Yasuhiko Jimbo

Spontaneous periodic synchronized bursting during formation of mature patterns of connections in cortical cultures.

Long-term recording of spontaneous activity in cultured cortical neuronal networks was carried out using substrates containing multi-electrode arrays. Spontaneous uncorrelated firing appeared within the first 3 days and transformed progressively into synchronized bursting within a week. By 30 days from the establishment of the culture, the network exhibited a complicated non-periodic, synchronized activity pattern which showed no changes for more than 2 months and thus represented the mature state of the network. Pharmacological inhibition of activity only during the period when regular synchronized bursting was observed was capable of producing a different mature activity pattern from the control. These results suggest that periodic synchronized bursting plays a critical role in the development of synaptic connections.

Electrical stimulation and recording from cultured neurons using a planar electrode array

Abstract Planar electrode arrays were fabricated using modern semiconductor technology. Mouse and chick dorsal root ganglion cells were successfully cultured on the surface of indium tin oxide electrode patterns. Contact between the neurites and the substrate electrodes was established by exploiting the surface properties of the culture substrates to guide neurite outgrowth. The guided neurites were stimulated electrically using the substrate electrodes and cell responses were recorded intracellularly and extracellularly. Depolarization of the cell membrane as well as generation of action potentials in response to the stimulation could be observed. The results suggest that this type of planar electrode array could be a useful tool for non-invasive electrophysiological measurements.

The dynamics of a neuronal culture of dissociated cortical neurons of neonatal rats

Abstract. Neuronal networks of dissociated cortical neurons from neonatal rats were cultured over a multielectrode dish with 64 active sites, which were used both for recording the electrical activity and for stimulation. After about 4 weeks of culture, a dense network of neurons had developed and their electrical activity was studied. When a brief voltage pulse was applied to one extracellular electrode, a clear electrical response was evoked over almost the entire network. When a strong voltage pulse was used, the response was composed of an early phase, terminating within 25 ms, and a late phase which could last several hundreds of milliseconds. Action potentials evoked during the early phase occurred with a precise timing with a small jitter and the electrical activity initiated by a localized stimulation diffused significantly over the network. In contrast, the late phase was characterized by the occurrence of clusters of electrical activity with significant spatio-temporal fluctuations. The late phase was suppressed by adding small amounts of d(−)-2-amino-5-phosphonovaleric acid to the extracellular medium, or by increasing the amount of extracellular Mg2+. The electrical activity of the network was substantially increased by the addition of bicuculline to the extracellular medium. The results presented here show that the neuronal network may exist in two different dynamical states: one state in which the neuronal network behaves as a non-chaotic deterministic system and another state where the system exhibits large spatio-temporal fluctuations, characteristic of stochastic or chaotic systems.

Strengthening of synchronized activity by tetanic stimulation in cortical cultures: application of planar electrode arrays

Rat cortical neurons were cultured on planar electrode arrays with 64 embedded electrodes. Whole-cell recording from single neurons and multisite extracellular recording were carried out simultaneously in the cultured cortical networks, and the effects of focal tetanic stimulation of the culture were studied. Both the number of action potentials and the propagation velocity of stimulated bursts were increased after tetanic stimulation. These changes were associated with a marked increase in the number of late components in the synaptic current, but with little or no increase in the early peak synaptic current. The effects of tetanic stimulation were consistent with a widespread increase in the reliability of monosynaptic transmission.

Activity-dependent enhancement in the reliability of correlated spike timings in cultured cortical neurons

Abstract. To study the use-dependent modification of activity in neural networks, we investigated the spike timing by simultaneously recording activity at multiple sites in a network of cultured cortical neurons. We used dynamical analysis to study the temporal structure of spike trains and the activity-dependent changes in the reliability and reproducibility of spike patterns evoked by a stimulus. We also used cross-correlation analysis to evaluate the interactions of neuron pairs. Our main conclusions are that even when no obvious change in spike numbers can be seen, use-dependent modification occurs, either enhancing or reducing in the reliability and reproducibility of spike trains evoked by a stimulus, and the fine temporal structure of stimulus-evoked spike trains and interactions between neurons are also modified by tetanic stimulation.

Spatio-temporal cholinergic modulation in cultured networks of rat cortical neurons: spontaneous activity.

