscillations. However, little is known about how the properties of bursting neurons affect the emergence of gamma oscillation, its waveforms, and its synchronized characteristics, especially when subjected to stochastic fluctuations. In this study, we proposed a bursting neuron model that can analyze the bursting ratio and the phase response function. Then we theoretically analyzed the neuronal population dynamics composed of bursting excitatory neurons, mixed with inhibitory neurons. The bifurcation analysis of the equivalent Fokker-Planck equation exhibits three types of gamma oscillations of unimodal firing, bimodal firing in the inhibitory population, and bimodal firing in the excitatory population under different interaction strengths. The analyses of the macroscopic phase response function by the adjoint method of the Fokker-Planck equation revealed that the inhibitory doublet facilitates synchronization of the high-frequency oscillations. When we keep the strength of interactions constant, decreasing the bursting ratio of the individual neurons increases the relative high-gamma component of the populational phase-coupling functions. This also improves the ability of the neuronal population model to synchronize with faster oscillatory input. The analytical frameworks in this study provide insight into nontrivial dynamics of the population of bursting neurons, which further suggest that bursting neurons have an important role in rhythmic activities., (© 2023 Massachusetts Institute of Technology.)

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.

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.

The serotonin 5-HT 2A receptor (5-HT 2A R) has been receiving increasing attention because its genetic variants have been associated with a variety of neurological diseases. To elucidate the pathogenesis of the neurological diseases associated with 5-HT 2A R gene (HTR2A) variants, we have previously established a protocol to induce HTR2A-expressing neurons from human-induced pluripotent stem cells (hiPSCs). Here, we investigated the maturation stages and electrophysiological properties of HTR2A-positive neurons induced from hiPSCs and constructed an HTR2A promoter-specific reporter lentivirus to label the neurons. We found that neuronal maturity increased over time and that HTR2A expression was induced at the late stage of neuronal maturation. Furthermore, we demonstrated successful labelling of the HTR2A-positive neurons, which had fluorescence and generated repetitive action potentials in response to depolarizing currents and an inward current during the application of TCB-2, a selective agonist of 5-HT 2A Rs, respectively. These results indicated that our in vitro model mimicked the in vivo dynamics of 5-HT 2A R. Therefore, in vitro monitoring of the function of HTR2A-positive neurons induced from hiPSCs could help elucidate the pathophysiological mechanisms of neurological diseases associated with genetic variations of the HTR2A gene., (© 2021. The Author(s).)

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.

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 GABA A 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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

It is still unclear how the activity of sympathetic and parasympathetic neurons influences the activity of cardiomyocytes in culture. We developed a device for co-culturing sympathetic neurons, parasympathetic neurons, and cardiomyocytes using micro-fabrication techniques. Morphological connections between each type of autonomic neuron and the cardiomyocytes were observed by immunostaining. The inter-beat-interval (IBI) of the cardiomyocytes was modulated after electrically stimulating each type of autonomic neuron. Modulation of the IBI was blocked by the addition of pharmacological blockers to the culture medium. These results suggest that the co-culture device can be utilized to understand how the activity of sympathetic neurons and parasympathetic neurons influences the activity of cardiomyocytes in the culture environment.

Blind source separation is the computation underlying the cocktail party effect--a partygoer can distinguish a particular talker's voice from the ambient noise. Early studies indicated that the brain might use blind source separation as a signal processing strategy for sensory perception and numerous mathematical models have been proposed; however, it remains unclear how the neural networks extract particular sources from a complex mixture of inputs. We discovered that neurons in cultures of dissociated rat cortical cells could learn to represent particular sources while filtering out other signals. Specifically, the distinct classes of neurons in the culture learned to respond to the distinct sources after repeating training stimulation. Moreover, the neural network structures changed to reduce free energy, as predicted by the free-energy principle, a candidate unified theory of learning and memory, and by Jaynes' principle of maximum entropy. This implicit learning can only be explained by some form of Hebbian plasticity. These results are the first in vitro (as opposed to in silico) demonstration of neural networks performing blind source separation, and the first formal demonstration of neuronal self-organization under the free energy principle.

Objective: Simplified neuronal circuits are required for investigating information representation in nervous systems and for validating theoretical neural network models. Here, we developed patterned neuronal circuits using micro fabricated devices, comprising a micro-well array bonded to a microelectrode-array substrate., Approach: The micro-well array consisted of micrometre-scale wells connected by tunnels, all contained within a silicone slab called a micro-chamber. The design of the micro-chamber confined somata to the wells and allowed axons to grow through the tunnels bidirectionally but with a designed, unidirectional bias. We guided axons into the point of the arrow structure where one of the two tunnel entrances is located, making that the preferred direction., Main Results: When rat cortical neurons were cultured in the wells, their axons grew through the tunnels and connected to neurons in adjoining wells. Unidirectional burst transfers and other asymmetric signal-propagation phenomena were observed via the substrate-embedded electrodes. Seventy-nine percent of burst transfers were in the forward direction. We also observed rapid propagation of activity from sites of local electrical stimulation, and significant effects of inhibitory synapse blockade on bursting activity., Significance: These results suggest that this simple, substrate-controlled neuronal circuit can be applied to develop in vitro models of the function of cortical microcircuits or deep neural networks, better to elucidate the laws governing the dynamics of neuronal networks.

