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Biomedical subjects

G Moonen

Publications and source records attributed to G Moonen.

At least 127 records · Page 7Linked to original sources

Plasminogen activators in developing peripheral nervous system, cellular origin and mitogenic effect.

Newborn rat dorsal root ganglia release two different plasminogen activators (PAs): the urokinase (UK) and the tissue (tPA) type. The former is secreted by neurons while the latter is secreted by Schwann cells. tPA release by Schwann cells is modulated by choleratoxin, a known mitogen for these cells. UK but not tPA stimulates in a dose-dependent fashion the proliferation of Schwann cells. This effect is observed in the absence of plasminogen, suggesting that the substrate for PAs in the developing nervous system is not plasminogen. Since UK is secreted by neurons, our data suggest a new mechanism for neuronal control of Schwann cell proliferation.

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Plasminogen activator is a mitogen for astrocytes in developing cerebellum.

Newborn rat cerebellum microexplants cultured in Minimal Essential Medium with glucose and insulin released plasminogen activator (PA), which was detected in living cultures by a substrate overlay assay. Gel electrophoresis of cerebellum-conditioned medium followed by zymography resolved PA activity in two separate bands of 48,000 and 75,000 daltons apparent mol. wt. Using specific antisera, these bands were shown to be respectively urokinase and tissue-type PA. Cerebellum conditioned medium as well as purified human urokinase induced the proliferation and outgrowth of glial fibrillary acid protein-positive cells from newborn cerebellar microexplants. The effect was suppressed by the serine protease inhibitor phenyl methanesulfonylfluoride. Since PAs are most likely of neuronal origin, we suggest that at least one of these proteases acts as a neuronoglial mitogenic signal during development.

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Laminin promotes cerebellar granule cells migration in vitro and is synthesized by cultured astrocytes.

Newborn rat cerebellum microexplants have been used as a model to study neuronal migration. Laminin in a substrate-bound form modifies extensively the migratory behavior of the neurones, an effect which is blocked if antilaminin antibodies are present during the assay. 35S methionine incorporation followed by SDS-PAGE electrophoresis, fluorography and immunoprecipitation with antilaminin antibodies allowed to demonstrate that laminin is synthesized and secreted by cultured newborn rat cerebellum as well as by cultured newborn rat cerebral cortex astrocytes.

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Fetal calf serum-mediated inhibition of neurite growth from ciliary ganglion neurons in vitro.

Embryonic chick ciliary ganglion (CG) neurons cultured in fetal calf serum-containing medium have been previously reported to extend neurites on polyornithine (PORN) substrata precoated with a neurite-promoting factor (PNPF) from rat schwannoma-conditioned medium. On PORN substrata alone, however, no neuritic growth occurred. This was interpreted as evidence that PORN was an incompetent substratum for ciliary neuritic growth. In this study, we now find that an untreated PORN substratum allows neuritic growth in serum-free defined medium. When PNPF was added to PORN, a more rapid and extensive neuritic response occurred. After 5 hr of culture, a 60% neuritic response occurred on PNPF/PORN, whereas no neurons initiated neurites until 10-12 hr on PORN. The inhibitory effect of fetal calf serum noted above on PORN could be obtained in part by pretreating the substratum with serum for 1 hr. Maximal inhibitory effects in the PORN pretreatment were achieved after 30 min and were not further improved by treatments up to 4 hr. Bovine serum albumin was also found to inhibit neurite growth on PORN to about 60% of the inhibition obtained by an equivalent amount of serum protein. Fetal calf serum was shown to cause a 15% reduction in the percentage of neurons bearing neurites after its addition to 18-hr serum-free PORN cultures and to cause statistically significant reductions in neurite lengths measured 2 hr later.

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Cerebellar macroneurons in serum-free cultures: evidence for intrinsic neuronotrophic and neuronotoxic activities.

Cerebellar macroneurones survive and differentiate for at least 10 days in Eagle's minimum essential medium with insulin as the only supplement when cultured either as microexplants or in high-density dissociated cultures, while they do not survive if cultured in low density. The survival is related to the extracellular release of neuronotrophic factor(s). Using a quantitative bioassay of the neuronotrophic effect, it is possible to demonstrate that the effect is concentration dependent. The analysis of the dose-response curves suggests that the neuronotrophic activity is associated with a neuronotoxic activity. The two activities can be segregated using a simple physical method, allowing direct demonstration of the neuronotoxic activity.

