Akio Kawana

The mechanisms of generation and propagation of synchronized bursting in developing networks of cortical neurons

The characteristics and mechanisms of synchronized firing in developing networks of cultured cortical neurons were studied using multisite recording through planar electrode arrays (PEAs). With maturation of the network (from 3 to 40 d after plating), the frequency and propagation velocity of bursts increased markedly (approximately from 0.01 to 0.5 Hz and from 5 to 100 mm/sec, respectively), and the sensitivity to extracellular magnesium concentration (0–10 mM) decreased. The source of spontaneous bursts, estimated from the relative delay of onset of activity between electrodes, varied randomly with each burst. Physical separation of synchronously bursting networks into several parts using an ultraviolet laser, divided synchronous bursting into different frequencies and phases in each part. Focal stimulation through the PEA was effective at multiple sites in eliciting bursts, which propagated over the network from the site of stimulation. Stimulated bursts exhibited both an absolute refractory period and a relative refractory period, in which partially propagating bursts could be elicited. Periodic electrical stimulation (at 1 to 30 sec intervals) produced slower propagation velocities and smaller numbers of spikes per burst at shorter stimulation intervals. These results suggest that the generation and propagation of spontaneous synchronous bursts in cultured cortical neurons is governed by the level of spontaneous presynaptic firing, by the degree of connectivity of the network, and by a distributed balance between excitation and recovery processes.

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.

Simultaneous measurement of intracellular calcium and electrical activity from patterned neural networks in culture

Multisite extracellular electrical activity and intracellular calcium were recorded simultaneously. Electrical signals were measured using microelectrode array substrates. A novel cell positioning technique was combined with a method for controlling neurite outgrowth, which allowed cell-electrode contacts to be established easily, thus facilitating the electrical recording. Intracellular calcium was measured optically using the indicator fluo-3. Under low-magnesium conditions, cultured rat cortical neurons showed periodic transients of fluo-3 fluorescence, which were synchronized with the periodic bursting observed electrically. The intervals between bursts could be determined by electrical stimulation through the substrate electrodes. The results suggest that functional synaptic connections are formed in the culture system.<<ETX>>

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.

Modification of parallel activity elicited by propagating bursts in developing networks of rat cortical neurones

Networks of cultured cortical neurones exhibit regular, synchronized, propagating bursts which are synaptically mediated, and which are hypothesized to play a part in activity‐dependent formation of connections during development in vivo. The relationship between the strength of synaptic connections and the characteristics of synchronized propagating bursting, however, is unclear. Modification of synchronized activity in cortical cultures in response to electrical stimulation was examined using multisite electrode array recording. By measuring the response of the network to weak, localized, test stimulation (TS), we observed a potentiation of activity following a relatively stronger inducing stimulation (IS). This potentiation was evident as an increased probability of eliciting bursts by TS, an increased frequency of spontaneous bursts and number of spikes per burst, and increased speed of burst propagation, and it lasted for at least 20 min. Changing the parameters of IS revealed that high frequency tetanic stimulation is not necessary to induce potentiation, while it is essential for IS to produce a regeneratively propagating burst. The results provide a direct demonstration of modification of both the spatial and temporal characteristics of synchronized network activity, and suggest an important physiological role for propagating synchronized bursting, as a mechanism for inducing plastic modifications in the developing cortex.

Experimental analysis of neuronal dynamics in cultured cortical networks and transitions between different patterns of activity

Abstract. Experimental investigation of the dynamics of biological networks is a fundamental step towards understanding how the nervous system works. Spontaneous activity in cultured networks of cortical neurons has been investigated by using a multisite recording technique with planar electrode arrays. In these networks, the spatiotemporal firing patterns were studied in the presence of different extracellular solutions. Transitions from asynchronous firing dynamics to synchronous firing dynamics were observed when the extracellular Ca2+ concentration was increased from 0.1 mM to 1 mM. Addition of extracellular Mg2+ reduced the spontaneous activity at any Ca2+ concentration, and an increase in the extracellular K+ concentration enhanced the frequency of periodical synchronous bursts. N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor antagonists inhibited synchronous activity. A spatiotemporal analysis of the data has been performed, and the properties of the network such as the synchronization and the periodicity have been quantified in order to clarify how variations of intrinsic parameters of the network can induce structural transitions in the neural dynamics. This experimental study is a possible approach to investigate the computational properties of a neuronal network.

Recognition of artificial microstructures by sensory nerve fibers in culture

Dissociated culture of adult mouse dorsal root ganglion cells on glass plates, on which grating-associated microstructures (a repetition of microgrooves [mGRV] and microsteps [mSTP] of 0.1-10 micron) are fabricated by the conventional lithographic techniques, represents a remarkable bi-directional growth of their nerve fibers in the axial direction of the grating. Microscopical observation shows that the nerve fibers prefer to grow in the mGRV (70%), while their growth cones exhibit an even distribution onto the mGRV and mSTP. The efficiency of the nerve fibers to grow along the grating-axis are highly sensitive to a fine alteration of the width and depth of the mGRV. The preferential growth of the nerve fibers is thus due to a mechanical recognition of the microstructures by the growth cones and neurites.

Selective growth of sensory nerve fibers on metal oxide pattern in culture

Metal oxides were used to study how the sensory nerve fibers recognize surface properties. Neurites selectively grow on the metal oxides deposited on silica glass, being guided along the axial direction of the patterns. The guiding ability depends on the electronegativity of the metal in metal oxide. Aluminum oxide or indium oxide patterns showed a remarkable ability to guide the growth direction. Neurites recognize the differences in surface properties (which are reflected by electronegativity) between metal oxides when the metal oxide substrata are only 1 micron in width.