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Effect of treatment with difluoromethylornithine on polyamine and spectrin breakdown levels in neonatal rat brain
Developmental Brain Research, 63 (1991) 287-289 © Elsevier Science Publishers B.V. All rights reserved. 0165-3806/91/$03.50 ADONIS 0165380691694246
287
BRESD 60424
Effect of treatment with difluoromethylornithine on polyamine and spectrin breakdown levels in neonatal rat brain Imad Najm 1, Peter Vanderklish 2, Gary Lynch 2 and Michel Baudry 1 tNeuroscience Program, USC, Los Angeles, CA 90089-2520 (U.S.A.) and 2CNLM, U.C., lrvine, CA 92717 (U.S.A.)
(Accepted 23 July 1991) Key words: Polyamine; Growth; Degeneration; Calpain; Hippocampus
Impairment of polyamine synthesis by treatment with difluoromehtylornithine(DFMO), an irreversible inhibitor of ornithine decarboxylase, has been shown to alter normal brain development. In the present study we determined the effect of DFMO treatment during a discrete developmental period on polyamine levels and on the in situ activity of calpain, as reflected by the level of degradation of spectrin, in various brain regions of rat pups. DFMO treatment from postnatal days 5 to 10 produced a marked decrease in putrescine levels in every brain structure and a significant decrease in spectrin breakdown levels in hippocampus and cortex but not in cerebellum. The results indicate that the ODC/polyamine pathway partly regulates the in situ activity of calpain and that polyamines may play a role in both growth and degeneration phenomena. The polyamines, putrescine, spermidine and spermine, play an essential role in tissue growth and differentiation 1'6'~4. Numerous studies have shown that polyamine levels as well as the activity of ornithine decarboxylase (ODC), the limiting enzyme in their synthesis, are highest in rapidly proliferating tissues and decline significantly after growth processes stop 18'21. Moreover inhibition of polyamine synthesis during the developmental period by injection of the irreversible ODC inhibitor, difluoromethylornithine (DFMO), impairs the development of different tissues, including some areas of the brain 3'19'2°. In the brain, the developmental period is also accompanied by higher levels of proteolyic activity and rapid turnover of cell constituents 8. It is generally admitted that the extent of growth is a function of the difference between rates of protein synthesis and protein degradation, and that growth ceases when the rates of these two processes become equal. Clearly then, normal development requires very precise adaptation and regulation of growth and degradation processes and it is of importance to identify factors that regulate the balance between them. We have recently shown that polyamines potentiate calcium-dependent proteolysis in endogenous fractions from rat brain, and we have obtained evidence that the interaction between polyamines and calpain is probably mediated through the interaction of polyamines with an endogenous calpain activator H. Furthermore, a variety of experimental manipulations that increase in situ
calpain activity, as evidenced by the accumulation of spectrin breakdown products (bdp) 15'17, also result in the induction of ODC and increased polyamine levels4'12. In particular, kainic acid (KA)-induced seizure activity is accompanied by increased levels of both putrescine and spectrin bdp. Pretreatment of the animals with DFMO reduces the effect of KA-induced seizure activity on both parameters 1°. These experiments indicated that polyamines might, in addition to their role in growth processes, exert an important role in the regulation of proteolytic processes. The present study further tested this hypothesis by determining the effect of chronic DFMO treatment on spectrin bdp levels in different brain areas during the postnatal period. Pregnant Sprague-Dawley rats (Charles River) were housed individually in breeding cages and allowed food and water ad libitum. Pups from all litters were randomized at birth and redistributed to the nursing mothers with litter sizes kept between 10 and 12. Pups from different litters were selected from both sexes. Five-day-old pups (n = 8) were given DFMO (500 mg/kg; i.p.) daily for 5 consecutive days. Control animals (n = 8) received equal volumes of saline solution. Pups were sacrificed at postnatal day 11 and their brains removed and chilled in ice-cold dissection buffer (10 mM Tris-HC1, pH 7.4 containing sucrose 0.32 M, EDTA 2 mM, EGTA 1 mM, leupeptin 0.1 mM and TPCK 1/zg/1). Brains were divided into two halves and each part was dissected into olfactory bulb, cerebellum, piriform cortex, hippocampus and
Correspondence: M. Baudry, HNB311, USC, Los Angeles, CA 90089-2520, U.S.A. Fax: (1) (213) 746-2863.
