G.J.A. Ramakers

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.

NETMORPH: A Framework for the Stochastic Generation of Large Scale Neuronal Networks With Realistic Neuron Morphologies

We present a simulation framework, called NETMORPH, for the developmental generation of 3D large-scale neuronal networks with realistic neuron morphologies. In NETMORPH, neuronal morphogenesis is simulated from the perspective of the individual growth cone. For each growth cone in a growing axonal or dendritic tree, its actions of elongation, branching and turning are described in a stochastic, phenomenological manner. In this way, neurons with realistic axonal and dendritic morphologies, including neurite curvature, can be generated. Synapses are formed as neurons grow out and axonal and dendritic branches come in close proximity of each other. NETMORPH is a flexible tool that can be applied to a wide variety of research questions regarding morphology and connectivity. Research applications include studying the complex relationship between neuronal morphology and global patterns of synaptic connectivity. Possible future developments of NETMORPH are discussed.

Conditional firing probabilities in cultured neuronal networks: a stable underlying structure in widely varying spontaneous activity patterns

To properly observe induced connectivity changes after training sessions, one needs a network model that describes individual relationships in sufficient detail to enable observation of induced changes and yet reveals some kind of stability in these relationships. We analyzed spontaneous firing activity in dissociated rat cortical networks cultured on multi-electrode arrays by means of the conditional firing probability. For all pairs (i, j) of the 60 electrodes, we calculated conditional firing probability (CFP(i,j)[tau]) as the probability of an action potential at electrode j at t = tau, given that one was detected at electrode i at t = 0. If a CFP(i,j)[tau] distribution clearly deviated from a flat one, electrodes i and j were considered to be related. For all related electrode pairs, a function was fitted to the CFP-curve to obtain parameters for strength and delay (i.e. maximum and latency of the maximum of the curve) of each relationship. In young cultures the set of identified relationships changed rather quickly. At 16 days in vitro (DIV) 50% of the set changed within 2 days. Beyond 25 DIV this set stabilized: during a week more than 50% of the set remained intact. Most individual relationships developed rather gradually. Moreover, beyond 25 DIV relational strength appeared quite stable, with coefficients of variation (100 x SD/mean) around 25% in periods of approximately 10 h. CFP analysis provides a robust method to describe the underlying probabilistic structure of highly varying spontaneous activity in cultured cortical networks. It may offer a suitable basis for plasticity studies, in the case of changes in the probabilistic structure. CFP analysis monitors all pairs of electrodes instead of just a selected one. Still, it is likely to describe the network in sufficient detail to detect subtle changes in individual relationships.

The role of calcium signaling in early axonal and dendritic morphogenesis of rat cerebral cortex neurons under non-stimulated growth conditions.

The effects of depolarizing stimuli on neurite outgrowth have been shown to depend on an influx of extracellular calcium. However, the role of calcium under non-stimulated growth conditions is less well established. Here we investigated the contribution of calcium signaling to early neuronal morphogenesis of rat cerebral cortex neurons at three levels by blocking L-type voltage sensitive calcium channels, by depleting intracellular calcium or by blocking myosin light chain kinase. Detailed quantitative morphological analysis of neurons treated for 1 day revealed that depletion of intracellular calcium strongly decreased the density of filopodia, arrested axonal outgrowth and strongly decreased dendritic branching. Preventing calcium influx through L-type voltage sensitive calcium channels and blocking of myosin light chain kinase activity selectively decreased dendritic branching. Our observations support an essential role for basal intracellular calcium levels in axonal elongation. Furthermore, under non-stimulated conditions calcium entry through L-type voltage sensitive calcium channels and myosin light chain kinase play an important role in dendritic branching.

Competition for tubulin between growing neurites during development

Abstract At its tip—called growth cone—a neurite is elongated by the assembly of tubulin into microtubules. We present a model of elongation (extended from Van Veen and Van Pelt, Bull. Math. Biol. 56 (1994) 249–273) in which tubulin is produced in the soma and transported to the growth cone by diffusion and active transport. The model accounts for competition observed between growing neurites of the same neuron and for ‘dormant growth cones’, and shows that cessation of growth in one neurite—e.g., when it encounters a target—can trigger the growth of the other neurites. The model makes testable predictions for the time course of outgrowth and the concentration of tubulin during competition.

DEPOLARIZATION STIMULATES LAMELLIPODIA FORMATION AND AXONAL BUT NOT DENDRITIC BRANCHING IN CULTURED RAT CEREBRAL CORTEX NEURONS

Electric activity is known to have profound effects on growth cone morphology and neurite outgrowth, but the nature of the response varies strongly between neurons derived from different species or brain areas. To establish the role of electric activity in neurite outgrowth and neuronal morphogenesis of rat cerebral cortex neurons, cultured neurons were depolarized for up to 72 h and quantitatively analyzed for changes in axonal and dendritic morphology. Depolarization with 25 mM potassium chloride induced a rapid increase in lamellipodia in almost all growth cones and along both axons and dendrites. Lamellipodia formation was dependent on an influx of extracellular calcium through L-type voltage-sensitive calcium channels. Prolonged depolarization for 24 h induced an increase in total axonal length, mainly due to an increase in branching. After three days of depolarization axonal outgrowth was largely the same as in control neurons, suggesting accommodation of the growth cones to chronic depolarization. Dendrites showed very little change during the first three days in culture, and dendritic length or branching were not affected by depolarization. Thus, in early cerebral cortex neurons depolarization specifically stimulates axonal outgrowth through increased branching. This increase in branching may be a consequence of the earlier increase in lamellipodia formation. In contrast, early dendrites seem to be unable to translate the increase in lamellipodia into changes in outgrowth or branching. This difference between axons and dendrites could be due to differences in the stabilization of the tubulin cytoskeleton.

Characterizing neuromorphologic alterations with additive shape functionals

The complexity of a neuronal cell shape is known to be related to its function. Specifically, among other indicators, a decreased complexity in the dendritic trees of cortical pyramidal neurons has been associated with mental retardation. In this paper we develop a procedure to address the characterization of morphological changes induced in cultured neurons by over-expressing a gene involved in mental retardation. Measures associated with the multiscale connectivity, an additive image functional, are found to give a reasonable separation criterion between two categories of cells. One category consists of a control group and two transfected groups of neurons, and the other, a class of cat ganglionary cells. The reported framework also identified a trend towards lower complexity in one of the transfected groups. Such results establish the suggested measures as an effective descriptors of cell shape.

Influence of dendritic morphology on axonal competition

Abstract The development of nerve connections involves competition among axons for survival promoting factors, or neurotrophins, which are released by the axons’ target cells. We have extended our model of axonal competition (van Ooyen and Willshaw, Proc. R. Soc. B. 266 (1999) 883–892) to study the influence of the targets dendritic tree on competition. We show that spatial separation of innervating axons on the targets dendrites mitigates competition and permits the coexistence of axons. The model accounts for the finding that in many types of neurons a positive correlation exists between the size of the dendritic tree and the number of innervating axons surviving into adulthood (Hume and Purves, Nature 293 (1981) 469–471; Purves and Hume, J. Neurosci. 1 (1981) 441–452). Our results emphasize the importance of postsynaptic dendritic morphology in the development of specific patterns of nerve connections.

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.