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Anisomycin and cycloheximide protect cerebellar neurons in culture from anoxia
Brain Research, 581 (1992) 323-326 t~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
323
BRES 25186
Anisomycin and cycloheximide protect cerebellar neurons in culture from anoxia Fr6d6ric Dessi, Christiane Charriaut-Marlangue and Yehezkel Ben-Ari INSERM U29, Paris (France)
(Accepted 11 February 1992) Key words: Anoxia; Protein synthesis inhibitor; Granule cell culture
Protein synthesis inhibitors have recently been shown to protect from ischemia-induced neuronal death in the rat hippocampus in vivo. In an attempt to further investigate the mechanism of neuronal death resulting from anoxia, cerebellar neurons grown in culture were exposed to an anoxic atmosphere in the presence of protein synthesis inhibitors. Anisomycin and cycloheximide (100/~g/ml) offered, respectively, a 97+4% and 26+13% protection against anoxia-induced neuronal death. It is well known that neurons are very sensitive to oxygen deprivation. In vitro anoxia in cultured neurons or in vivo ischemia in experimental animals lead to a neuronal death which is typically observed about 24 h after the insult 7. The molecular and cellular events involved in this delayed neuronal death have not been completely elucidated. The current excitotoxic hypothesis 1'2 points to the release of the excitatory amino acid glutamate and excessive activation of the N-methyl-o-aspartate receptor, a subtype of glutamate receptors, as a major event. The resulting increase of intracellular calcium would induce the death of the neuron 24 h later by a series of mechanisms including the activation of proteases. Activation of a programmed cell death has been suggested in the delayed neuronal death induced by nerve growth factor deprivation in cultured sympathetic neurons, and protein synthesis inhibitors protect from this delayed neuronal death 1°. A similar mechanism might be effective in the anoxia-induced neuronal death and could underlie the delayed neuronal death. Protein synthesis inhibitors have recently been shown to protect from ischemia-induced neuronal death in the rat hippocampus in v i v o 6'13 and to reduce the deleterious effects of anoxia in hippocampal s l i c e s 11 . This prompted us to investigate the effects of a similar treatment on neuronal death induced in cultured cerebellar neurons by anoxia. Granule cell cultures were prepared as described previously4. In brief, cerebella were dissected from the brains of 8-day-old rats under sterile conditions and were placed in 0.1% trypsin 0.1% DNAse (Sigma) in phosphate-buffered saline (PBS; Gibco) supplemented with 0.6% glucose for 15 min at room temperature and then
in 1% soybean trypsin inhibitor (Sigma) for 5 min. The cerebella were then washed three times with PBS and resuspended in culture medium containing equal volume of H a m ' s F-12 and Dulbecco's modified Eagle's medium (DMEM-F12, Gibco) supplemented glucose (30 mM), KC1 (25 mM), penicillin (5 U/ml) and gentamycin (5 pg/ ml). The cells were then dissociated with Pasteur pipettes with flame-narrowed tips, centrifuged at 250 × g for 5 min, resuspended in the same culture medium except for supplementation with 10% fetal calf serum (Seromed) and plated on 16-ram dished (Nunc) (500 pl per well) coated with poly-L-lysine (10 pg/ml) (Sigma) to a density of 5 x 105 cells per well. The cultures were maintained at 37°C in humidified 5% CO2/95% 0 2 atmosphere. Cytosine arabinoside (40 pM) was added to the culture medium after 24 h. The culture medium was not changed thereafter. Cultures were used between 12 and 18 days in vitro (DIV). Anoxia was carried out without a n y change of the culture medium by placing the cultures in a hermetic chamber filled with 95% N2/5% CO 2 in an humidified atmosphere at 37°C for 16 to 24 h. Protein synthesis inhibitors or other drugs were eventually added in the culture medium immediately before the onset of anoxia. Neuronal cell injury was assessed after the end of the anoxia by examination of cultures with phase-contrast microscopy and with bright-field examination after Trypan blue staining (0.4% 5 min). Viable cells were counted in triplicate from 9 representative microscopic fields (magnification x200). Methionine incorporation assay was carried out essentially as described previously 3. Each culture well was in-
Correspondence: Y. Ben-Ari, INSERM U29, 123 Bd. de Port-Royal, 75674 Paris, France.