288 30 800 i"] Control •
[] Put Control • Put DFMO 600
DFMO
[] Spd Cont
20"
• Sod D F M O [] Spn Cont • Spn D F M O
10 •~
200
0
Hippocampus
PlrlformC o r t e x
Cerebellum
Cortex
Fig. 1. Effect of DFMO treatment on levels of polyamines in various brain regions in rat pups. Five-day-old pups were given DFMO (500 mg]kg; i.p.) daily for 5 consecutive days while control animals were treated with equal volumes of saline. Pups were sacrificed at postnatal day 11 and the brains were rapidly dissected and processed for analyses of polyamine levels as described in the text. Results are the means -+ S.E.M. of 4 experiments. White bars: saline-treated animals; black bars: DFMO-treated animals. Open bar: putrescine levels; hatched bars: spermidine levels: dotted bars: spermine levels. neocortex. Dissected brain areas were either homogenized in 5 vols. of dissection buffer for O D C and spectrin assays or homogenized in 5 vols. of 0.2 N perchloric acid for polyamine analyses. O D C activity in dissected brain regions was measured using [14C]ornithine (NEN, Boston, M A ) as substrate as previously described 2 and the results were expressed as pmoles of 14CO2 formed per mg of protein per hour. Polyamine levels were assayed by reversed phase high performance liquid chromatography using the dansylation method previously described by Desiderio et al. 5 with minor modifications. In situ calpain activity was assayed by determining the amount of spectrin bdp generated from calpain-mediated proteolysis of spectrin 15. After boiling the samples, they were subjected to electrophoresis, transferred to nitrocellulose paper, incubated with affinity purified spectrin antibodies and stained using anti-rabbit IgG-alkaline phosphatase conjugated with the 5-bromo-4-chloro-indolyl-phosphate/nitroblue tetrazolium substrate system of detection. O D C activity was significantly decreased in every brain area studied in DFMO-treated pups as compared to saline-treated animals (data not shown). The effect of D F M O treatment on tissue polyamine concentrations is shown in Fig. 1. D F M O treatment decreased the levels of putrescine substantially and uniformly in all 5 brain areas studied with the greatest degree of decrease in cerebellum. D F M O also produced substantial reductions in spermidine concentrations, although the effect was not as pronounced as for putrescine, especially in piriform cortex. In contrast, spermine levels were largely unaffected by D F M O treatment.
Hippocampus
Plriform Cortex
Cerebellum
Cortex
Fig. 2. Effect of DFMO treatment on amount of spectrin breakdown products in various brain regions in rat pups. Five-day-oldpups were given DFMO (500 mg/kg; i.p.) daily for 5 consecutive days while control animals were treated with equal volumes of saline. Pups were sacrificed at postnatal day I1 and the brains were rapidly dissected and processed for analyses of spectrin degradation as described in the text. For spectrin breakdown products the results were calculated as the amount of the main degradation product of spectrin (M r 150 kDa) expressed as a percent of total spectrin immunoreactive material. Results are the means ± S.E.M. of 8 experiments. Open bars: saline-treated animals; dark bars: DFMO-treated animals. *P < 0.05 (Student's t-test). The effect of D F M O treatment on spectrin degradation was assessed by an lmmunoassay based on the identification of spectrin and its breakdown product (Mr 150 kDa) by spectrin antibodies on Western blots. Blots stained by spectrin antibodies were scanned by reflective densitometry and the areas under the peaks corresponding to spectrin and its breakdown product were analyzed. Daily D F M O treatment for 5 days from postnatal day 5 to postnatal day 10, decreased significantly the relative amount of spectrin breakdown product in hippocampus, piriform cortex and the rest of cortex as compared to control levels (Fig. 2). However. no significant effect on the relative amount of spectrin breakdown was found in cerebellum. The present results indicate that D F M O treatment from postnatal days 5 to 11. produces a marked reduction in putrescine and spermidine levels throughout the brain as well as a significant decrease in the levels of spectrin bdp in hippocampus, piriform cortex and cortex. As we previously showed that polyamines potentiate calpain activity in brain soluble and membrane fractions 11, and that D F M O treatment reduces the levels of putrescine and spectrin bdp measured in hippocampus and piriform cortex 16 h following KA-induced setzure activity in adult rats t°. these results support the idea that polyamines regulate the in situ activity of calcium-dependent proteases. However. the lack of effect of D F M O treatment on spectrin bdp in cerebellum, despite the massive reduction in polyamine levels, indicate that the in vivo interaction between polyamines and calpain dur-
