P.S. Wolters

Long-term characterization of firing dynamics of spontaneous bursts in cultured neural networks

Extracellular action potentials were recorded from developing dissociated rat neocortical networks continuously for up to 49 days in vitro using planar multielectrode arrays. Spontaneous neuronal activity emerged toward the end of the first week in vitro and from then on exhibited periods of elevated firing rates, lasting for a few days up to weeks, which were largely uncorrelated among different recording sites. On a time scale of seconds to minutes, network activity typically displayed an ongoing repetition of distinctive firing patterns, including short episodes of synchronous firing at many sites ( network bursts). Network bursts were highly variable in their individual spatio-temporal firing patterns but showed a remarkably stable underlying probabilistic structure (obtained by summing consecutive bursts) on a time scale of hours. On still longer time scales, network bursts evolved gradually, with a significant broadening (to about 2 s) in the third week in vitro, followed by a drastic shortening after about one month in vitro. Bursts at this age were characterized by highly synchronized onsets reaching peak firing levels within less than ca. 60 ms. This pattern persisted for the rest of the culture period. Throughout the recording period, active sites showed highly persistent temporal relationships within network bursts. These longitudinal recordings of network firing have, thus, brought to light a reproducible pattern of complex changes in spontaneous firing dynamics of bursts during the development of isolated cortical neurons into synaptically interconnected networks.

Longterm stability and developmental changes in spontaneous network burst firing patterns in dissociated rat cerebral cortex cell cultures on multielectrode arrays

Spontaneous action potentials were recorded longitudinally for 4-7 weeks from dissociated rat occipital cortex cells cultured on planar multi-electrode plates, during their development from isolated neurons into synaptically connected neuronal networks. Activity typically consisted of generalized bursts lasting up to several seconds, separated by variable epochs of sporadic firing at some of the active sites. These network bursts displayed discharge patterns with age-dependent firing rate profiles, and durations significantly increasing in the 3rd week in vitro and decreasing after about 1 month in vitro, when they evolved into short events with prompt onsets. These findings indicate that after about a month in vitro these cultured neuronal networks have developed a degree of excitability that allows almost instantaneous triggering of generalized discharges. Individual neurons tend to fire in specific and persistent temporal relationships to one another within these network bursts, suggesting that network connectivity maintains a core topology during its development.

Dynamics and plasticity in developing neuronal networks in vitro

When dissociated cortical tissue is brought into culture, neurons readily grow out by forming axonal and dendritic arborizations and synaptic connections. These developing neuronal networks in vitro display spontaneous firing activity from about the end of the first week in vitro. When cultured on multielectrode arrays firing activity can be recorded from many neurons simultaneously over long periods of time. These experimental approaches provide valuable data for studying firing dynamics in neuronal networks in relation to an ongoing development of neurons and synaptic connectivity in the network. This chapter summarizes recent findings on the characteristics and developmental changes in the spontaneous firing dynamics. These changes include long-lasting transient periods of increased firing at individual sites on a time scale of days to weeks, and an age-specific repetitive pattern of synchronous network firing (network bursts) on a time scale of seconds. Especially the spatio-temporal organization of firing within network bursts showed great stability over many hours. In addition, a progressive day-to-day evolution was observed, with an initial broadening of the burst firing rate profile during the 3rd week in vitro (WIV) and a pattern of abrupt onset and precise spike timing from the 5th WIV onwards. These developmental changes are discussed in the light of structural changes in the network and activity-dependent plasticity mechanisms. Preliminary findings are presented on the pattern of spike sequences within network burst, as well as the effect of external stimulation on the spatio-temporal organization within network bursts.

Low-frequency stimulation induces stable transitions in stereotypical activity in cortical networks

Reverberating spontaneous synchronized brain activity is believed to play an important role in neural information processing. Whether and how external stimuli can influence this spontaneous activity is poorly understood. Because periodic synchronized network activity is also prominent in in vitro neuronal cultures, we used cortical cultures grown on multielectrode arrays to examine how spontaneous activity is affected by external stimuli. Spontaneous network activity before and after low-frequency electrical stimulation was quantified in several ways. Our results show that the initially stable pattern of stereotypical spontaneous activity was transformed into another activity pattern that remained stable for at least 1 h. The transformations consisted of changes in single site and culture-wide network activity as well as in the spatiotemporal dynamics of network bursting. We show for the first time that low-frequency electrical stimulation can induce long-lasting alterations in spontaneous activity of cortical neuronal networks. We discuss whether the observed transformations in network activity could represent a switch in attractor state.