324
L~
e
7
Fig. 1. Anisomycin prevents anoxia-induced neuronal death. Phase-contrast photomicrographs of representative fields of cultured granule cells. A: control experiment. B: 20 h anoxia. C: 20 h anoxia in the presence of cycloheximide (100 ~g/ml). D: 20 h anoxia in the presence of anisomycin (100 ~g/ml).
cubated with 20 ~Ci/ml [35S]methionine (Amersham) in culture medium for 20 h with cycloheximide or anisomycin at different concentrations. The cultures were then washed three times with cold culture medium and lysed with 1% Triton. The lysates were spotted to dry glass fiber filters and the proteins were precipitated with icecold 10% trichloracetic acid (TCA) by filtration. TCA precipitable counts were expressed as percentages relative to [35S]methionine incorporated by control neurons. Presumptive granule cells grown for 12-18 D I V typically appeared as a homogeneous population of round phase-bright cells sending out an extensive network of neurites (Fig. 1A). A few larger fiat and polygonal cells presumed to be astrocytes developed in spite of early exposure to cytosine arabinoside (40/~M). Granule cell cultures exposed to an anoxic atmosphere for 16, 20, or 24 h exhibited similar morphological changes which were already apparent under phase-contrast microscopy 16 h after the onset of anoxia. Most if not all the neurons were clearly degenerated, the cell bodies were replaced by debris or ghosts of cell bodies and the neurites were
disintegrated (Fig. 1B). Cell counts performed in triplicate on 9 representative microscopic fields in separate experiments showed that 97___2% (S.E.M.) of neurons were damaged or destroyed after exposure to an anoxic atmosphere for 20 h (Fig. 2). In contrast exposure to the anoxic atmosphere for 4 or 8 h did not induce significant morphological changes. This is likely to be due to remaining oxygen dissolved in the culture medium. Addition into the culture medium of either magnesium (i0 mM) or the specific N M D A receptor antagonist MK-801 (10/xM) prevented the dramatic neuronal degeneration induced by a 20 h anoxic episode (Fig. 2). To investigate the effects of protein synthesis inhibitors, anisomycin was added to the culture medium at different concentrations ranging from 1 to 100/~g/ml immediately before an anoxia of 20 h duration. The morphological changes were assessed 20 h after the onset of the hypoxic injury. No protective effect of anisomycin was apparent at 1 and 10 #g/ml whereas 50 and 100 ~tg/ml anisomycin offered respectively a 16___2 and 97___4% protection against the hypoxic insult (Figs. 1D and 2).
325
120 100 0 u
80 --o =~ u
60 40 2o o N2
Mg
MK801
CHX
AM
Fig. 2. Anisomycin prevents anoxia-induced neuronal death. Percent neuronal survival after 20 h anoxia (N2), in the presence of Mg (10 mM), MK-801 (10 #M) cycloheximide (CHX) (100/~g/ml), and anisomycin (AM) (100 ~g/ml). Viable neuronal cells were counted in triplicate from 9 representative microscopic fields under phase contrast microscopy. Errors bars represent S.E.M. Values of MK-801, Mg and anisomycin were not significantly different from that of control (P > 0.01 by Student's test). *Significantly different from anoxia (P < 0.05 by Student's test). **Significantly different from control (P < 0.0001 by Student's test).
