NAD causes dissociation of neural networks into subpopulations of neurons by inhibiting the network synchronous hyperactivity evoked by ammonium ions

The mechanisms of hyperexcitability of neuronal networks by ammonium ions and inhibition of this activity by coenzyme NAD were investigated on mixed neuro-glial cultures of rat hippocampus. Ammonium ions cause activation of silent or spontaneously active neuronal networks inducing a bursting electrical activity of neurons and high-frequency synchronous calcium oscillations. In control conditions NAD completely inhibits spontaneous activity of the neuronal network. NAD added after NH₄Cl disrupts synchronous oscillation in neurons and splits the network into five populations of neurons. In 4% of cells NAD decreased the amplitude of Ca²⁺ oscillations, preserving initial mode of oscillations. In 32% of cells, a transient suppression of the neuronal oscillations was observed: inhibition was followed by restoration of the synchronous periodic activity. In 10% of cells, NAD produced a gradual decrease of Ca²⁺ oscillations down to a complete termination of the initial periodic activity induced by ammonium. Fast and total inhibition of Ca²⁺ oscillations by NAD was observed in two small groups of neurons. First group (A) participated in the initial spontaneous network activity (5% of cells) with a period of 66–100 s. Second group (B), on the contrary, did not participate in the spontaneous activity. This group of neurons began to pulse with a high frequency (with a period of 6–8 s) synchronously with other neurons in the network after the addition of NH₄Cl. Based on the comparison of calcium responses of different cell groups to the depolarization caused by KCl and NH₄Cl and to the application of domoic acid, as well as on the results obtained in experiments with fluorescent antibodies against GAD 65/67, parvalbumin, calretinin, and calbindin, we propose that neurons of populations (A) and (B) may belong to GABAergic neurons containing calbindin and parvalbumin, respectively. Further analysis of specificity of the NAD effect on these neuronal groups may allow identification of the main targets of the ammonium toxic action in the brain. Thus, we have shown that NAD selectively inhibits neuronal activity and high-frequency synchronous Ca²⁺ oscillations in GABAergic neurons containing calcium-binding proteins. The inhibition is accompanied by desynchronization of oscillations and dissociation of neuronal network into several populations.     

Bifurcations of emergent bursting in a neuronal network

Complex neuronal networks are an important tool to help explain paradoxical phenomena observed in biological recordings. Here we present a general approach to mathematically tackle a complex neuronal network so that we can fully understand the underlying mechanisms. Using a previously developed network model of the milk-ejection reflex in oxytocin cells, we show how we can reduce a complex model with many variables and complex network topologies to a tractable model with two variables, while retaining all key qualitative features of the original model. The approach enables us to uncover how emergent synchronous bursting can arise from a neuronal network which embodies known biological features. Surprisingly, the bursting mechanisms are similar to those found in other systems reported in the literature, and illustrate a generic way to exhibit emergent and multiple time scale oscillations at the membrane potential level and the firing rate level.     

On the dynamics of the spontaneous activity in neuronal networks

Most neuronal networks, even in the absence of external stimuli, produce spontaneous bursts of spikes separated by periods of reduced activity. The origin and functional role of these neuronal events are still unclear. The present work shows that the spontaneous activity of two very different networks, intact leech ganglia and dissociated cultures of rat hippocampal neurons, share several features. Indeed, in both networks: i) the inter-spike intervals distribution of the spontaneous firing of single neurons is either regular or periodic or bursting, with the fraction of bursting neurons depending on the network activity; ii) bursts of spontaneous spikes have the same broad distributions of size and duration; iii) the degree of correlated activity increases with the bin width, and the power spectrum of the network firing rate has a 1/f behavior at low frequencies, indicating the existence of long-range temporal correlations; iv) the activity of excitatory synaptic pathways mediated by NMDA receptors is necessary for the onset of the long-range correlations and for the presence of large bursts; v) blockage of inhibitory synaptic pathways mediated by GABA(A) receptors causes instead an increase in the correlation among neurons and leads to a burst distribution composed only of very small and very large bursts. These results suggest that the spontaneous electrical activity in neuronal networks with different architectures and functions can have very similar properties and common dynamics.     