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Kainic acid sensitivity of mammalian Purkinje cells in monolayer cultures.

Purkinje cells from fetal mouse cerebellum were studied in a monolayer culture system. These neurons, labeled by intracellular iontophoresis of HRP, showed one or more thick, spine-covered dendrites and an axon whose swellings contacted other neuronal processes and Purkinje cell somata. In short-term experiments, kainic acid was cytotoxic to Purkinje neurons and not to granule cells in monolayer cultures of fetal rat cerebellum.

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Cerebellar macroneurons in microexplant cell culture. Methodology, basic electrophysiology, and morphology after horseradish peroxidase injection.

Cerebellar macroneurons, including Purkinje cells, survive and differentiate in long-term monolayer cultures, which are prepared by a partial dissociation procedure we refer to as a microexplant technique. Intracellular recording demonstrated that these neurons were functional, showing spontaneous spiking activity and electrical excitability, and both spontaneous and evoked synaptic activity. In order to further characterize cell types, light and electron microscopic studies were performed after intracellular iontophoresis of horseradish peroxidase. Purkinje neurons were identified by their form of dendritic arborization and numerous dendritic spines. Cortical granule cells and macroneurons derived from the deep nuclei could also be demonstrated.

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Cerebellar macroneurons in microexplant cell culture. Postsynaptic amino acid pharmacology.

Cerebellar neurons derived from 17- to 19-day-old fetal rats have been grown in a monolayer in microexplant cell culture, and intracellular recording coupled with iontophoresis of amino acid neurotransmitters has been employed to characterize their amino acid chemosensitivity. Although these cultures contain at least 3 different neuronal cell types, intracellular recordings were obtained from large neurons (diameter greater than 15 microns) with 1-5 dendritic shafts and fine dendritic arborizations and which could, on morphological grounds, be identified as Purkinje cells. All neurons with resting membrane potentials greater than 25 mV and with action potentials evoked by intracellular stimulation, responded to iontophoretically applied glutamate and GABA. There was essentially no chemosensitivity to glycine, beta-alanine or taurine. Aspartate application evoked only small responses at high iontophoretic currents. GABA reversibly increased membrane conductance and produced hyperpolarization at resting membrane potential with reversal potentials between -50 and -40 mV (5-10 mV more negative than resting membrane potential). Glutamate reversibly increased membrane conductance and produced depolarizing responses with extrapolated reversal potentials between 0 and -10 mV. Aspartate augmented glutamate responses at low iontophoretic currents which did not directly alter membrane potential or conductance. Thus Purkinje cells grown in the absence of parallel fiber and climbing fiber input develop autonomous neuropharmacologic specificity similar to that of Purkinje cells in vivo.

Amino Acids↗

Cerebellar macroneurons in microexplant cell culture: ultrastructural morphology.

Microexplant cell cultures of fetal rat cerebellum contain essentially monolayer networks of Purkinje cells, occasional granule cells and neurons from the deep nuclei. The neurons and occasional filament-packed glial cells develop on top of a sheet of flattened, non-neuronal cells. In the absence of extrinsic input to the cerebellum and greatly reduced numbers of granule cells, the Purkinje cells develop a stunted and non-oriented dendritic arbor similar to that observed in agranular cerebella. The Purkinje cell dendritic branches, however, are spine-covered. Although the spines are not enveloped by glia and are only rarely contacted by a presynaptic bouton, most spines display a patch of electron-dense material resembling a postsynaptic membrane specialization. The Purkinje cells develop synaptic interactions among themselves and with granule cells. The ultrastructural morphology of boutons derived from both Purkinje cells and large neurons of the deep nuclei, identified after intracellular injection of horseradish peroxidase, is consistent with that observed in vivo. The present study indicates that cerebellar Purkinje cells survive and differentiate in a culture system in which individual neurons are accessible for electrophysiological and morphological analyses.

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Comparative ultrastructural study of the effects of serum-free medium and dibutyryl-cyclic AMP on newborn rat astroblasts.

Modifications of cell shape induced in cultured newborn rat astroblasts by serum deprivation or dibutyryladenosine 3'-5' monophosphate (dBcAMP) are described. Serum suppression modifies the adhesitivity of the cells to the substrate, but this modification is not consistent with a true differentiation. The main ultrastructural feature of dBcAMP-treated astroblasts is the presence of an extensive system of 90 A microfilaments, while control cells are relatively devoid of these structures.

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