The EDs0 was calculated to be 65 pg/ml. Similar experiments were performed with the protein synthesis inhibitor cycloheximide. No protective effect of cycloheximide was apparent at 1 and 10/~g/ml whereas 100/~g/ml anisomycin offered a 26+13% protection (Figs. 1C and 2). Since high concentration of protein synthesis inhibitors was required in order to protect the cells from anoxia, a methionine incorporation assay was carried out with different concentrations of anisomycin or cycloheximide. Protein synthesis was inhibited by 94.8___0.2% and 92+0.3% with concentration of anisomycin or cycloheximide as low as 1 pg/ml, respectively, and a 98.6+0.1% (P < 0.05 from 1 #g/ml) protein synthesis inhibition was achieved with anisomycin at concentration 100 btg/ml. As already reported for cultures of cortical 5, hippocampaP 2 or cerebellar ]5 neurons, this study confirms that anoxia-induced cell death can be prevented by magne-
1 Choi, D.W., Cerebral hypoxia: some new approaches and unanswered questions, J. Neurosci., 10 (1990) 2493-2501. 2 Choi, D.W. and Rothman, S.M., The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death, Annu. Rev. Neurosci., 13 (1990) 171-182. 3 Eichler, M.E. and Rich, K.M., Death of sensory ganglion neurons after acute withdrawal of nerve growth factor in dissociated cell cultures, Brain Res., 482 (1989) 340-346. 4 Gallo, V., Ciotti, M.T., Coletti, A., Aloisi, E and Levi, G., Selective release of glutamate from cerebellar granule cells differentiating in culture, Proc. Natl. Acad. Sci. USA, 79 (1982) 7919-7923. 5 Goldberg, M.P., Weiss, J.H., Pham, P.C. and Choi, D.W., N-Methyl-D-aspartate receptors mediate hypoxic neuronal in-
sium and the non-competitive N M D A receptor antagonist MK-801 in cultured cerebellar neurons. Assuming both procedures only block N M D A currents, this observation provides support for the view that anoxic injury in culture is mediated by activation of N M D A receptors. Furthermore this study suggests that anisomycin and cycloheximide can offer a substantial protection against anoxia-induced cell death in cultured cerebellar neurons. Indeed protein synthesis inhibitors have recently been shown to protect from ischemia-induced neuronal death in the rat hippocampus in v i v o 6't1'13 and from N G F deprivation-induced neuronal death in cultured sympathetic neurons 1°. A preliminary report has also demonstrated a protective effect of protein synthesis inhibitors against excitatory amino acid-induced neuronal death in cultured cerebellar neurons x4. However, in our study the concentration of protein synthesis inhibitors (100 #g/ml) which offered a substantial protection against anoxia was 100 times higher than that required to inhibit more than 90% of protein synthesis. Furthermore cycloheximide was found to be less potent than anisomycin in protecting neuronal cell death although both drugs inhibit protein synthesis to a similar extent. This observation suggests that the reported protective effect of anisomycin and cycloheximide might be due to an action other than protein synthesis inhibition. In fact anisomycin and cycloheximide were recently found to stimulate phosphorylation of histone H3 and of another chromatin-associated protein of 33 kDa 9 and this effect was demonstrated to be dissociable of protein synthesis inhibition 8. A n alternative hypothesis might be that the synthesis of a few proteins which are not completely inhibited at 1 #g/ml of protein synthesis inhibitors but fully inhibited at 100 /~g/ml could play a significant part in the observed neuroprotection. Two-dimensional electrophoreses are currently being carried out in order to further elucidate the role of protein synthesis in anoxia-induced neuronal death in cultured cerebellar neurons. We are grateful to S. Guidasci for the photographs and to Fidia for support.
6
7 8 9
10
jury in neuronal culture, J. Pharmacol. Exp. Ther., 243 (1990) 784-791. Goto, K., Ishige, A., Sekigushi, K., huka, S., Sugimoto, A., Yuzurihara, M., Aburada, M., Hosoya, E. and Kogure, K., Effects of cycloheximide on delayed neuronal death in rat hippocampus, Brain Res., 534 (1990) 299-302. Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57-69. Mahadevan, L.C., Signalling and superinduction, Nature, 349 (1991) 747-748. Mahadevan, L.C., Willis, A.C. and Barratt, M.J,, Rapid histone H3 phosphorylation in response to growth factors, phorbol esters, okadaic acid and protein synthesis inhibitors, Cell, 65 (1991) 775-783. Martin, D.P., Schmidt, R.E., Distefano, P.S., Lowry, O.H.,
326 Carter, J-G. and Johnson, E.M., Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation, J. Cell Biol., 106 (1988) 829-844. 11 Papas, S., Hasboun, D., Crepel, V., Chinestra, P. and Ben-Ari, Y., Cycloheximide offers protection against the deleterious influences of anoxia in the rat, Soc. Neurosci. Abstr., 17 (1991) 425.7. 12 Rothman, S.M., Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death, J, Neurosci., 4 (1984) 1884-1891.
13 Shigeno, T., Yamasaki, Y., Kato, G., Kusaka, K., Mima, T., Takakura, K., Graham, D.I. and Furukawa, S., Reduction of delayed neuronal death by inhibition of protein synthesis, Neurosci. Lett., 120 (1990) 117-119. 14 Skaper, S., Morandi, A., Facci, L., Milani, D. and Leon, A., Activation of a translational death program in CNS neurons by excitatory amino acids, Soc. Neurosci. Abstr., 16 (1990). 15 Skaper, S.D., Facci, L., Milani, D. and Leon, A., Monosialoganglioside GM1 protects against anoxia-induced neuronal death in vitro, Exp. Neurol., 106 (1989) 297-305.