Synchronous and asynchronous bursting states: role of intrinsic neural dynamics

Brain signals such as local field potentials often display gamma-band oscillations (30-70 Hz) in a variety of cognitive tasks. These oscillatory activities possibly reflect synchronization of cell assemblies that are engaged in a cognitive function. A type of pyramidal neurons, i.e., chattering neurons, show fast rhythmic bursting (FRB) in the gamma frequency range, and may play an active role in generating the gamma-band oscillations in the cerebral cortex. Our previous phase response analyses have revealed that the synchronization between the coupled bursting neurons significantly depends on the bursting mode that is defined as the number of spikes in each burst. Namely, a network of neurons bursting through a Ca(2+)-dependent mechanism exhibited sharp transitions between synchronous and asynchronous firing states when the neurons exchanged the bursting mode between singlet, doublet and so on. However, whether a broad class of bursting neuron models commonly show such a network behavior remains unclear. Here, we analyze the mechanism underlying this network behavior using a mathematically tractable neuron model. Then we extend our results to a multi-compartment version of the NaP current-based neuron model and prove a similar tight relationship between the bursting mode changes and the network state changes in this model. Thus, the synchronization behavior couples tightly to the bursting mode in a wide class of networks of bursting neurons.     

Cell Type-Specific Properties of Subicular GABAergic Currents Shape Hippocampal Output Firing Mode

GABAergic function of the subiculum is central to the regulation of hippocampal output activity. Subicular neuronal networks are indeed under potent control by local inhibition. However, information about the properties of GABAergic currents generated by neurons of this parahippocampal area in normal tissue is still missing. Here, we describe GABA_A receptor (GABA_AR)-mediated phasic and tonic currents generated by principal cells (PCs) and interneurons (INs) of the rat subiculum. We show that in spite of similar synaptic current densities, INs generate spontaneous IPSCs (sIPSCs) that occur less frequently and exhibit smaller charge transfer, thus receiving less synaptic total current than PCs. Further distinction of PCs between intrinsically bursting (IB) and regular-spiking (RS) neurons suggested that sIPSCs generated by the two PC sub-types are likely to be similar. PCs and INs are also controlled by a similar tonic inhibition. However, whereas a comparable tonic current density is found in RS cells and INs, IB neurons are constrained by a greater inhibitory tone. Finally, pharmacological blockade of GABA_AR did not promote functional switch of RS neurons to IB mode, but influenced the bursting propensity of IB cells and released fast spiking activity in INs. Our findings reveal differences in GABAergic currents between PCs and INs as well as within PC sub-types. We propose that GABAergic inhibition may shape hippocampal output activity by providing cell type-specific fine-tuning of subicular excitatory and inhibitory drives.     

Changing excitation and inhibition in simulated neural networks: effects on induced bursting behavior

 The development of synchronous bursting in neuronal ensembles represents an important change in network behavior. To determine the influences on development of such synchronous bursting behavior we study the dynamics of small networks of sparsely connected excitatory and inhibitory neurons using numerical simulations. The synchronized bursting activities in networks evoked by background spikes are investigated. Specifically, patterns of bursting activity are examined when the balance between excitation and inhibition on neuronal inputs is varied and the fraction of inhibitory neurons in the network is changed. For quantitative comparison of bursting activities in networks, measures of the degree of synchrony are used. We demonstrate how changes in the strength of excitation on inputs of neurons can be compensated by changes in the strength of inhibition without changing the degree of synchrony in the network. The effects of changing several network parameters on the network activity are analyzed and discussed. These changes may underlie the transition of network activity from normal to potentially pathologic (e.g., epileptic) states. Received: 21 May 2002 / Accepted in revised form: 3 December 2002 / Published online: 7 March 2003 Correspondence to: P. Kudela (e-mail: pkudela@jhmi.edu) Acknowledgements. This research was supported by NIH grant NS 38958.     

Stimulation of bursting in pre-Bötzinger neurons by Epac through calcium release and modulation of TRPM4 and K-ATP channels

The exchange factor directly activated by cAMP (Epac) can couple cAMP production to the activation of particular membrane and cytoplasmic targets. Using patch-clamp recordings and calcium imaging in organotypic brainstem slices, we examined the role of Epac in pre-B?tzinger complex, an essential part of the respiratory network. The selective agonist 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT) sensitized calcium mobilisation from inositol-1,4,5-trisphosphate-sensitive internal stores that stimulated TRPM4 (transient receptor potential cation channel, subfamily M, Melastatin) channels and potentiated the bursts of action potentials. 8-pCPT actions were abolished after inhibition of phospholipase C with U73122 and depletion of calcium stores with thapsigargin. Caffeine-sensitive release channels were not modulated by 8-pCPT. Epac inhibited ATP-sensitive K(+) channels that also led to the enhancement of bursting by 8-pCPT. Bursting activity, spontaneous calcium transients and activity of TRPM4 and ATP-sensitive K(+) channels were potentiated after brief exposures to bradykinin and incubation with wortmannin produced opposite effects that can be explained by changes in phosphatidylinositol 4,5-bisphosphate levels. 8-pCPT stimulated the respiratory motor output in functionally intact preparations and the effects of bradykinin and wortmannin were identical to those observed in organotypic slices. The data thus indicate a novel pathway of controlling bursting activity in pre-B?tzinger complex neurons through Epac that can involved in reinforcement of the respiratory activity by cAMP.     