289 ing development might be region-specific. In particular, additional factors participate in calpain regulation and D F M O treatment might affect these factors differently in various brain regions. Thus, we previously showed that polyamines and in particular spermine, regulate calcium transport in mitochondria in a regionally specific manner, and it is conceivable that this effect participates in calpain regulation 7. Furthermore, it has been shown that N M D A receptor stimulation is also linked to calpain activation 16, and the N M D A receptor exhibits a polyamine site that presumably modulates the functional state of the receptor 13. As the N M D A receptors exhibit marked regional variations in their density 9, it is likely that alterations of polyamine levels would produce regionally specific effects. Finally, it should also be stressed that different brain regions exhibit different patterns of
postnatal development and that we have selected only a very short treatment with D F M O . Had we selected a longer period of drug administration, we might well have been able to observe a more general correlation between polyamine levels and spectrin bdp. In particular, a longer period of D F M O treatment has been shown to produce a marked reduction in the number of and an impairmant in the migration of granule cells in the cerebellum 3. Considering the postulated roles of calpain in cell death and motility it is tempting to attribute this effect to a prolonged reduction of in situ calpain activity. Independently of the precise mechanism involved in the differential effect of D F M O treatment on spectrin bdp, it remains that the present results add further support for a role of polyamines in the regulation of protein degradation.
1 Abraham, A.K. and Pihl, A., Role of polyamines in macromolecular synthesis, Trends Biochem. Sci. 6 (1981) 106-107. 2 Arai, A., Baudry, M., Staubli, U., Lynch, G. and Gall, C., Induction of omithine decarboxylase by subseizure stimulation in the hippocampus in vivo, MoL Brain Res., 7 (1990) 167-169. 3 Bartolome, J.V., Schweitzer, L., Slotkin, T.A. and Nadler, J.V., Impaired development of cerebellar cortex in rats treated postnatally with a-difluoromethyloruithine, Neuroscience, 15 (1985) 203-213. 4 Baudry, M., Lynch G. and Gall, C., Induction of ornithine decarboxylase as a possible mechanism for seizure-elicited changes in genomic expression in rat hippocampus, J. Neurosci., 6 (1986) 3430-3435. 5 Desiderio, M.A., Davalli, P. and Perin, A., Simultaneous determination of ~,-aminobutyric acid and polyamines by high performance liquid chromatography, J. Chromatogr., 419 (1987) 285-290. 6 Janne, J., Poso, H. and Raina, A., Polyamines in rapid growth and cancer, Biochim. Biophys. Acta, 473 (1978) 241-293. 7 Jensen, J., Lynch, G. and Baudry, M., Regional differences in the activation of synaptosomal mitochondrial Ca2÷ uptake by spermine in rat brain, Brain Res., 523 (1990) 313-315. 8 Lajtha, A. and Dunlop, D., Turn-over of proteins in the nervous system, Life Sci., 29 (1981) 755-767. 9 Monaghan, D.T. and Cotman, C.W., Distribution of N-methylo-aspartate-sensitive L-[3H]glutamate-binding sites in rat brain, J. Neurosci., 5 (1985) 2909-2919. 10 Najm, I., Vanderklish, P., Etebari, A., Lynch, G. and Baudry, M., Posssible involvement of the ODC/polyamine pathway in neuronal degeneration, Abstracts of Society for Neuroscience