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.

Investigating the mechanisms of the neuron-glia interaction is important in the basic research of neuroscience and neural transplantation. Synaptic transmission is modulated by astrocyte activation in the pre- and post-synaptic terminals, and this phenomenon is spread to the surrounding astrocytes through gap junctions. However, the modulation of network-wide neuronal activity dependent on extensive astrocyte activation is not well understood. In this study, we show network-wide neuronal modulation associated with a newly developed three-dimensional neuronal and astrocytic network co-culture method. To establish widespread neuronal and astrocytic network interactions in vitro, we performed integration of magnetic nanoparticle-injected astrocytes (Mag-AS) onto the matured monolayer of neuronal networks using an external magnetic force. The neuronal electrical activity was dynamically synchronized at 24 h after integration of the Mag-AS network. In addition, Mag-AS network activation using a caged calcium compound rapidly induced suppression and subsequent synchronization of neuronal electrical activity. These results indicate that the high-density astrocytic network integration onto the neuronal network can induce widespread neuronal modulation, and our in vitro co-culture method contributes to the advancement of neuronal and astrocytic transplantation research.

Gamma oscillations of the local field potential are organized by collective dynamics of numerous neurons and have many functional roles in cognition and/or attention. To mathematically and physiologically analyse relationships between individual inhibitory neurons and macroscopic oscillations, we derive a modification of the theta model, which possesses voltage-dependent dynamics with appropriate synaptic interactions. Bifurcation analysis of the corresponding Fokker-Planck equation (FPE) enables us to consider how synaptic interactions organize collective oscillations. We also develop the adjoint method (infinitesimal phase resetting curve) for simultaneous equations consisting of ordinary differential equations representing synaptic dynamics and a partial differential equation for determining the probability distribution of the membrane potential. This method provides a macroscopic phase response function (PRF), which gives insights into how it is modulated by external perturbation or internal changes of parameters. We investigate the effects of synaptic time constants and shunting inhibition on these gamma oscillations. The sensitivity of rising and decaying time constants is analysed in the oscillatory parameter regions; we find that these sensitivities are not largely dependent on rate of synaptic coupling but, rather, on current and noise intensity. Analyses of shunting inhibition reveal that it can affect both promotion and elimination of gamma oscillations. When the macroscopic oscillation is far from the bifurcation, shunting promotes the gamma oscillations and the PRF becomes flatter as the reversal potential of the synapse increases, indicating the insensitivity of gamma oscillations to perturbations. By contrast, when the macroscopic oscillation is near the bifurcation, shunting eliminates gamma oscillations and a stable firing state appears. More interestingly, under appropriate balance of parameters, two branches of bifurcation are found in our analysis of the FPE. In this case, shunting inhibition can effect both promotion and elimination of the gamma oscillation depending only on the reversal potential.

Regenerative medicine is expected to be a potent therapeutic option for the disorders or injuries of the central nervous system. However, little is known about how the newly formed neurons derived from grafted stem cells integrate into the host established tissue. The aims of this study are to make functional connection between primary neurons and stem cell-derived neurons via chemical synapses and maintain the connection for a long time in in vitro. We employed an in vitro co-culture device to cultivate two different neuronal populations and evaluate the interaction between them. Mouse cortical neurons and P19 cell-derived neurons were co-cultured in the co-culture device. The synchronous activities were maintained for at least 4 weeks. Evoked responses to electrical stimulation suggested that bi-directional connections were formed between cortical and P19-derived neurons. The responses were changed after pharmacological treatment. These results showed that cortical neurons and P19 cell-derived neurons formed bidirectional synaptic connections via glutamate receptors and the connection was maintained for at least 4 weeks.

Induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CMs) has been expected as a cell source for therapy of serious heart failure. However, it is unclear whether the function of iPS-CMs is modulated by the host sympathetic nervous system. Here we developed a device for co-culture of sympathetic neurons and iPS-CMs using microfabrication technique. The device consisted of a culture chamber and a microelectrode-array (MEA) substrate. The superior cervical ganglion (SCG) neurons were co-cultured with iPS-CMs in a microfabricated device, which had multiple compartments. Several days after seeding, synapses were formed between SCG neurons and iPS-CMs, as confirmed by immunostaining. Spontaneous electrical activities of the SCG neurons and the iPS-CMs were observed from the electrode of the MEA substrate. The beat rate of iPS-CMs increased after electrical stimulation of the co-cultured SCG neurons. Such changes in the beat rate were prevented in the presence of propranolol, a β-adrenoreceptor antagonist. These results suggest that the microfabricated device will be utilized for studying the functional modulation of iPS-CMs by connected sympathetic neurons.