Theta oscillations by synaptic excitation in a neocortical circuit model

Neocortical theta-band oscillatory activity is associated with cognitive tasks involving learning and memory. This oscillatory activity is proposed to originate from the synchronization of interconnected layer V intrinsic bursting (IB) neurons by recurrent excitation. To test this hypothesis, a sparsely connected spiking circuit model based on empirical data was simulated using Hodgkin-Huxley-type bursting neurons and use-dependent depressing synaptic connections. In response to a heterogeneous tonic current stimulus, the model generated coherent and robust oscillatory activity throughout the theta-band (4-12 Hz). These oscillations were not, however, self-sustaining without a driving current, and not dependent on N-methyl-D-aspartate receptor synaptic currents. At realistic connection strengths, synaptic depression was necessary to avoid instability and expanded the basin of attraction for theta oscillations by controlling the gain of recurrent excitation. These results support the hypothesis that IB neuron networks can generate robust and coherent theta-band oscillations in neocortex.     

Management of synchronized network activity by highly active neurons

Increasing evidence supports the idea that spontaneous brain activity may have an important functional role. Cultured neuronal networks provide a suitable model system to search for the mechanisms by which neuronal spontaneous activity is maintained and regulated. This activity is marked by synchronized bursting events (SBEs)--short time windows (hundreds of milliseconds) of rapid neuronal firing separated by long quiescent periods (seconds). However, there exists a special subset of rapidly firing neurons whose activity also persists between SBEs. It has been proposed that these highly active (HA) neurons play an important role in the management (i.e. establishment, maintenance and regulation) of the synchronized network activity. Here, we studied the dynamical properties and the functional role of HA neurons in homogeneous and engineered networks, during early network development, upon recovery from chemical inhibition and in response to electrical stimulations. We found that their sequences of inter-spike intervals (ISI) exhibit long time correlations and a unimodal distribution. During the network's development and under intense inhibition, the observed activity follows a transition period during which mostly HA neurons are active. Studying networks with engineered geometry, we found that HA neurons are precursors (the first to fire) of the spontaneous SBEs and are more responsive to electrical stimulations.     

Coemergence of regularity and complexity during neural network development

With the growing recognition that rhythmic and oscillatory patterns are widespread in the brain and play important roles in all aspects of the function of our nervous system, there has been a resurgence of interest in neuronal synchronized bursting activity. Here, we were interested in understanding the development of synchronized bursts as information-bearing neuronal activity patterns. For that, we have monitored the morphological organization and spontaneous activity of neuronal networks cultured on multielectrode-arrays during their self-executed evolvement from a mixture of dissociated cells into an active network. Complex collective network electrical activity evolved from sporadic firing patterns of the single neurons. On the system (network) level, the activity was marked by bursting events with interneuronal synchronization and nonarbitrary temporal ordering. We quantified these individual-to-collective activity transitions using newly-developed system level quantitative measures of time series regularity and complexity. We found that individual neuronal activity before synchronization was characterized by high regularity and low complexity. During neuronal wiring, there was a transient period of reorganization marked by low regularity, which then leads to coemergence of elevated regularity and functional (nonstochastic) complexity. We further investigated the morphology-activity interplay by modeling artificial neuronal networks with different topological organizations and connectivity schemes. The simulations support our experimental results by showing increased levels of complexity of neuronal activity patterns when neurons are wired up and organized in clusters (similar to mature real networks), as well as network-level activity regulation once collective activity forms.     