Activation of the cholinergic innervation of the cortex has been implicated in sensory processing, learning, and memory. At the cellular level, acetylcholine both increases excitability and depresses synaptic transmission, and its effects on network firing are hard to predict. We studied the effects of carbachol, a cholinergic agonist, on network firing in cultures of rat cortical neurons, using electrode arrays to monitor the activity of large numbers of neurons simultaneously. These cultures show stable spontaneous synchronized burst firing which propagates through dense synaptic connections. Carbachol (10-50 microM), acting through muscarinic receptors, was found to induce a switch to asynchronous single-spike firing and to result in a loss of regularity and fragmentation of the burst structure. To obtain a quantitative measure of cholinergic actions on cortical networks, we applied a cluster Poisson-process model to sets of paralleled spike-trains in the presence and absence of carbachol. This revealed that the time series can be well-characterized by such a simple model, consistent with the observed 1/f(b)-like spectra (0.04<b<2.08). After applying higher concentrations of carbachol the property of the spectra shifted toward a Poisson-process (white) spectrum. These results indicate that cholinergic neurotransmitters have a strong and reliable desynchronizing action on cortical neural activity.

Modification of a neuronal network direction using stepwise photo-thermal etching of an agarose architecture.

Control over spatial distribution of individual neurons and the pattern of neural network provides an important tool for studying information processing pathways during neural network formation. Moreover, the knowledge of the direction of synaptic connections between cells in each neural network can provide detailed information on the relationship between the forward and feedback signaling. We have developed a method for topographical control of the direction of synaptic connections within a living neuronal network using a new type of individual-cell-based on-chip cell-cultivation system with an agarose microchamber array (AMCA). The advantages of this system include the possibility to control positions and number of cultured cells as well as flexible control of the direction of elongation of axons through stepwise melting of narrow grooves. Such micrometer-order microchannels are obtained by photo-thermal etching of agarose where a portion of the gel is melted with a 1064-nm infrared laser beam. Using this system, we created neural network from individual Rat hippocampal cells. We were able to control elongation of individual axons during cultivation (from cells contained within the AMCA) by non-destructive stepwise photo-thermal etching. We have demonstrated the potential of our on-chip AMCA cell cultivation system for the controlled development of individual cell-based neural networks.

Individual-cell-based electrophysiological measurement of a topographically controlled neuronal network pattern using agarose architecture with a multi-electrode array

We have developed a new type of individual-cell-based electrophysiological measurement method using an on-chip multi-electrode array (MEA) cell-cultivation system with an agarose microchamber (AMC) array for topographical control of the network patterns of a living neuronal network. The advantages of this method are that it allows the recording of the firing of multiple cells simultaneously for weeks without contamination using the MEA, and that it allows control of the cell positions and numbers, and their connections for cultivation using AMCs with microchannels fabricated by photothermal etching where a portion of the agarose layer is melted with a 1480 nm infrared laser beam. Using this method, we formed an individual-cell-based neural network pattern of Rat hippocampal cells within the AMC array without cells escaping from the electrode positions in the microchamber during a thirteen-day cultivation, and could record the cell firing of lined-up hippocampal cells in response to 20 µA, 5 kHz stimulation via an electrode. This demonstrated the potential of our on-chip AMC/MEA cell cultivation method for long-term single-cell-based electrophysiological measurement of a neural network system for understanding the topographical meaning of neuronal network patterns.

Real-time multisite observation of glutamate release in rat hippocampal slices

A multichannel glutamate sensor was fabricated that consists of enzyme modified electrodes and has a high sensitivity and selectivity to glutamate. We placed a rat hippocampal slice on the sensor and monitored the current at four electrodes resulting from the stimulation with muscimol, a gamma-aminobutyric acid(A) (GABA(A)) receptor agonist. We obtained different glutamate concentration increases at the different positions, suppressed by bicuculline, a GABA(A) receptor antagonist. This demonstrated that the sensor can monitor the glutamate released via GABA(A) receptors pathways, and the difference in the concentrations may indicate differences in the distribution of GABA(A) receptor as well as diverse receptor functions. This multichannel sensor may be useful for non-invasive, real-time monitoring of glutamate distribution, which would make it a valuable tool for pharmacological analysis.

Light-addressable electrode with hydrogenated amorphous silicon and low-conductive passivation layer for stimulation of cultured neurons

The authors propose a light-addressable planar electrode with a simple three-layer laminated structure that can induce pinpoint neuronal activation on the culture substrate. The structure consists of a tin oxide (SnO2), hydrogenated amorphous silicon (a-Si:H), and passivation layer. The passivation layer was a spin-coated low-conductive zinc antimonate (ZnOSb2O5)-dispersed epoxy, which was proved to be effective for preventing penetration of culture medium and thus avoiding deterioration of a-Si:H layer. Illumination to the electrode locally elevated the conductivity with 60-fold stimulus charge density. The fluo-4 calcium imaging of neurons cultured on the developed electrode showed that the neuronal activation was confined around the illuminated location, thus demonstrating the light-addressing capability of the proposed electrode.