Although stem cell-derived cardiomyocytes have great potential for the therapy of heart failure, it is unclear whether their function after grafting can be controlled by the host sympathetic nervous system, a component of the autonomic nervous system (ANS). Here we demonstrate the formation of functional connections between rat sympathetic superior cervical ganglion (SCG) neurons and pluripotent (P19.CL6) cell-derived cardiomyocytes (P19CMs) in compartmentalized co-culture, achieved using photolithographic microfabrication techniques. Formation of synapses between sympathetic neurons and P19CMs was confirmed by immunostaining with antibodies against β-3 tubulin, synapsin I and cardiac troponin-I. Changes in the beat rate of P19CMs were triggered after electrical stimulation of the co-cultured SCG neurons, and were affected by the pulse frequency of the electrical stimulation. Such changes in the beat rate were prevented when propranolol, a β-adrenoreceptor antagonist, was added to the culture medium. These results suggest that the beat rate of differentiated cardiomyocytes can be modulated by electrical stimulation of connected sympathetic neurons.

Regeneration of damaged central nervous systems (CNS) is an important topic in neuroscience and neuroengineering. Grafting new neurons derived from pluripotent stem cells into damaged regions can be done to restore functions after injury. Little is known, however, about network-wide interactions between stem-cell-derived neurons and CNS neurons. In this study, we developed a co-culture method of stem cell-derived neuronal networks and CNS networks and observed spontaneous activity in the co-culture samples. By using a microfabricated poly(dimethylsiloxane) device having two culture compartments and 20 connecting microconduits, we are able to compartmentalize P19-derived neurons and mouse cortical neurons and connect them via the microconduits. Furthermore, we combined the co-culture device and a microelectrode array (MEA)-based recording system and recorded spontaneous activity in the co-cultured networks. We found that periodic synchronized bursting spreading over both neuronal networks occurred during the second week in vitro and that P19-derived neurons in the co-cultured networks had different developmental processes compared with those grown in monoculture. These findings suggest that functional interactions form between P19-dervived neurons and mouse cortical neurons and that the co-culture method is useful for exploring the network-wide integrations between stem cell-derived neurons and CNS neurons., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Rat superior cervical ganglion (SCG) neurons and ventricular myocytes (VMs) were co-cultured separately in a minichamber placed on a microelectrode-array (MEA) substrate. The minichamber, fabricated photolithographically using polydimethylsiloxane (PDMS), had 2 compartments, 16 microcompartments and 8 microconduits. The SCG neurons were seeded into one of the compartments and all of the microcompartments using a glass pipette controlled by a micromanipulator and a microinjector. The VMs were seeded into the other compartment. Three days after seeding of the VMs, the SCG neurons were still confined to one compartment and all of the microcompartments, and the neurites of the SCG neurons had connected with the VMs via the microconduits. Constant-voltage stimulation, using a train of biphasic square pulses (1 ms at +1 V, followed by -1 ms at 1 V), was applied to the SCG neurons in the microcompartments using 16 electrodes. Evoked responses were observed in several electrodes while electrical stimulation was applied to the SCG neurons. Two-way analysis of variance (ANOVA) revealed that the frequency of the stimulation pulses had significant effects in increasing the beat rate of the VMs, and that the interaction between the frequency and the number of the pulses also had a significant effect on the ratio. No significant increases in the beat rate were observed when propranolol, a β-adrenergic receptor antagonist, was added to the culture medium. These results suggest that synaptic pathways were formed between the SCG neurons and the VMs, and that this co-culture device can be utilized for studies of network-level interactions between sympathetic neurons and cardiomyocytes., (This journal is © The Royal Society of Chemistry 2011)

Magnetic stimulation is able to modulate the neuronal network activity using the non-invasive magnetically induced current. However, it is unknown how stimulation modulates the neuronal network activity. Therefore, we considered that precise stimulation and evaluation of the modulation of network activities in the vicinity of stimulated sites is required. Here, to establish precisely magnetic stimulation, we developed a Mu-metal that has high magnetic permeability soft magnetic material based localized magnetic stimulation (LMS) system with micro-fabricated dual cell-culture chambers. And, combining this device with a microelectrode array (MEA) permitted the evaluation of the stimulus effects at the stimulated and non-stimulated sites. Here, the dual cell-culture chambers were arranged in a concentric circle manner. Between the inner and outer chambers, 4, 8 and 12 connecting microfluid channels were fabricated using polydimethylsiloxane (PDMS). Rat cortical neurons were separately cultured in outer and inner chambers. Through the micro-conduits, functional synaptic connections were formed. Mu-metal was aligned along the outer circle, which allowed us of focal magnetic stimulation to the cells in the outer chamber. Applying low frequency magnetic field to the Mu-metal, induced currents were generated and the electrical activity of the cells in the outer chamber was modified depending on the stimulation intensity. Following the modified activity in the outer circles, the cells in the inner chamber also showed slightly depressed activity patterns. These results suggested that our system would be promising for highly regulated neural stimulation.