Electrical connection between two bursting neurons inHelix pomatia

In some preparations of the CNS ofHelix pomatia, two neurons with bursting activity may be present in the right parietal ganglion, where usually there is only one bursting neuron RPal. If electrical activity of these neurons is recorded simultaneously, fluctuations of membrane potential are almost completely synchronized. Artificial depolarization and hyperpolarization of the membrane of one neuron caused depolarization or hyperpolarization of the other neuron. During long-term recording of the activity of both neurons synchronous modulation of their bursting activity was observed. Modulating factor (a peptide fraction obtained from the water-soluble part of snail brain homogenate) led to potentiation of the bursting activity of both neurons. It is concluded from the results of these experiments that two bursting RPal neurons, connected electrically with one another, may exist in the snail nervous system. In cases when the parameters of pacemaker activity of these two neurons are closely similar, electrical connection guarantees synchronization of their bursting activity and ensures a common frequency of changes in their membrane potential.     

Innate synchronous oscillations in freely-organized small neuronal circuits

Background

Information processing in neuronal networks relies on the network''s ability to generate temporal patterns of action potentials. Although the nature of neuronal network activity has been intensively investigated in the past several decades at the individual neuron level, the underlying principles of the collective network activity, such as the synchronization and coordination between neurons, are largely unknown. Here we focus on isolated neuronal clusters in culture and address the following simple, yet fundamental questions: What is the minimal number of cells needed to exhibit collective dynamics? What are the internal temporal characteristics of such dynamics and how do the temporal features of network activity alternate upon crossover from minimal networks to large networks?

Methodology/Principal Findings

We used network engineering techniques to induce self-organization of cultured networks into neuronal clusters of different sizes. We found that small clusters made of as few as 40 cells already exhibit spontaneous collective events characterized by innate synchronous network oscillations in the range of 25 to 100 Hz. The oscillation frequency of each network appeared to be independent of cluster size. The duration and rate of the network events scale with cluster size but converge to that of large uniform networks. Finally, the investigation of two coupled clusters revealed clear activity propagation with master/slave asymmetry.

Conclusions/Significance

The nature of the activity patterns observed in small networks, namely the consistent emergence of similar activity across networks of different size and morphology, suggests that neuronal clusters self-regulate their activity to sustain network bursts with internal oscillatory features. We therefore suggest that clusters of as few as tens of cells can serve as a minimal but sufficient functional network, capable of sustaining oscillatory activity. Interestingly, the frequencies of these oscillations are similar those observed in vivo.     

Alteration of neuronal activity in response to cyclic nucleotide agents in Aplysia

Responsiveness of Aplysia neurons to agents that affect cyclic nucleotide levels was not limited to neurons exhibiting a spontaneous bursting activity pattern. Analyses of the I-V relationship elicited by triangular current ramps within cells exposed to different agents presumably causing elevated cyclic nucleotide levels showed qualitatively similar alterations. These included an increased slope conductance at more negative potentials, possibly related to anomalous rectification, and the induction of a hysteresis in response to a triangular ramp. The paired metacerebral giant cells showed induction of synchronous bursting when exposed to phosphodiesterase inhibitors, and we examined this phenomenon more closely. Classical methods to inactivate a presynaptic source did not eliminate the induction of synchronous bursting. Intracellular injection of a phosphodiesterase inhibitor into a metacerebral giant cell caused changes in the current-voltage relationship similar to those described above for other cells. Subsequent perfusion with the inhibitor caused an enhancement of these effects and the induction of bursting. The alteration of the current-voltage plot in the metacerebral cells and abdominal ganglion cells is qualitatively similar to that induced in the similarly treated bursting neuron R15, suggesting a similar mechanism of action in both burster and nonburster neurons. The implications of these results for cyclic nucleotide mediation of neuronal events are discussed.     

Pacemaker and network mechanisms of rhythm generation: cooperation and competition

The origin of rhythmic activity in brain circuits and CPG-like motor networks is still not fully understood. The main unsolved questions are (i) What are the respective roles of intrinsic bursting and network based dynamics in systems of coupled heterogeneous, intrinsically complex, even chaotic, neurons? (ii) What are the mechanisms underlying the coexistence of robustness and flexibility in the observed rhythmic spatio-temporal patterns? One common view is that particular bursting neurons provide the rhythmogenic component while the connections between different neurons are responsible for the regularisation and synchronisation of groups of neurons and for specific phase relationships in multi-phasic patterns. We have examined the spatio-temporal rhythmic patterns in computer-simulated motif networks of H-H neurons connected by slow inhibitory synapses with a non-symmetric pattern of coupling strengths. We demonstrate that the interplay between intrinsic and network dynamics features either cooperation or competition, depending on three basic control parameters identified in our model: the shape of intrinsic bursts, the strength of the coupling and its degree of asymmetry. The cooperation of intrinsic dynamics and network mechanisms is shown to correlate with bistability, i.e., the coexistence of two different attractors in the phase space of the system corresponding to different rhythmic spatio-temporal patterns. Conversely, if the network mechanism of rhythmogenesis dominates, monostability is observed with a typical pattern of winnerless competition between neurons. We analyse bifurcations between the two regimes and demonstrate how they provide robustness and flexibility to the network performance.     