by neurotransmitters, drugs and chemical modification, Life Sci., 47 (1990) 1785-1792. 14 Russell, D.H. and Durie, B.G.M., Polyamines as biochemical markers of normal and malignant growth. In Progress in Cancer Research and Therapy, Vol. 8, Raven, New York, 1978. 15 Seubert, P., Ivy, G., Larson, J., Lee, J., Shahi, K., Baudry, M. and Lynch, G., Lesions of entorhinal cortex produce a calpainmediated degradation of brain spectrin in denatate gyrus: I. Biochemical studies, Brain Res., 459 (1988) 226-232. 16 Seubert, P., Larson, J., Oliver, M., Jung, M.W., Baudry, M. and Lynch, G., Stimulation of NMDA receptors induces proteolysis of spectrin in hippocampus, Brain Res., 460 (1988) 189194. 17 Seubert, P., Lee, K. and Lynch, G., Ischemia triggers NMDA receptor-linked cytoskeletal proteolysis in hippocampus, Brain Res., 492 (1989) 1-2. 18 Slotkin, T.A. and Bartolome, J., Role of ornithine decarboxylase and the polyamines in nervous system development: a review, Brain Res. Bull., 17 (1986) 307-320. 19 Slotkin, T.A., Grignolo, A., Whitmore, W.L., Lerea, L., Trepanier, P.A., Barnes, G.A., Weigel, S.J., Seidler, F.J. and Bartolome, J., Impaired development of central and peripheral catecholamine neurotransmitter systems in preweanling rats treated with a-difluoromethylornithine, a specific irreversible inhibitor of ornithine decarboxylase, J. Pharmacol. Exp. Ther., 222 (1982) 746-751. 20 Slotkin, T.A., Seidler, F.J., Trepanier, P.A., Whitmore, W.L., Lerea, L., Barnes, G.A., Weigel, S.J. and Bartolome, J., Ornithine decarboxylase and polyamines in tissues of the neonatal rat: effects of a-difluoromethylornithine, a specific, irreversible inhibitor of ornithine decarboxylase, J. Pharm. Exp. Ther., 222 (1982) 741-745. 21 Slotkin, T.A., Seidler, F.J., Whitmore, W.L., Lerea, L., Barnes, G.A., Weigel, S.J. and Bartolome, J.V., Critical periods for the role of ornithine decarboxylase and the polyamines in growth and development of the rat: effects of exposure to a-difluoromethylornithine during discrete prenatal or postnatal intervals, Int. J. Dev. Neurosci., 2 (1982) 113-127.
Meeting, New Orleans, 1991, Vol. 17.
I1 Najm, I., Vanderklish, P., Etebari, A., Lynch, G. and Baudry, M., Complex interactions between polyamines and calpain-mediated proteolysis in rat brain, J. Neurochem., in press. 12 Paschen, W., Schmidt, K.R., Djuricic, B., Meese, C., Linn, F. and Hossmann, K.A, Polyamine changes in reversible cerebral ischemia, J. Neurochem, 49 (1987) 35-37. 13 Reynolds, I.J., Modulation of NMDA receptor responsiveness
287
BRESD 60424
Effect of treatment with difluoromethylornithine on polyamine and spectrin breakdown levels in neonatal rat brain Imad Najm 1, Peter Vanderklish 2, Gary Lynch 2 and Michel Baudry 1 tNeuroscience Program, USC, Los Angeles, CA 90089-2520 (U.S.A.) and 2CNLM, U.C., lrvine, CA 92717 (U.S.A.)
(Accepted 23 July 1991) Key words: Polyamine; Growth; Degeneration; Calpain; Hippocampus
Impairment of polyamine synthesis by treatment with difluoromehtylornithine(DFMO), an irreversible inhibitor of ornithine decarboxylase, has been shown to alter normal brain development. In the present study we determined the effect of DFMO treatment during a discrete developmental period on polyamine levels and on the in situ activity of calpain, as reflected by the level of degradation of spectrin, in various brain regions of rat pups. DFMO treatment from postnatal days 5 to 10 produced a marked decrease in putrescine levels in every brain structure and a significant decrease in spectrin breakdown levels in hippocampus and cortex but not in cerebellum. The results indicate that the ODC/polyamine pathway partly regulates the in situ activity of calpain and that polyamines may play a role in both growth and degeneration phenomena. The polyamines, putrescine, spermidine and spermine, play an essential role in tissue growth and differentiation 1'6'~4. Numerous studies have shown that polyamine levels as well as the activity of ornithine decarboxylase (ODC), the limiting enzyme in their synthesis, are highest in rapidly proliferating tissues and decline significantly after growth processes stop 18'21. Moreover inhibition of polyamine synthesis during the developmental period by injection of the irreversible ODC inhibitor, difluoromethylornithine (DFMO), impairs the development of different tissues, including some areas of the brain 3'19'2°. In the brain, the developmental period is also accompanied by higher levels of proteolyic activity and rapid turnover of cell constituents 8. It is generally admitted that the extent of growth is a function of the difference between rates of protein synthesis and protein degradation, and that growth ceases when the rates of these two processes become equal. Clearly then, normal development requires very precise adaptation and regulation of growth and degradation processes and it is of importance to identify factors that regulate the balance between them. We have recently shown that polyamines potentiate calcium-dependent proteolysis in endogenous fractions from rat brain, and we have obtained evidence that the interaction between polyamines and calpain is probably mediated through the interaction of polyamines with an endogenous calpain activator H. Furthermore, a variety of experimental manipulations that increase in situ
