Michael A. Corner

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.

Sleep and Wakefulness During Early Life in the Domestic Chicken, and Their Relationship to Hatching and Embryonic Motility

Publisher Summary With recent demonstrations that suggest that considerable movement takes place during sleep as well as during wakefulness, the possibility must be seriously entertained that spontaneous embryonic motility—rather than being a specifically prenatal type of behavior or an early sign of “arousal”—has something to do with the development of sleep mechanisms. Wakefulness is a state which, in precocial species, develops with forward reference to its later function as the conditions for its manifestation are not normally present until some time after the potentiality has appeared. The localization of lower arousal circuits might be approached by means of direct brain stimulation, combined with defect experiments employing the same stereotactically positioned electrodes for both purposes. Specific patterns of electrical activity would then be predicted to be recorded at such sites according to the predominating source of the input: from sensory pathways, from forebrain excitation, or from reciprocal inhibition from the sleep systems.

Homeostatically regulated spontaneous neuronal discharges protect developing cerebral cortex networks from becoming hyperactive following prolonged blockade of excitatory synaptic receptors

In order to further examine the role of spontaneous action potential (SAP) discharges in neocortical development, amino-acid-mediated synaptic transmission was selectively blocked in an improved organotypic neocortex culture preparation. Contralateral occipital cortex slices from neonatal rats were co-cultured for several weeks in a ventricle-to-ventricle orientation known to greatly enhance cyto-morphological and electrophysiological maturation. Such preparations are highly resistant to attempts to suppress neuronal firing by blocking ionotropic glutamate receptors: not only can kainate receptors partly substitute for NMDA- and AMPA-mediated neurotransmission when these receptors are pharmacologically blocked, but (muscarinic) cholinergic receptors also begin to drive SAP activity when the kainate receptors, too, are chronically blocked. Only tetrodotoxin proved able to eliminate SAPs altogether in these co-cultures, while GABAergic receptor blockade (using bicucculine) led to persistent epileptiform discharges. Treatment effects were assayed upon transfer to control medium by means of a quantitative analysis of spontaneously occurring polyneuronal spike trains. Total suppression of action potentials for several weeks (by tetrodotoxin treatment) led, as in earlier experiments, to strongly intensified burst firing upon transfer to control medium. Chronic glutamate receptor blocked cultures, on the other hand, showed only minor deviations from control firing levels and patterns when assayed in normal medium. Protection against the development of hyperactivity despite partial blockade of synaptic transmission was roughly proportional to the degree to which spontaneous firing during the treatment period approximated normal SAP levels. This homeostatic response to chronically reduced excitatory drive thus differs from earlier results using isolated organotypic cortex cultures, in which the restoration of SAP activity failed to prevent the development of network hyperactivity. Chronic bicucculine treatment, in contrast, had little or no homeostatic effect on SAP firing patterns; on the contrary, opposite to earlier findings using isolated occipital cortex explants, paroxysmal discharges persisted even after transfer to control medium.

Some functional effects of suppressing bioelectric activity in fetal mouse spinal cord-dorsal root ganglion explants

Organotypic explants of fetal mouse spinal cord-dorsal root ganglia were grown for 3 weeks in the presence of 10 mM magnesium ion, which effectively eliminated all recordable bioelectric activity throughout the culturing period. When tested in minimal essential medium, the chronically silenced explants had significantly fewer points from which spontaneous neuronal activity could be recorded. In addition, fewer points could be found that showed dorsal root ganglion-evoked responses, resulting from a greater tendency for the spinal cord activity to be restricted to the vicinity of the dorsally entering DRG fibers. These findings, therefore, support the hypothesis that spontaneous bioelectric activity is required for functional as well as structural maturation of neural networks.

Central neuronal responsiveness to sensory ganglion stimulation is correlated with the incidence of spontaneous bioelectric activity in developing spinal cord cultures.

In spinal cord explants co-cultured with dorsal root ganglion cells for 3–4 weeks in a (horse)serum-containing medium, the spread of ganglion-evoked action potentials from monosynaptic innervation sites (“polysynaptic excitability index”) was not correlated with the incidence of neuronal “background” discharges. Moreover, chronic exposure of serum-grown cultures to tetrodotoxin (TTX) in a dose sufficient to reversibly block bioelectric activity, failed to significantly affect this index. For explants grown in a chemically defined medium (CDM) similar excitability scores were obtained only if a low level of spontaneous activity was measured. The most active preparations scored considerably higher, with intermediate values being found in the moderately active cultures. Chronic TTX-exposure in developing CDM-grown cultures reduced their excitability scores to the level found in weakly active, untreated, explants despite a normal incidence of spontaneous activity. The present study indicates that low levels of spontaneous activity in untreated explants were associated with a similar sluggishness of DRG-evoked responses as previously observed after chronic treatment with TTX. These results give additional grounds for confidence that this reduced responsiveness of spinal cord neurons to sensory input is indeed attributable to prolonged reduction of centrally generated excitation during development in vitro.

Activity-dependent regulation of neuronal network excitability

Electric activity plays a major role in the fine-tuning of neuronal connections during development. Since alterations in connectivity will in turn affect network activity it is clear that neuronal network formation is the result of reciprocal interactions between the activity and the structure of the network. To investigate the role of electric activity in neuronal network development we use primary cultures of dissociated fetal rat cerebral cortex. Following prolonged suppression of spontaneous electric activity in culture neuronal firing showed a strong increase in stereotyped burst firing at the expense of variable non-burst firing. This mode of firing could be mimicked by blocking GABAergic inhibition indicating that chronic suppression of electric activity induced a shift in the balance between excitation and inhibition resulting in overexcitation. Chronic silencing induced a disproportionate decrease in GABA content while the release of glutamate and aspartate was facilitated in early cultures. As a result the ratio of stimulated release of excitatory versus inhibitory neurotransmitter was increased in line with our hypothesis. Conversely prolonged depolarization increased GABAergic staining intensity without affecting the density of GABAergic neurons. These data suggest that during brain development compensatory mechanisms may operate which serve to keep the level and/or pattern of electric activity within physiological limits. We propose a negative feed-back loop whereby electric activity stimulates the synthesis and release of BDNF which through trkB receptors on GABAergic neurons stimulates GABAergic network activity restraining overall network activity.

Long-lasting transients of activation in neural networks

Abstract The question has been investigated whether long-lasting transients of activation (i.e. slow waves), observed to occur in the intact cerebral cortex (EEG ‘delta’ waves and ‘K’ complexes) as well as an isolated tissue cultured in vitro, can also emerge in a simplified neural network model of interconnected excitatory and inhibitory cells. It is shown that slow waves can indeed occur even if the cells in the network do not have explicitly built-in slow processes. The mechanism underlying the termination of transient activity depends crucially upon the presence of a refractory period and random activity, rather than upon inhibitory suppression. A wide range of characteristic unit firing patterns is associated with transient population activities, even though all the cells in the network model have identical response properties.