Activity dynamics and propagation of synchronous spiking in locally connected random networks

Random network models have been a popular tool for investigating cortical network dynamics. On the scale of roughly a cubic millimeter of cortex, containing about 100,000 neurons, cortical anatomy suggests a more realistic architecture. In this locally connected random network, the connection probability decreases in a Gaussian fashion with the distance between neurons. Here we present three main results from a simulation study of the activity dynamics in such networks. First, for a broad range of parameters these dynamics exhibit a stationary state of asynchronous network activity with irregular single-neuron spiking. This state can be used as a realistic model of ongoing network activity. Parametric dependence of this state and the nature of the network dynamics in other regimes are described. Second, a synchronous excitatory stimulus to a fraction of the neurons results in a strong activity response that easily dominates the network dynamics. And third, due to that activity response an embedding of a divergent-convergent feed-forward subnetwork (as in synfire chains) does not naturally lead to a stable propagation of synchronous activity in the subnetwork; this is in contrast to our earlier findings in isolated subnetworks of that type. Possible mechanisms for stabilizing the interplay of volleys of synchronous spikes and network dynamics by specific learning rules or generalizations of the subnetworks are discussed.     

Network bursts in cortical cultures are best simulated using pacemaker neurons and adaptive synapses

One of the most specific and exhibited features in the electrical activity of dissociated cultured neural networks (NNs) is the phenomenon of synchronized bursts, whose profiles vary widely in shape, width and firing rate. On the way to understanding the organization and behavior of biological NNs, we reproduced those features with random connectivity network models with 5,000 neurons. While the common approach to induce bursting behavior in neuronal network models is noise injection, there is experimental evidence suggesting the existence of pacemaker-like neurons. In our simulations noise did evoke bursts, but with an unrealistically gentle rising slope. We show that a small subset of ‘pacemaker’ neurons can trigger bursts with a more realistic profile. We found that adding pacemaker-like neurons as well as adaptive synapses yield burst features (shape, width, and height of the main phase) in the same ranges as obtained experimentally. Finally, we demonstrate how changes in network connectivity, transmission delays, and excitatory fraction influence network burst features quantitatively.     

Long-term Relationships between Cholinergic Tone, Synchronous Bursting and Synaptic Remodeling

Cholinergic neuromodulation plays key roles in the regulation of neuronal excitability, network activity, arousal, and behavior. On longer time scales, cholinergic systems play essential roles in cortical development, maturation, and plasticity. Presumably, these processes are associated with substantial synaptic remodeling, yet to date, long-term relationships between cholinergic tone and synaptic remodeling remain largely unknown. Here we used automated microscopy combined with multielectrode array recordings to study long-term relationships between cholinergic tone, excitatory synapse remodeling, and network activity characteristics in networks of cortical neurons grown on multielectrode array substrates. Experimental elevations of cholinergic tone led to the abrupt suppression of episodic synchronous bursting activity (but not of general activity), followed by a gradual growth of excitatory synapses over hours. Subsequent blockage of cholinergic receptors led to an immediate restoration of synchronous bursting and the gradual reversal of synaptic growth. Neither synaptic growth nor downsizing was governed by multiplicative scaling rules. Instead, these occurred in a subset of synapses, irrespective of initial synaptic size. Synaptic growth seemed to depend on intrinsic network activity, but not on the degree to which bursting was suppressed. Intriguingly, sustained elevations of cholinergic tone were associated with a gradual recovery of synchronous bursting but not with a reversal of synaptic growth. These findings show that cholinergic tone can strongly affect synaptic remodeling and synchronous bursting activity, but do not support a strict coupling between the two. Finally, the reemergence of synchronous bursting in the presence of elevated cholinergic tone indicates that the capacity of cholinergic neuromodulation to indefinitely suppress synchronous bursting might be inherently limited.     

