Histogenesis of mouse cerebellum in microwell cultures. Cell reaggregation and migration, fiber and synapse formation.

Trenkner, Ekkhart; Sidman, Richard L.

doi:10.1083/jcb.75.3.915

Cited by 141 publications

(60 citation statements)

References 52 publications

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“…1). Tetrodotoxin sensitivity emerges in a manner consistent with evolution of functional synapses (ϳ3-5 days) (Trenkner and Sidman, 1977) and coincides with a wave of programmed cell death in these cultures (Gallo et al, 1987). The observation that some neurons are vulnerable to tetrodotoxin, and even more to ionotropic glutamate receptor antagonists, suggests that glutamate is released by action potential-dependent and -independent modes.…”

Section: Discussionmentioning

confidence: 93%

“…1D, the addition of tetrodotoxin to immature neurons has no effect (5 mM, 97.9 Ϯ 1.2%; 25 mM, 95.8 Ϯ 1.2%; n ϭ 3) compared with vehicle-treated controls (set at 100%). However, the addition of tetrodotoxin after a critical period of ϳ4 days, in parallel with synapse formation (Trenkner and Sidman, 1977), leads to a significant decrease in MTT reductive capacity in neurons cultured in 5 mM KCl (5-8 DIV, 81.4 Ϯ 2.6%; 1-8 DIV, 73.9 Ϯ 2.6%; n ϭ 3) but not 25 mM KCl (5-8 DIV, 94.6 Ϯ 3.1%; 1-8 DIV, 99.2 Ϯ 3.1%; n ϭ 3). The integral synaptic vesicle protein synaptophysin is the most widely used immunohisotchemical marker of synapse density (Valtorta et al, 2004).…”

Section: Resultsmentioning

confidence: 99%

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Roscovitine Triggers Excitotoxicity in Cultured Granule Neurons by Enhancing Glutamate Release

Monaco

Vallano²

2005

Mol Pharmacol

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Cerebellar granule neurons are highly susceptible to injury in vivo and in vitro, and primary cultures are widely used to characterize relevant receptors and signaling pathways. However, there are problems associated with their use. In particular, cultures are typically grown in medium supplemented with elevated KCl levels because it improves survival, but accumulating evidence indicates that this causes profound neuroadaptations. For example, growth in elevated KCl levels renders neurons electrically silent. Thus, they cannot be used to examine excitotoxicity of synaptic origins. On the other hand, cultures grown in physiological medium are rarely studied because a proportion undergoes apoptosis. Herein, we provide evidence that mature neurons cultured in physiological KCl develop spontaneous action potentials that support survival through N-methyl-D-aspartate (NMDA) receptor-mediated mechanisms. Furthermore, the cdk inhibitor roscovitine enhances the coupling between tetrodotoxin-sensitive action potentials and P/Q-type voltage-dependent calcium channels (VDCCs), thereby converting this survival program to excitotoxicity of synaptic origin. Therefore, roscovitine-triggered necrosis requires spontaneous Na ϩ -based action potentials (tetrodotoxin inhibits, (Ϯ)-2-amino-4-phosphonobutyric acid enhances), P/Q-type VDCC currents (-agatoxin-IVA and -conotoxin-MVIIC inhibit, but not -conotoxin-GVIA), intact vesicle fusion processes (tetanus toxin inhibits), and transmitter-filled vesicles (concanamycin and bafilomycin inhibit). From a postsynaptic standpoint, roscovitine-mediated excitotoxicity requires the functionally linked activation of ␣-amino-3-hydroxy-5-methyl-isoxazole-4-propionate/kainate (AMPA/ KA) and NMDA receptors, which is consistent with evidence that activated AMPA/KA receptors relieve the voltage-dependent Mg 2ϩ block of NMDA receptors, resulting in excitotoxic Ca 2ϩ influx. In the end, NMDA receptor-linked pathways transduce excitotoxicity. On the other hand, L-type VDCC blockers are not protective. Further characterization of this new model is expected to provide important insights about excitotoxicity of synaptic origins and about roscovitine as a selective modulator of this process.Glutamate-mediated excitotoxicity is extensively studied in cell cultures and animals to model key events underlying neuronal loss in human neurodegeneration (Aarts and Tymianski, 2004). Numerous experimental paradigms support the theory that sustained NMDA receptor activation leads to toxic intracellular Ca 2ϩ deregulation and death by necrosis and/or apoptosis. Therefore, NMDA receptor antagonists afford protection and provide a rationale for their use in clinical trials in humans. Unfortunately, these trials were not correspondingly successful, prompting investigators to re-evaluate the predictive value of existing models and stimulating renewed efforts to identify novel therapeutic targets.The "source-specificity hypothesis" of excitotoxicity posits that discrete Ca 2ϩ microdomains, as opposed to alteration...

