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P S Wolters

Publications and source records attributed to P S Wolters.

12 recordsLinked to original sources
PubMed2004

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.

Action Potentials↗
PubMed2002

Physiological effects of sustained blockade of excitatory synaptic transmission on spontaneously active developing neuronal networks--an inquiry into the reciprocal linkage between intrinsic biorhythms and neuroplasticity in early ontogeny.

Spontaneous bioelectric activity (SBA) taking the form of extracellularly recorded spike trains (SBA) has been quantitatively analyzed in organotypic neonatal rat visual cortex explants at different ages in vitro, and the effects investigated of both short- and long-term pharmacological suppression of glutamatergic synaptic transmission. In the presence of APV, a selective NMDA receptor blocker, 1-2- (but not 3-)week-old cultures recovered their previous SBA levels in a matter of hours, although in imitation of the acute effect of the GABAergic inhibitor picrotoxin (PTX), bursts of action potentials were abnormally short and intense. Cultures treated either overnight or chronically for 1-3 weeks with APV, the AMPA/kainate receptor blocker DNQX, or a combination of the two were found to display very different abnormalities in their firing patterns. NMDA receptor blockade for 3 weeks produced the most severe deviations from control SBA, consisting of greatly prolonged and intensified burst firing with a strong tendency to be broken up into trains of shorter spike clusters. This pattern was most closely approximated by acute GABAergic disinhibition in cultures of the same age, but this latter treatment also differed in several respects from the chronic-APV effect. In 2-week-old explants, in contrast, it was the APV+DNQX treated group which showed the most exaggerated spike bursts. Functional maturation of neocortical networks, therefore, may specifically require NMDA receptor activation (not merely a high level of neuronal firing) which initially is driven by endogenous rather than afferent evoked bioelectric activity. Putative cellular mechanisms are discussed in the context of a thorough review of the extensive but scattered literature relating activity-dependent brain development to spontaneous neuronal firing patterns.

Action Potentials↗
PubMed1993

A developmental decrease in NMDA-mediated spontaneous firing in cultured rat cerebral cortex.

In primary cultures of fetal rat cerebral cortex chronic manipulation of the level and/or pattern of bioelectric activity leads to plastic changes in bioelectric activity, opposite to those seen during the manipulation. This suggests the presence of adaptive mechanisms which regulate functional development in the neuronal network. Since NMDA receptors play an important role in early postnatal bioelectric activity and have been implicated in activity-dependent plasticity in vivo, the involvement of NMDA and non-NMDA receptors in spontaneously occurring bioelectric activity was investigated in cultured rat cerebral cortex by assaying the effects of NMDA and non-NMDA antagonists on neuronal firing. In addition, the physiological consequences of chronic suppression of bioelectric activity were investigated following development in the presence of tetrodotoxin. NMDA receptors appeared at all ages to be more crucial for spontaneous bioelectric activity than non-NMDA receptors, although their relative importance decreased during the first 3 weeks. Whereas the NMDA antagonist APV strongly reduced burst firing, the non-NMDA antagonist DNQX tended to increase burst firing slightly. Following chronic suppression of bioelectric activity, non-variable burst firing was increased, thus replicating previous findings in cerebral cortex culture grown under different conditions. The prominence of NMDA receptor activation in spontaneous bioelectric activity in early cultures suggests a role for these receptors in activity-dependent functional plasticity, as found in vivo.

2-Amino-5-phosphonovalerate↗
PubMed1991

Developmental changes in B-50 (GAP-43) in primary cultures of cerebral cortex: B-50 immunolocalization, axonal elongation rate and growth cone morphology.

Changes in neurite outgrowth parameters and in the immunolocalization of the neuronal growth-associated protein B-50 (GAP-43) were studied in cultured neocortex as a function of development. In addition, we studied the effects of chronic blockade of bioelectric activity (BEA) with tetrodotoxin (TTX) on these parameters. Axonal outgrowth rate in control cultures reached a maximum at 8 days in vitro (DIV) and declined to a low level at 21 DIV. B-50 staining shifted from the perikaryon to the axons and growth cones during the first 3 DIV. In axons the intensity of B-50 staining increased towards the growth cone. Within growth cones, the central/basal region and filopodia were intensely stained, whereas lamellipodia showed only marginal staining. Growth cone size gradually decreased after 3 DIV, due to the successive loss of lamellipodia and filopodia, and became club-shaped during the second week, until by 21 DIV growth cones were completely lost, and axons started retracting and degenerated. In the central area of the cultures, growth cones also decreased in size with time, but became stabilized as presynaptic elements onto other neurons. Acute addition of TTX did not affect the outgrowth rate at 6 DIV. Chronic TTX treatment led to an earlier retraction and degeneration of axons than in control cultures and to a loss of B-50-stained cells and varicosities during the third week, but did not affect growth cone morphology or B-50 staining. The regressive phenomena are probably due to an increased neuronal cell death shown to occur after chronic TTX treatment. The developmental changes in axonal elongation rate and growth cone morphology may be related to developmental changes in the content and/or phosphorylation of B-50 (GAP-43, which are studied in the same cultures in the following paper (Ramakers et al. (1991) Int. J. Devl Neurosci. 9, 231-241].