Manifestation of function-follow-form in cultured neuronal networks

We expose hidden function-follow-form schemata in the recorded activity of cultured neuronal networks by comparing the activity with simulation results of a new modeling approach. Cultured networks grown from an arbitrary mixture of neuron and glia cells in the absence of external stimulations and chemical cues spontaneously form networks of different sizes (from 50 to several millions of neurons) that exhibit non-arbitrary complex spatio-temporal patterns of activity. The latter is marked by formation of a sequence of synchronized bursting events (SBEs)--short time windows (approximately 200 ms) of rapid neuron firing, separated by longer time intervals (seconds) of sporadic neuron firing. The new dynamical synapse and soma (DSS) model, used here, has been successful in generating sequences of SBEs with the same statistical scaling properties (over six time decades) as those of the small networks. Large networks generate statistically distinct sub-groups of SBEs, each with its own characteristic pattern of neuronal firing ('fingerprint'). This special function (activity) motif has been proposed to emanate from a structural (form) motif--self-organization of the large networks into a fabric of overlapping sub-networks of about 1 mm in size. Here we test this function-follow-form idea by investigating the influence of the connectivity architecture of a model network (form) on the structure of its spontaneous activity (function). We show that a repertoire of possible activity states similar to the observed ones can be generated by networks with proper underlying architecture. For example, networks composed of two overlapping sub-networks exhibit distinct types of SBEs, each with its own characteristic pattern of neuron activity that starts at a specific sub-network. We further show that it is possible to regulate the temporal appearance of the different sub-groups of SBEs by an additional non-synaptic current fed into the soma of the modeled neurons. The ability to regulate the relative temporal ordering of different SBEs might endow the networks with higher plasticity and complexity. These findings call for additional mechanisms yet to be discovered. Recent experimental observations indicate that glia cells coupled to neuronal soma might generate such non-synaptic regulating currents.     

Population oscillations in a discrete model of neural networks of the brain

A discrete model of biological neural networks is used to find out how synchronized firing of neurons emerges in a randomly connected neural population. The objective is to understand the mechanisms underlying brain waves and to find and characterize conditions which support spontaneous switching from disordered to rhythmic population activity as in case of an epileptic seizure. The model is kept as simple as possible to achieve on one hand a fast performance of computer simulations of networks with up to 10,000 neurons and to keep on the other hand an overview of parameter dependences. Dynamics of the model can be classified into different regimes: random fluctuations, rhythmic oscillations and silence. When the ratio of the inhibitory/excitatory connectivity is raised the system crosses from the fluctuating regime through the rhythmic oscillating region to the silence regime. Close to the boundary between the fluctuating and the oscillating regimes the network shows spontaneous bursting of high amplitude rhythmic oscillations, which is characteristic of epileptiform behavior. The simulation results are in agreement with recent theories saying that focal epilepsy after injury of the brain could result from axonal sprouting of GABAergic neurons in the injured region.     

Cellular physiology of epileptogenic phenomena and its application to therapy against intractable epilepsy.

1. During pentylenetetrazol-induced bursting activity which is characteristic intracellular potential change of seizure discharge, intracellular stored calcium is released and moved toward the inner surface of the cell membrane. Calcium is released from lysosome-like granules with morphological changes. During bursting activity, the intracellular free calcium level was higher than the normal state. During bursting activity, intracellular protein of 5 kDa and 15 kDa was changed qualitatively and quantitatively. 2. Primary cultured cerebral cortical neurons of rats and mice showed spontaneous regular firing, and by PTZ application, showed bursting activity. A single potassium channel showed the random open-close state in the normal state and showed burst type open-close state after PTZ application. 3. Primary cultured cerebral cortical neurons of the E1 mouse, the epilepsy animal model, showed developmental defects in neurite extension and content of gangliosides, in addition to their very high susceptibility to convulsions. 4. A new antiepileptic drug, TJ-960, which originates from a mixture of nine herbal drugs, normalized the above-mentioned seizure-related, calcium-related intracellular pathological phenomena. TJ-960 normalized cytochalasin-B-induced looping phenomena and protected the neuron damage induced by cytochalasin B in addition to anticonvulsant action. TJ-960 also completely normalized the cobalt-induced EEG changes and also protected against neuron damage in the hippocampus induced by cobalt application to the cerebral cortex. TJ-960 normalized the developmental defects of the E1 mouse neuron. 5. For better therapy of epilepsy, it is probably necessary to normalize the developmental defects and to protect against neuron damage in addition to inhibition of seizure activity.