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Section: Discussionmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Roscovitine Triggers Excitotoxicity in Cultured Granule Neurons by Enhancing Glutamate Release

Monaco

Vallano²

2005

Mol Pharmacol

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“…For human neuroblastoma cultures, 10 5 SHSY5Y cells were seeded and grown for 4 days prior to use. Murine cerebellar cells were prepared essentially as described (13) and were plated onto poly(L-lysine)-coated 35-mm-diameter Petri * This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 284/B5. The costs of publication of this article were defrayed in part by the payment of page charges.…”

Section: Methodsmentioning

confidence: 99%

Demonstration of Direct Glycosylation of Nondegradable Glucosylceramide Analogs in Cultured Cells

Schwarzmann

Hofmann

Pütz

et al. 1995

Journal of Biological Chemistry

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After uptake by various cells (human skin fibroblasts, rat neuroblastoma B 104, human neuroblastoma SHSY5Y, murine cerebellar cells), a radioactive and a fluorescent analog of a nondegradable glucosylceramide with sulfur in the glycosidic link were glycosylated to a cell-specific pattern of glycolipid analogs. These results, for the first time, show that a glucosylceramide analog can be conveyed from the plasma membrane of cultured cells to those Golgi compartments that function in the early glycosylation steps of glycolipids. This observation is further confirmed by the fact that the cationic ionophore monensin, known to impede membrane flow from proximal to distal Golgi cisternae, inhibited the formation of complex ganglioside analogs but not those of lactosylceramide, sialyl lactosylceramide (G M3 ), and disialyl lactosylceramide (G D3 ).

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“…Cell Culture-Granule cells were cultured from cerebella of 6-day-old mice according to the method of Trenkner and Sidman (20). Cells were isolated by mild trypsinization (0.05%, w/v) and dissociated by repeated passage through a constricted Pasteur pipette in a DNase solution (0.1%, w/v).…”

Section: Methodsmentioning

confidence: 99%

1-Methylthiodihydroceramide, a Novel Analog of Dihydroceramide, Stimulates Sphinganine Degradation Resulting in Decreased de Novo Sphingolipid Biosynthesis

Echten‐Deckert¹,

Giannis²,

Schwarz³

et al. 1998

Journal of Biological Chemistry

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1-Methylthiodihydroceramide (10 M) decreased de novo ceramide biosynthesis by about 90% in primary cultured cerebellar neurons. Accordingly, de novo formation of sphingomyelin and of glycosphingolipids, all of which contain ceramide in their backbone, was reduced in a time-and concentration-dependent manner by up to 80%. Complex sphingolipid synthesis was restored upon addition of dihydroceramide or ceramide, in micromolar concentrations, to the culture medium, suggesting that none of the glycosyltransferases involved in glycosphingolipid biosynthesis is inhibited by this analog. Assays of the enzymes catalyzing sphinganine biosynthesis, as well as its N-acylation to form dihydroceramide, revealed that they were also not affected. In contrast, there was a 2.5-fold increase in the activity of sphinganine kinase. Reduction of de novo sphingolipid biosynthesis by 1-methylthiodihydroceramide is therefore due to its ability to deplete cells of newly formed free sphinganine. As a consequence of depletion of sphinganine levels, 1-methylthiodihydroceramide disrupted axonal growth in cultured hippocampal neurons in a manner similar to that reported for direct inhibitors of sphingolipid synthesis; thus, there was essentially no axon growth after incubation with 1-methylthiodihydroceramide between days 2 and 3, and co-incubation with short acyl chain analogs of ceramide (5 M) antagonized the inhibition of growth. Interestingly, the D-erythro and the L-threo isomere were equally effective, but the corresponding free base as well as other structurally related compounds did not affect either sphingolipid biosynthesis or neuronal growth.

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Histogenesis of mouse cerebellum in microwell cultures. Cell reaggregation and migration, fiber and synapse formation.

Cited by 141 publications

References 52 publications

Roscovitine Triggers Excitotoxicity in Cultured Granule Neurons by Enhancing Glutamate Release

Roscovitine Triggers Excitotoxicity in Cultured Granule Neurons by Enhancing Glutamate Release

Demonstration of Direct Glycosylation of Nondegradable Glucosylceramide Analogs in Cultured Cells

1-Methylthiodihydroceramide, a Novel Analog of Dihydroceramide, Stimulates Sphinganine Degradation Resulting in Decreased de Novo Sphingolipid Biosynthesis

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