Chronic blockade of glutamate-mediated bioelectric activity in long-term organotypic neocortical explants differentially effects pyramidal/non-pyramidal dendritic morphology

Dendritic/axonal growth has been examined in long-term organotypic neocortical explants taken from neonatal rat pups and grown either as isolated slices or as co-cultures. The quantitative light microscopic measurement of dendritic and axonal branching patterns within both types of explants was carried out on Golgi-stained materials. Spontaneous bioelectric activity (SBA) was blocked within both types of explants using a combination of APV and DNQX, NMDA and non-NMDA receptor antagonists, respectively. No extracellularly measurable SBA was observed to occur in the silenced explants in the presence of both antagonists but reappeared following wash-out with control medium. In both control and silenced explants, the overall cellular organization of the slice was maintained throughout the culturing period, with distinguishable pyramidal and non-pyramidal neurons located within the same layers and with the same orientations as observed in situ. The major findings of the present study show the following. (i) Pyramidal neurones chronically exposed to APV/DNQX exhibited no basal dendritic growth in co-cultured explants, while growth of apical dendritic lengths was similar to control values in the absence of SBA. (ii) Pyramidal neurones, nonetheless, exhibited significant terminal segment growth under SBA blockade which was correlated with a concomitant decrease in number of basal dendrites. (iii) Axonal growth in co-cultures was not sustained in silenced pyramidal neurones. (iv) Non-pyramidal neurones showed significant total dendritic and axonal growth in co-cultures following APV/DNQX treatment. (v) Non-pyramidal cells in co-cultures experienced an increase in terminal segment length at 2 weeks which declined in the third week. This increase-decrease was correlated with a decrease-increase in the total number of dendritic segments during the second and third weeks, respectively. (vi) In isolated explants the only departure from control growth curves was a significant increase in terminal segment length which was offset by a similar decrease in number of dendritic segments under APV/DNQX growth conditions. Thus the chronic loss of glutamate-mediated SBA differentially effected pyramidal and non-pyramidal neurones in isolated and co-cultured explants, with pyramidal neurones experiencing the more pronounced effects. We conclude that SBA effects the dynamics of neuritic elongation and branching and that these changes are most dramatically seen in co-cultures which cross-innervate one another, presumably via pyramidal axons. We hypothesize that the activity-dependent changes associated with reduction in pyramidal dendritic and axonal growth may be associated with neurotrophin receptor production/maturation.

Long-term multielectrode registration of neuronal firing activity from rat cerebral cortex tissue in-vitro

Activity-dependent processes are involved in neurite outgrowth and synaptogenesis. The authors expect that during neural network formation neuronal morphogenesis and synaptic connectivity are reciprocally dependent on the emerging bioelectric activity in the network. The authors want to study whether and how bioelectric activity is involved in the formation of network structure. A multielectrode recording facility has been constructed for the long-term registration of action potentials of individual neurons during network development in both organotypic and dissociated rat cerebral cortex tissue cultures. Long-term recordings of action potentials with good signal-to-noise ratios have been obtained. Experiments to correlate these activity levels with quantitative data on neuronal morphological development are in progress. Uncorrelated periodic fluctuations at a time scale of about ten minutes have been observed.

Effect of Low-Frequency Stimulation on Spontaneous Firing in Cultured Neuronal Networks

Cultured neuronal networks from dissociated rat cortical tissue show spontaneous firing activity from about the end of the first week in vitro. Multielectrode recordings have shown slow developmental changes in the firing activity at the individual electrode sites. Here we report that a short period of low-frequency electrical stimulation is able to induce lasting changes in the spontaneous firing activity, significantly larger than developmental changes over similar periods of time.