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Biomedical subjects

G J Ramakers

Publications and source records attributed to G J Ramakers.

11 recordsLinked to original sources
PubMed1998

Regulation of astrocyte morphology by RhoA and lysophosphatidic acid.

Astrocytes in the CNS undergo morphological changes and start to proliferate after breakdown of the blood-brain barrier. In culture, proliferating astrocytes have a flat, polygonal shape. When treated with cAMP-raising agents, astrocytes adopt a stellate, process-bearing morphology resembling their in vivo appearance. Stellation is accompanied by loss of actin stress fibers and focal adhesions. Lysophosphatidic acid (LPA), a blood-borne mitogen that signals through its cognate G protein-coupled receptor, stimulates DNA synthesis in astrocytes and causes rapid reversal of cAMP-induced stellation. LPA reversal of stellation is initiated by f-actin reassembly and tyrosine phosphorylation of focal adhesion proteins such as paxillin. Botulinum C3 toxin, which inactivates the Rho GTPase, mimics cAMP-raising agents in inducing stellation, f-actin disassembly, paxillin dephosphorylation, and growth arrest. However, unlike cAMP-induced stellation, C3-induced stellation cannot be reversed by LPA. Conversely, astrocytes expressing activated RhoA fail to undergo cAMP-induced stellation. Thus, RhoA controls astrocyte morphology in that active RhoA directs LPA reversal of stellation, while inactivation of RhoA is sufficient to induce stellation.

ADP Ribose Transferases↗
PubMed1998

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.

Animals↗
PubMed1994

Activity-dependent plasticity of inhibitory and excitatory amino acid transmitter systems in cultured rat cerebral cortex.

Chronic suppression of spontaneous bioelectric activity in cultures of dissociated fetal rat cerebral cortex increases neuronal cell death and results in electrophysiological changes which indicate an altered balance between excitatory and inhibitory neurotransmission in culture. To delineate whether alterations in neurotransmitter release could underlie this imbalance, we investigated the effects of chronic tetrodotoxin (TTX) treatment on the content and release of glutamate, aspartate and gamma-aminobutyric acid (GABA) in culture. Chronic TTX treatment decreased the content of all amino acids investigated. However, only GABA was decreased relative to the neuronal marker NSE (neuron-specific enolase), indicating a disproportionate loss of GABA production following chronic silencing. Depolarization-induced release of GABA, glutamate and aspartate increased about 10-fold between 7 and 21 days in control cultures. Chronic TTX treatment significantly increased the depolarization-induced release of glutamate and aspartate at 7 days in vitro relative to control levels. At all ages it caused a two-fold increase in the ratio of evoked excitatory amino acid release to that of GABA. These observations suggest that chronic silencing of developing neocortex cell cultures increases the ratio of excitatory to inhibitory synaptic activity either by differential cell death or by reduced synaptic efficiency, on which a decrease in GABA neurotransmission appears to play a major role. Since similar mechanisms may be involved in activity-dependent plasticity in vivo, these cultures provide a useful model to analyse this phenomenon at the cell biological and molecular level.

Animals↗
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↗
PubMed1992

Spontaneous firing as an epigenetic factor in brain development--physiological consequences of chronic tetrodotoxin and picrotoxin exposure on cultured rat neocortex neurons.

Functional consequences of either suppressing or intensifying spontaneous neuronal firing have been studied in developing rat cerebral cortex cultures using, respectively, tetrodotoxin (TTX) and picrotoxin (PTX) added chronically to the growth medium. Simple measures derived from the interspike interval histogram were able to powerfully discriminate between age and treatment groups. After return to control medium, most TTX-treated neurons spontaneously displayed stereotyped clustering of action potentials ('phasic' firing) which closely resembled the characteristic firing patterns seen acutely in the presence of PTX. The 'TTX-syndrome' thus suggests that GABAergic synaptic inhibition is ineffective in cortical networks grown under conditions which prevent the expression of bioelectric activity. In contrast, after return to control medium, neurons which had been partially disinhibited throughout development (by continuous exposure to PTX) had even less phasic firing than was measured in age-matched controls. Based upon these and previous findings, a two (main) factor model is put forth which can economically account for the major effects. The working hypothesis embodied in this model is that phasic neuronal discharges not only accelerate the maturation of excitatory connections within the neocortex but, even more important, are crucial for the development of adequate inhibitory synaptic transmission.

Animals↗
PubMed1992

Immunolocalization of B-50 (GAP-43) in the mouse olfactory bulb: predominant presence in preterminal axons.