323
BRES 25186
Anisomycin and cycloheximide protect cerebellar neurons in culture from anoxia Fr6d6ric Dessi, Christiane Charriaut-Marlangue and Yehezkel Ben-Ari INSERM U29, Paris (France)
(Accepted 11 February 1992) Key words: Anoxia; Protein synthesis inhibitor; Granule cell culture
Protein synthesis inhibitors have recently been shown to protect from ischemia-induced neuronal death in the rat hippocampus in vivo. In an attempt to further investigate the mechanism of neuronal death resulting from anoxia, cerebellar neurons grown in culture were exposed to an anoxic atmosphere in the presence of protein synthesis inhibitors. Anisomycin and cycloheximide (100/~g/ml) offered, respectively, a 97+4% and 26+13% protection against anoxia-induced neuronal death. It is well known that neurons are very sensitive to oxygen deprivation. In vitro anoxia in cultured neurons or in vivo ischemia in experimental animals lead to a neuronal death which is typically observed about 24 h after the insult 7. The molecular and cellular events involved in this delayed neuronal death have not been completely elucidated. The current excitotoxic hypothesis 1'2 points to the release of the excitatory amino acid glutamate and excessive activation of the N-methyl-o-aspartate receptor, a subtype of glutamate receptors, as a major event. The resulting increase of intracellular calcium would induce the death of the neuron 24 h later by a series of mechanisms including the activation of proteases. Activation of a programmed cell death has been suggested in the delayed neuronal death induced by nerve growth factor deprivation in cultured sympathetic neurons, and protein synthesis inhibitors protect from this delayed neuronal death 1°. A similar mechanism might be effective in the anoxia-induced neuronal death and could underlie the delayed neuronal death. Protein synthesis inhibitors have recently been shown to protect from ischemia-induced neuronal death in the rat hippocampus in v i v o 6'13 and to reduce the deleterious effects of anoxia in hippocampal s l i c e s 11 . This prompted us to investigate the effects of a similar treatment on neuronal death induced in cultured cerebellar neurons by anoxia. Granule cell cultures were prepared as described previously4. In brief, cerebella were dissected from the brains of 8-day-old rats under sterile conditions and were placed in 0.1% trypsin 0.1% DNAse (Sigma) in phosphate-buffered saline (PBS; Gibco) supplemented with 0.6% glucose for 15 min at room temperature and then
in 1% soybean trypsin inhibitor (Sigma) for 5 min. The cerebella were then washed three times with PBS and resuspended in culture medium containing equal volume of H a m ' s F-12 and Dulbecco's modified Eagle's medium (DMEM-F12, Gibco) supplemented glucose (30 mM), KC1 (25 mM), penicillin (5 U/ml) and gentamycin (5 pg/ ml). The cells were then dissociated with Pasteur pipettes with flame-narrowed tips, centrifuged at 250 × g for 5 min, resuspended in the same culture medium except for supplementation with 10% fetal calf serum (Seromed) and plated on 16-ram dished (Nunc) (500 pl per well) coated with poly-L-lysine (10 pg/ml) (Sigma) to a density of 5 x 105 cells per well. The cultures were maintained at 37°C in humidified 5% CO2/95% 0 2 atmosphere. Cytosine arabinoside (40 pM) was added to the culture medium after 24 h. The culture medium was not changed thereafter. Cultures were used between 12 and 18 days in vitro (DIV). Anoxia was carried out without a n y change of the culture medium by placing the cultures in a hermetic chamber filled with 95% N2/5% CO 2 in an humidified atmosphere at 37°C for 16 to 24 h. Protein synthesis inhibitors or other drugs were eventually added in the culture medium immediately before the onset of anoxia. Neuronal cell injury was assessed after the end of the anoxia by examination of cultures with phase-contrast microscopy and with bright-field examination after Trypan blue staining (0.4% 5 min). Viable cells were counted in triplicate from 9 representative microscopic fields (magnification x200). Methionine incorporation assay was carried out essentially as described previously 3. Each culture well was in-
Correspondence: Y. Ben-Ari, INSERM U29, 123 Bd. de Port-Royal, 75674 Paris, France.