calpain activity, as evidenced by the accumulation of spectrin breakdown products (bdp) 15'17, also result in the induction of ODC and increased polyamine levels4'12. In particular, kainic acid (KA)-induced seizure activity is accompanied by increased levels of both putrescine and spectrin bdp. Pretreatment of the animals with DFMO reduces the effect of KA-induced seizure activity on both parameters 1°. These experiments indicated that polyamines might, in addition to their role in growth processes, exert an important role in the regulation of proteolytic processes. The present study further tested this hypothesis by determining the effect of chronic DFMO treatment on spectrin bdp levels in different brain areas during the postnatal period. Pregnant Sprague-Dawley rats (Charles River) were housed individually in breeding cages and allowed food and water ad libitum. Pups from all litters were randomized at birth and redistributed to the nursing mothers with litter sizes kept between 10 and 12. Pups from different litters were selected from both sexes. Five-day-old pups (n = 8) were given DFMO (500 mg/kg; i.p.) daily for 5 consecutive days. Control animals (n = 8) received equal volumes of saline solution. Pups were sacrificed at postnatal day 11 and their brains removed and chilled in ice-cold dissection buffer (10 mM Tris-HC1, pH 7.4 containing sucrose 0.32 M, EDTA 2 mM, EGTA 1 mM, leupeptin 0.1 mM and TPCK 1/zg/1). Brains were divided into two halves and each part was dissected into olfactory bulb, cerebellum, piriform cortex, hippocampus and
Correspondence: M. Baudry, HNB311, USC, Los Angeles, CA 90089-2520, U.S.A. Fax: (1) (213) 746-2863.
288 30 800 i"] Control •
[] Put Control • Put DFMO 600
DFMO
[] Spd Cont
20"
• Sod D F M O [] Spn Cont • Spn D F M O
10 •~
200
0
Hippocampus
PlrlformC o r t e x
Cerebellum
Cortex
Fig. 1. Effect of DFMO treatment on levels of polyamines in various brain regions in rat pups. Five-day-old pups were given DFMO (500 mg]kg; i.p.) daily for 5 consecutive days while control animals were treated with equal volumes of saline. Pups were sacrificed at postnatal day 11 and the brains were rapidly dissected and processed for analyses of polyamine levels as described in the text. Results are the means -+ S.E.M. of 4 experiments. White bars: saline-treated animals; black bars: DFMO-treated animals. Open bar: putrescine levels; hatched bars: spermidine levels: dotted bars: spermine levels. neocortex. Dissected brain areas were either homogenized in 5 vols. of dissection buffer for O D C and spectrin assays or homogenized in 5 vols. of 0.2 N perchloric acid for polyamine analyses. O D C activity in dissected brain regions was measured using [14C]ornithine (NEN, Boston, M A ) as substrate as previously described 2 and the results were expressed as pmoles of 14CO2 formed per mg of protein per hour. Polyamine levels were assayed by reversed phase high performance liquid chromatography using the dansylation method previously described by Desiderio et al. 5 with minor modifications. In situ calpain activity was assayed by determining the amount of spectrin bdp generated from calpain-mediated proteolysis of spectrin 15. After boiling the samples, they were subjected to electrophoresis, transferred to nitrocellulose paper, incubated with affinity purified spectrin antibodies and stained using anti-rabbit IgG-alkaline phosphatase conjugated with the 5-bromo-4-chloro-indolyl-phosphate/nitroblue tetrazolium substrate system of detection. O D C activity was significantly decreased in every brain area studied in DFMO-treated pups as compared to saline-treated animals (data not shown). The effect of D F M O treatment on tissue polyamine concentrations is shown in Fig. 1. D F M O treatment decreased the levels of putrescine substantially and uniformly in all 5 brain areas studied with the greatest degree of decrease in cerebellum. D F M O also produced substantial reductions in spermidine concentrations, although the effect was not as pronounced as for putrescine, especially in piriform cortex. In contrast, spermine levels were largely unaffected by D F M O treatment.