Aging↗
PubMed1988

Hypoxia preferentially destroys GABAergic neurons in developing rat neocortex explants in culture.

The hypothesis that hypoxic ischemia before or during the human birth process preferentially destroys GABAergic nerve cells, particularly in the neocortex, was tested in a tissue culture model system. To that end, rat neocortex explants dissected from 6-day-old rat pups and cultured to a developmental stage approximately comparable to that of the newborn human neocortex were exposed to hypoxia for different periods. Quantitative light microscopic and immunocytochemical evaluation of the cultures demonstrated that GABAergic neurons were indeed the first neurons to die during hypoxia.

Animals↗
PubMed1984

Towards an improved serum-free, chemically defined medium for long-term culturing of cerebral cortex tissue.

The present study describes a series of experiments which have led to a substantially improved serum-free, chemically defined medium (CDM) for long-term culturing of reaggregated fetal rat cerebral cortex tissue. A reduction of the original medium concentrations of the hormones insuline, T3 and corticosterone, on the one hand, and an enrichment of the medium with the vitamins A, C and E, the unsaturated fatty acids linoleic and linolenic acid, and biotin, L-carnitine, D(+)-galactose, glutathione (reduced) and ethanolamine, on the other hand, formed the most important chemical adjustments of the medium. With the aid of this CDM (encoded R12), the light- and electron microscopic architecture of the tissue could be kept in a good condition (superior to that seen earlier in serum-supplemented medium) up to 23 days in vitro. From that time on, the neuronal network lying between the reaggregates degenerated for the largest part, while a portion of the large neurons (probably pyramidal cells) plus some of the neuronal network within the reaggregates degenerated too. This degeneration process continued during the following weeks, but the reaggregates nevertheless retained most of their mass, so that both small and large neuronal cell bodies (visible in transparent regions at the edge of the reaggregates) remained in good condition up to at least 103 DIV. Stout, thick nerve bundles interconnecting the reaggregates, also survived up to this point. Electron microscopic evaluation of such 'aged' reaggregates revealed degenerating as well as healthy regions. The latter had indeed retained healthy-looking pyramidal and non-pyramidal neurons, embedded within a dense neuropil which was often traversed by myelinated axons. The numerical synapse density in such selected, healthy tissue regions reached its maximum during the sixth week in vitro, followed by a rapid decrease and a stabilization at about half the peak values. The present culture system has opened the possibility for performing controlled quantitative studies on the relationship between structure and function of cerebral cortex tissues during development and aging, on its dependence on nutrients, hormones and drugs, and on special factors synthesized by the tissue and released into the nutrient medium.

Animals↗
PubMed1981

A quantitative electron microscopic study on synapse formation in dissociated fetal rat cerebral cortex in vitro.

Occipital cortex of 19-day-old fetal rats was dissociated and cultured in vitro for 2-5 weeks in horse serum supplemented Eagle's MEM. The outgrowing neurons rapidly formed a dense network which started to degenerate after 3 weeks in vitro. By means of electron microscopy the numerical development of 6 different categories of synapses was followed during the time in culture. This approach revealed a sigmoid growth curve emerging over the first 3 weeks for the category of axo-dendritic synapses (which comprised the bulk of all synapses counted). Thereafter a decline in number set in. Chronic exposure of these dissociated cerebral cortex cultures to 50 microgram/ml xylocaine (a concentration adequate to block all measurable bioelectrical activity) did not prevent the formation of functional synaptic networks having normal synaptic ultrastructure. These results are in agreement with previous studies on fetal cerebral explants in culture. However, in some groups of our cultures, xylocaine led to a retardation in neurite outgrowth and in numerical synapse formation. Since these xylocaine effects were dose-related at a concentration double that required to silence the cultures, the growth retardation was probably not due to selective suppression of bioelectric activity, but rather to some general cytotoxic effect of the drug. In one of the xylocaine-treated groups (50 microgram/ml) which showed an exceptionally rapid neuronal outgrowth by the use of horse serum obtained from a different source than in the other groups, no deficit was noted in the number of synapses formed. Extracellular recording in this latter drug-treated group of cultures during the third week revealed (after return to control medium) spontaneous isolated action potentials, burst patterns and slow waves which were indistinguishable from the bioelectric activity seen in the control cultures. It is concluded that bioelectric activity in dissociated cortex cultures is not a prerequisite for the formation of apparently normal, functional synaptic networks.

Animals↗
PubMed1981

Nerve outgrowth, synaptogenesis and bioelectric activity in fetal rat cerebral cortex tissue cultured in serum-free, chemically defined medium.

Dissociated and non-dissociated cerebral cortex of fetal rat was successfully cultured in a serum-free, chemically defined medium for at least 18 days without any preincubation in serum-supplemented medium. Neurite outgrowth, synapse formation and spontaneous bioelectric activity, in its qualitative and quantitative aspects, were essentially the same as was observed in medium containing 20% heat-inactivated horse serum.

Animals↗