324
L~
e
7
Fig. 1. Anisomycin prevents anoxia-induced neuronal death. Phase-contrast photomicrographs of representative fields of cultured granule cells. A: control experiment. B: 20 h anoxia. C: 20 h anoxia in the presence of cycloheximide (100 ~g/ml). D: 20 h anoxia in the presence of anisomycin (100 ~g/ml).
cubated with 20 ~Ci/ml [35S]methionine (Amersham) in culture medium for 20 h with cycloheximide or anisomycin at different concentrations. The cultures were then washed three times with cold culture medium and lysed with 1% Triton. The lysates were spotted to dry glass fiber filters and the proteins were precipitated with icecold 10% trichloracetic acid (TCA) by filtration. TCA precipitable counts were expressed as percentages relative to [35S]methionine incorporated by control neurons. Presumptive granule cells grown for 12-18 D I V typically appeared as a homogeneous population of round phase-bright cells sending out an extensive network of neurites (Fig. 1A). A few larger fiat and polygonal cells presumed to be astrocytes developed in spite of early exposure to cytosine arabinoside (40/~M). Granule cell cultures exposed to an anoxic atmosphere for 16, 20, or 24 h exhibited similar morphological changes which were already apparent under phase-contrast microscopy 16 h after the onset of anoxia. Most if not all the neurons were clearly degenerated, the cell bodies were replaced by debris or ghosts of cell bodies and the neurites were
disintegrated (Fig. 1B). Cell counts performed in triplicate on 9 representative microscopic fields in separate experiments showed that 97___2% (S.E.M.) of neurons were damaged or destroyed after exposure to an anoxic atmosphere for 20 h (Fig. 2). In contrast exposure to the anoxic atmosphere for 4 or 8 h did not induce significant morphological changes. This is likely to be due to remaining oxygen dissolved in the culture medium. Addition into the culture medium of either magnesium (i0 mM) or the specific N M D A receptor antagonist MK-801 (10/xM) prevented the dramatic neuronal degeneration induced by a 20 h anoxic episode (Fig. 2). To investigate the effects of protein synthesis inhibitors, anisomycin was added to the culture medium at different concentrations ranging from 1 to 100/~g/ml immediately before an anoxia of 20 h duration. The morphological changes were assessed 20 h after the onset of the hypoxic injury. No protective effect of anisomycin was apparent at 1 and 10 #g/ml whereas 50 and 100 ~tg/ml anisomycin offered respectively a 16___2 and 97___4% protection against the hypoxic insult (Figs. 1D and 2).
325
120 100 0 u
80 --o =~ u
60 40 2o o N2
Mg
MK801
CHX
AM
Fig. 2. Anisomycin prevents anoxia-induced neuronal death. Percent neuronal survival after 20 h anoxia (N2), in the presence of Mg (10 mM), MK-801 (10 #M) cycloheximide (CHX) (100/~g/ml), and anisomycin (AM) (100 ~g/ml). Viable neuronal cells were counted in triplicate from 9 representative microscopic fields under phase contrast microscopy. Errors bars represent S.E.M. Values of MK-801, Mg and anisomycin were not significantly different from that of control (P > 0.01 by Student's test). *Significantly different from anoxia (P < 0.05 by Student's test). **Significantly different from control (P < 0.0001 by Student's test).
The EDs0 was calculated to be 65 pg/ml. Similar experiments were performed with the protein synthesis inhibitor cycloheximide. No protective effect of cycloheximide was apparent at 1 and 10/~g/ml whereas 100/~g/ml anisomycin offered a 26+13% protection (Figs. 1C and 2). Since high concentration of protein synthesis inhibitors was required in order to protect the cells from anoxia, a methionine incorporation assay was carried out with different concentrations of anisomycin or cycloheximide. Protein synthesis was inhibited by 94.8___0.2% and 92+0.3% with concentration of anisomycin or cycloheximide as low as 1 pg/ml, respectively, and a 98.6+0.1% (P < 0.05 from 1 #g/ml) protein synthesis inhibition was achieved with anisomycin at concentration 100 btg/ml. As already reported for cultures of cortical 5, hippocampaP 2 or cerebellar ]5 neurons, this study confirms that anoxia-induced cell death can be prevented by magne-