Hippocampus
Plriform Cortex
Cerebellum
Cortex
Fig. 2. Effect of DFMO treatment on amount of spectrin breakdown products in various brain regions in rat pups. Five-day-oldpups were given DFMO (500 mg/kg; i.p.) daily for 5 consecutive days while control animals were treated with equal volumes of saline. Pups were sacrificed at postnatal day I1 and the brains were rapidly dissected and processed for analyses of spectrin degradation as described in the text. For spectrin breakdown products the results were calculated as the amount of the main degradation product of spectrin (M r 150 kDa) expressed as a percent of total spectrin immunoreactive material. Results are the means ± S.E.M. of 8 experiments. Open bars: saline-treated animals; dark bars: DFMO-treated animals. *P < 0.05 (Student's t-test). The effect of D F M O treatment on spectrin degradation was assessed by an lmmunoassay based on the identification of spectrin and its breakdown product (Mr 150 kDa) by spectrin antibodies on Western blots. Blots stained by spectrin antibodies were scanned by reflective densitometry and the areas under the peaks corresponding to spectrin and its breakdown product were analyzed. Daily D F M O treatment for 5 days from postnatal day 5 to postnatal day 10, decreased significantly the relative amount of spectrin breakdown product in hippocampus, piriform cortex and the rest of cortex as compared to control levels (Fig. 2). However. no significant effect on the relative amount of spectrin breakdown was found in cerebellum. The present results indicate that D F M O treatment from postnatal days 5 to 11. produces a marked reduction in putrescine and spermidine levels throughout the brain as well as a significant decrease in the levels of spectrin bdp in hippocampus, piriform cortex and cortex. As we previously showed that polyamines potentiate calpain activity in brain soluble and membrane fractions 11, and that D F M O treatment reduces the levels of putrescine and spectrin bdp measured in hippocampus and piriform cortex 16 h following KA-induced setzure activity in adult rats t°. these results support the idea that polyamines regulate the in situ activity of calcium-dependent proteases. However. the lack of effect of D F M O treatment on spectrin bdp in cerebellum, despite the massive reduction in polyamine levels, indicate that the in vivo interaction between polyamines and calpain dur-
289 ing development might be region-specific. In particular, additional factors participate in calpain regulation and D F M O treatment might affect these factors differently in various brain regions. Thus, we previously showed that polyamines and in particular spermine, regulate calcium transport in mitochondria in a regionally specific manner, and it is conceivable that this effect participates in calpain regulation 7. Furthermore, it has been shown that N M D A receptor stimulation is also linked to calpain activation 16, and the N M D A receptor exhibits a polyamine site that presumably modulates the functional state of the receptor 13. As the N M D A receptors exhibit marked regional variations in their density 9, it is likely that alterations of polyamine levels would produce regionally specific effects. Finally, it should also be stressed that different brain regions exhibit different patterns of
postnatal development and that we have selected only a very short treatment with D F M O . Had we selected a longer period of drug administration, we might well have been able to observe a more general correlation between polyamine levels and spectrin bdp. In particular, a longer period of D F M O treatment has been shown to produce a marked reduction in the number of and an impairmant in the migration of granule cells in the cerebellum 3. Considering the postulated roles of calpain in cell death and motility it is tempting to attribute this effect to a prolonged reduction of in situ calpain activity. Independently of the precise mechanism involved in the differential effect of D F M O treatment on spectrin bdp, it remains that the present results add further support for a role of polyamines in the regulation of protein degradation.