Spontaneous neuronal activity plays an important role in the development of the brain. Developmental changes in the spontaneous activity pattern of neuronal networks in vitro have been extensively studied by using the microelectrode array (MEA) recording system. However, little is known about the transition of spontaneous intracellular calcium dynamics, and the relationship between calcium transients and electrical activity during development. In the present paper, we carry out simultaneous recording of spontaneous electrical activity and intracellular calcium transients of rat cortical networks cultured on MEA. In one-week cultures, periodic synchronized bursts are observed and are followed by synchronized calcium transients. In three-week cultures, synchronized calcium transients are rarely observed despite the presence of highly complicated synchronized activity. Between these two states, in two-week cultures, slow, radial propagation of calcium waves independent of electrical activity is observed. Pharmacological treatments with the purinergic receptor antagonist suramin and gap junction blocker 18- beta glycyrrhetinic acid reveal that the spontaneous radial calcium waves are mediated by the astrocytic network, and suggest that the astrocytic calcium waves can influence the electrical firing patterns of networks by locally affecting neuronal signaling. These results indicate that the various dynamics of intracellular calcium transients regulate the network maturation processes.

Modulation of pluripotent stem cell differentiation by several environmental factors, such as physical stimulation, is important theme in tissue engineering. In this study, we report the effects of extremely low frequency magnetic fields (ELF-MFs) exposure (1 mT or 10 mT, 50 Hz, sinusoidal) on the neuronal differentiation process of P19 embryonal carcinoma cells (P19 cells). Here, during induction of differentiation, the ELF-MFs exposed to embryoid bodies (EBs). After neuronal differentiation, the effects of ELF-MFs were evaluated by morphological analysis, immunochemical analysis (MAP2, GFAP), and the developmental neuronal network activities recorded by the micro-electrode arrays (MEAs). As a result, the percentage of MAP2 positive cells and the spike frequencies were increased by 10 mT ELF-MF, and then the percentage of GFAP positive cells were reduced. However, these effects were not seen in 1 mT exposed cells. Therefore, these results suggested that the intensity of a magnetic field was important for affecting a characteristic of neuronal differentiation and a functional neuronal network property.

Rat superior cervical ganglion (SCG) neurons and ventricular myocytes (VMs) were co-cultured in chambers made of polydimethylsyloxane. The chambers were placed on a microelectrode-array (MEA) substrate and connected with a pathway. 24 hours after dissemination of the VMs, neuntes of the SCG neurons outgrew through the pathway and reached the VMs. Spontaneous electrical activities of the SCG neurons and the VMs were observed several days after the dissemination. Constant-voltage stimualtion (1 V, 1 ms, biphasic square pulses) was applied to the SCG neurons at the frequency of 10 Hz using 32 electrodes. Contraction rate of the VMs increased by 153 +/-110 % immediately after the stimulation to the SCG neurons was stopped. Then contraction rate gradually decreased and returned to almost the same rate as before the stimulation 5 minutes after the 1-min stimulation. Propranolol (beta-adrenergic receptor antagonist) prevented contraction rate of the VMs from increasing after electrical stimulation to the SCG neurons. These results suggest that neuromuscular junctions were formed between the SCG neurons and the VMs. Overall the semi-separated co-culture system in this study is available in research on changes in contraction rate of the VMs after applying electrical stimulation to the SCG neurons.

Precise control of differentiation processes of pluripotent stem cell is key component for realization of regenerative medicine. Electrical stimulation is one of the promising techniques, particularly for regulation of neuronal regeneration. In the present study, we developed substrates with embedded electrodes, which allowed ensemble electrical stimulation of embryoid bodies (EBs) of stem cells. Microcavity-array patterns were fabricated on substrates with embedded electrodes using standard photo-lithography. Uniform-size EBs of P19 cells were prepared, inserted one by one in each microcavity, and electrical stimulation was applied through substrate electrodes. Stimulus-induced intracellular calcium transients were successfully monitored by fluorescence imaging. The results suggested that this method would be useful for applying precisely-controlled electrical stimulation to a large number of EBs of stem cells.