1 Choi, D.W., Cerebral hypoxia: some new approaches and unanswered questions, J. Neurosci., 10 (1990) 2493-2501. 2 Choi, D.W. and Rothman, S.M., The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death, Annu. Rev. Neurosci., 13 (1990) 171-182. 3 Eichler, M.E. and Rich, K.M., Death of sensory ganglion neurons after acute withdrawal of nerve growth factor in dissociated cell cultures, Brain Res., 482 (1989) 340-346. 4 Gallo, V., Ciotti, M.T., Coletti, A., Aloisi, E and Levi, G., Selective release of glutamate from cerebellar granule cells differentiating in culture, Proc. Natl. Acad. Sci. USA, 79 (1982) 7919-7923. 5 Goldberg, M.P., Weiss, J.H., Pham, P.C. and Choi, D.W., N-Methyl-D-aspartate receptors mediate hypoxic neuronal in-
sium and the non-competitive N M D A receptor antagonist MK-801 in cultured cerebellar neurons. Assuming both procedures only block N M D A currents, this observation provides support for the view that anoxic injury in culture is mediated by activation of N M D A receptors. Furthermore this study suggests that anisomycin and cycloheximide can offer a substantial protection against anoxia-induced cell death in cultured cerebellar neurons. Indeed protein synthesis inhibitors have recently been shown to protect from ischemia-induced neuronal death in the rat hippocampus in v i v o 6't1'13 and from N G F deprivation-induced neuronal death in cultured sympathetic neurons 1°. A preliminary report has also demonstrated a protective effect of protein synthesis inhibitors against excitatory amino acid-induced neuronal death in cultured cerebellar neurons x4. However, in our study the concentration of protein synthesis inhibitors (100 #g/ml) which offered a substantial protection against anoxia was 100 times higher than that required to inhibit more than 90% of protein synthesis. Furthermore cycloheximide was found to be less potent than anisomycin in protecting neuronal cell death although both drugs inhibit protein synthesis to a similar extent. This observation suggests that the reported protective effect of anisomycin and cycloheximide might be due to an action other than protein synthesis inhibition. In fact anisomycin and cycloheximide were recently found to stimulate phosphorylation of histone H3 and of another chromatin-associated protein of 33 kDa 9 and this effect was demonstrated to be dissociable of protein synthesis inhibition 8. A n alternative hypothesis might be that the synthesis of a few proteins which are not completely inhibited at 1 #g/ml of protein synthesis inhibitors but fully inhibited at 100 /~g/ml could play a significant part in the observed neuroprotection. Two-dimensional electrophoreses are currently being carried out in order to further elucidate the role of protein synthesis in anoxia-induced neuronal death in cultured cerebellar neurons. We are grateful to S. Guidasci for the photographs and to Fidia for support.
6
7 8 9
10
jury in neuronal culture, J. Pharmacol. Exp. Ther., 243 (1990) 784-791. Goto, K., Ishige, A., Sekigushi, K., huka, S., Sugimoto, A., Yuzurihara, M., Aburada, M., Hosoya, E. and Kogure, K., Effects of cycloheximide on delayed neuronal death in rat hippocampus, Brain Res., 534 (1990) 299-302. Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57-69. Mahadevan, L.C., Signalling and superinduction, Nature, 349 (1991) 747-748. Mahadevan, L.C., Willis, A.C. and Barratt, M.J,, Rapid histone H3 phosphorylation in response to growth factors, phorbol esters, okadaic acid and protein synthesis inhibitors, Cell, 65 (1991) 775-783. Martin, D.P., Schmidt, R.E., Distefano, P.S., Lowry, O.H.,
326 Carter, J-G. and Johnson, E.M., Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation, J. Cell Biol., 106 (1988) 829-844. 11 Papas, S., Hasboun, D., Crepel, V., Chinestra, P. and Ben-Ari, Y., Cycloheximide offers protection against the deleterious influences of anoxia in the rat, Soc. Neurosci. Abstr., 17 (1991) 425.7. 12 Rothman, S.M., Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death, J, Neurosci., 4 (1984) 1884-1891.
13 Shigeno, T., Yamasaki, Y., Kato, G., Kusaka, K., Mima, T., Takakura, K., Graham, D.I. and Furukawa, S., Reduction of delayed neuronal death by inhibition of protein synthesis, Neurosci. Lett., 120 (1990) 117-119. 14 Skaper, S., Morandi, A., Facci, L., Milani, D. and Leon, A., Activation of a translational death program in CNS neurons by excitatory amino acids, Soc. Neurosci. Abstr., 16 (1990). 15 Skaper, S.D., Facci, L., Milani, D. and Leon, A., Monosialoganglioside GM1 protects against anoxia-induced neuronal death in vitro, Exp. Neurol., 106 (1989) 297-305.