1 Abraham, A.K. and Pihl, A., Role of polyamines in macromolecular synthesis, Trends Biochem. Sci. 6 (1981) 106-107. 2 Arai, A., Baudry, M., Staubli, U., Lynch, G. and Gall, C., Induction of omithine decarboxylase by subseizure stimulation in the hippocampus in vivo, MoL Brain Res., 7 (1990) 167-169. 3 Bartolome, J.V., Schweitzer, L., Slotkin, T.A. and Nadler, J.V., Impaired development of cerebellar cortex in rats treated postnatally with a-difluoromethyloruithine, Neuroscience, 15 (1985) 203-213. 4 Baudry, M., Lynch G. and Gall, C., Induction of ornithine decarboxylase as a possible mechanism for seizure-elicited changes in genomic expression in rat hippocampus, J. Neurosci., 6 (1986) 3430-3435. 5 Desiderio, M.A., Davalli, P. and Perin, A., Simultaneous determination of ~,-aminobutyric acid and polyamines by high performance liquid chromatography, J. Chromatogr., 419 (1987) 285-290. 6 Janne, J., Poso, H. and Raina, A., Polyamines in rapid growth and cancer, Biochim. Biophys. Acta, 473 (1978) 241-293. 7 Jensen, J., Lynch, G. and Baudry, M., Regional differences in the activation of synaptosomal mitochondrial Ca2÷ uptake by spermine in rat brain, Brain Res., 523 (1990) 313-315. 8 Lajtha, A. and Dunlop, D., Turn-over of proteins in the nervous system, Life Sci., 29 (1981) 755-767. 9 Monaghan, D.T. and Cotman, C.W., Distribution of N-methylo-aspartate-sensitive L-[3H]glutamate-binding sites in rat brain, J. Neurosci., 5 (1985) 2909-2919. 10 Najm, I., Vanderklish, P., Etebari, A., Lynch, G. and Baudry, M., Posssible involvement of the ODC/polyamine pathway in neuronal degeneration, Abstracts of Society for Neuroscience
by neurotransmitters, drugs and chemical modification, Life Sci., 47 (1990) 1785-1792. 14 Russell, D.H. and Durie, B.G.M., Polyamines as biochemical markers of normal and malignant growth. In Progress in Cancer Research and Therapy, Vol. 8, Raven, New York, 1978. 15 Seubert, P., Ivy, G., Larson, J., Lee, J., Shahi, K., Baudry, M. and Lynch, G., Lesions of entorhinal cortex produce a calpainmediated degradation of brain spectrin in denatate gyrus: I. Biochemical studies, Brain Res., 459 (1988) 226-232. 16 Seubert, P., Larson, J., Oliver, M., Jung, M.W., Baudry, M. and Lynch, G., Stimulation of NMDA receptors induces proteolysis of spectrin in hippocampus, Brain Res., 460 (1988) 189194. 17 Seubert, P., Lee, K. and Lynch, G., Ischemia triggers NMDA receptor-linked cytoskeletal proteolysis in hippocampus, Brain Res., 492 (1989) 1-2. 18 Slotkin, T.A. and Bartolome, J., Role of ornithine decarboxylase and the polyamines in nervous system development: a review, Brain Res. Bull., 17 (1986) 307-320. 19 Slotkin, T.A., Grignolo, A., Whitmore, W.L., Lerea, L., Trepanier, P.A., Barnes, G.A., Weigel, S.J., Seidler, F.J. and Bartolome, J., Impaired development of central and peripheral catecholamine neurotransmitter systems in preweanling rats treated with a-difluoromethylornithine, a specific irreversible inhibitor of ornithine decarboxylase, J. Pharmacol. Exp. Ther., 222 (1982) 746-751. 20 Slotkin, T.A., Seidler, F.J., Trepanier, P.A., Whitmore, W.L., Lerea, L., Barnes, G.A., Weigel, S.J. and Bartolome, J., Ornithine decarboxylase and polyamines in tissues of the neonatal rat: effects of a-difluoromethylornithine, a specific, irreversible inhibitor of ornithine decarboxylase, J. Pharm. Exp. Ther., 222 (1982) 741-745. 21 Slotkin, T.A., Seidler, F.J., Whitmore, W.L., Lerea, L., Barnes, G.A., Weigel, S.J. and Bartolome, J.V., Critical periods for the role of ornithine decarboxylase and the polyamines in growth and development of the rat: effects of exposure to a-difluoromethylornithine during discrete prenatal or postnatal intervals, Int. J. Dev. Neurosci., 2 (1982) 113-127.
Meeting, New Orleans, 1991, Vol. 17.
I1 Najm, I., Vanderklish, P., Etebari, A., Lynch, G. and Baudry, M., Complex interactions between polyamines and calpain-mediated proteolysis in rat brain, J. Neurochem., in press. 12 Paschen, W., Schmidt, K.R., Djuricic, B., Meese, C., Linn, F. and Hossmann, K.A, Polyamine changes in reversible cerebral ischemia, J. Neurochem, 49 (1987) 35-37. 13 Reynolds, I.J., Modulation of NMDA receptor responsiveness