Because the growth-associated protein B-50 (GAP-43) has been implicated in neurite outgrowth as well as in synaptic plasticity, we studied its light and electron microscopical distribution in the mouse olfactory bulb, an area of the nervous system which exhibits a high degree of synaptic plasticity. Immunofluorescent staining with monospecific affinity-purified anti-B-50 antibodies revealed that B-50 is most abundantly expressed in the olfactory nerve fibre layer and the granule cell layer neuropil, while little staining was observed in the external plexiform layer and in cell bodies. B-50 is absent from dendrites and myelinated axons as indicated by double labelling with monoclonal antibodies against microtubule-associated protein 2 and the large neurofilament protein, respectively. Using post-embedding immunogold labelling on ultrathin Lowicryl sections, B-50 was found to be highly concentrated in presumed growth cones in the olfactory nerve fibre layer and in thin unmyelinated axons and presynaptic terminals in the granule cell layer neuropil. Near background immunolabelling was seen in perikarya, dendrites and myelinated axons. In view of the implication of B-50 in plasticity-related phenomena, its abundance in the thin unmyelinated preterminal axons suggests that these are potential sites of extrasynaptic plasticity.

Animals↗
PubMed1991

Abnormalities in the spontaneous firing patterns of cultured rat neocortical neurons after chronic exposure to picrotoxin during development in vitro.

We have used the GABA-A antagonist picrotoxin (PTX) to investigate whether chronic disinhibition, leading to intensified neuronal firing, would induce a specific pattern of physiological alterations in cultured rat neocortex cells. Overall mean spontaneous discharge rates were little affected by 1 microM PTX but firing occurred mainly as repetitive high-frequency bursts of action potentials. This "phasic" pattern contrasted with the irregular, quasi-random, firing usually seen in control units. Neurons tested in normal growth medium after prolonged exposure to 1 microM PTX showed weaker interspike interval dependencies (Markov value) than in controls, along with reduced regularity in the occurrence of bursts. Since all physiological changes were opposite in direction to those reported earlier after chronic suppression of bioelectric activity, the results support the hypothesis that endogenous synaptic and/or action potentials are important for the maturation of neocortical networks. Since experimental alterations were found only in spike-train parameters which reflect ontogenetic changes in untreated control cultures, GABAergic inhibition (by preventing neuronal discharges from becoming too intense) presumably serves to constrain the rate of development within optimal limits.

Action Potentials↗
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↗
PubMed1991

Developmental changes in B-50 (GAP-43) in primary cultures of cerebral cortex: content and phosphorylation of B-50.

The content and phosphorylation of the neuronal growth-associated protein B-50 (GAP-43) were studied in cultured neocortex as a function of normal development and development in the presence of tetrodotoxin (TTX), a blocker of bioelectric activity (BEA). The observations were correlated with previous morphological findings on neurite outgrowth and B-50 immunolocalization in the same cultures. In control cultures, the concentration of B-50 reached a maximum at 7 days in vitro (DIV) and decreased thereafter, whereas the concentration of neuron specific enolase (NSE), which was used as a neuronal reference marker, rose till 28 DIV and leveled off towards 42 DIV. The degree of basal phosphorylation of B-50 (relative to that of total protein) decreased after the first week in vitro. Stimulation of B-50 phosphorylation by phorbol ester also decreased with age in vitro, indicating that changes in B-50 phosphorylation were mainly due to changes in protein kinase C (PKC) activity. The chronic presence of TTX led to a reduced content of B-50 and NSE after 14 DIV. The basal phosphorylation of B-50 was neither affected by acute nor chronic TTX treatment. However, upon stimulation of PKC with phorbol esters, some alterations of B-50 phosphorylation were revealed in cultures grown in TTX. These biochemical observations are in line with the absence of effects of TTX on neurite outgrowth during the first 2 weeks in culture, and later effects of TTX on neuronal survival. The developmental changes in B-50 concentration and phosphorylation largely correlate with previous morphological observations on axonal outgrowth and growth cone shape in the same cultures. We suggest that B-50 phosphorylation plays an important role in transducing extracellular signals into directed neurite outgrowth.

Animals↗
PubMed1990

Development in the absence of spontaneous bioelectric activity results in increased stereotyped burst firing in cultures of dissociated cerebral cortex.

Quantitative analysis of neuronal firing patterns was used to study the effects of chronic suppression of bioelectric activity (BEA) on functional development in primary cultures of fetal rat cerebral cortex. BEA was monitored with extracellular electrodes in active control cultures or, after return to control medium, in cultures chronically silenced with tetrodotoxin (TTX) at around 7, 14, 21 and 42 days in vitro. Spike trains of single neurons lasting up to 25 min duration were analyzed using a previously published set of computer programs. In control cultures, the main developmental trends seen in a previous study could be replicated. After development in the presence of TTX, activity levels were increased at all ages, and a high incidence was found of a single firing pattern characterized by stereotyped burst firing, while showing a low minute order variability in firing rate and low dependencies between successive intervals; conversely, the incidence of variable/non-burst firing was decreased relative to untreated cultures. The former firing pattern (i.e. non-variable bursting) could also be produced through acute addition of the GABA (A)-antagonist picrotoxin to control cultures, and resembled interictal burst firing observed in models of chronic epilepsy in vivo. These similarities suggest that chronic silencing of the cultures may have resulted in a functional disinhibition of the neuronal network; such disinhibition might be related to the increased cell death which we observed with chronic TTX-treatment in the same cultures.

Action Potentials↗