Epilepsy Res., 11 (1992) 3-7
3
Elsevier EPIRES 00448
Ornithine decarboxylase induction and polyamine synthesis in the kindling of seizures: the effect of a-difluoromethylornithine L.J. Herberg a, I.C. Rose a, J.S. de B e l l e r o c h e b and M. Mintz c aExperimental Psychology Laboratory, Instituteof Neurology, London, bDepartmentof Biochemistry, CharingCross Westminster Medical School, London (UK) and CDepartmentof Psychology, Universityof Tel A viv (Israel) (Received 17 July 1991; revision received 28 October 1991; accepted 4 November 1991
Key words: Amygdala; a-Difluoromethylomithine; Immediate-early genes; Kindling; Ornithine decarboxylase; Polyamines; Seizure
It has been suggested that the kindling of seizures may depend on the induction of genes encoding enzymes involved in neurotransmission. Experimental seizures are followed by an especially rapid and massive induction of brain omithine decarboxylase (ODC), an enzyme which catalyses the rate-limiting step in the synthesis of polyamines. The latter compounds have been shown to act as positive allosteric modulators of the NMDA receptor, and also to play an important role in cell growth and differentiation. The induction of ODC by seizures has accordingly been suggested to play a pivotal role in the changes in synaptic structure and function that underlie kindling. In the present study we examined the progress of kindling during treatment with a-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. We found that progressive increase in the duration and severity of kindled seizures and in the duration of local afterdischarges was unaffected by daily injections of DFMO in doses previously shown to cause substantial depression of brain ODC activity. Treatment with DFMO also failed to produce significant anticonvulsant or proconvulsant effects. Progressive increase in seizure activity during kindling is therefore unlikely to depend to any appreciable extent on enhanced synthesis of polyamines by ODC.
INTRODUCTION Recent studies suggest that long-term synaptic changes underlying learning and memory may be brought about by activation of immediate-early genes such as the proto-oncogenes c-los and c-jun. These genes encode transcription factors which regulate the activity of other, 'effector' genes, whose translated products are thought to produce
Correspondence to: Jacob Herberg MBBCh, DPhil, Experimental Psychology Lab., Institute of Neurology, Queen Square, London WC1N 3BG, UK.
long-term change in synaptic function 12,22. The kindling of seizures may depend on similar mechanisms. In the kindling procedure, daily administration of a brief electrical stimulus, initially without overt effect, leads to progressively longer local afterdischarges, culminating in generalised tonic-clonic convulsions at every trial n. It has been suggested that kindling involves a sequential activation of immediate-early competence genes, such as c-los and c-]un, encoding transcription factors that regulate the activity of delayed-early or late-effector genes (reviewed by Dragunow and colleaguest). Products of effector genes would be directly or indirectly responsible for the perma-
0920-1211/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved
nent changes in synaptic structure underlying the kindling process. Recent studies have in fact demonstrated sharp increases in c-los, c-jun and other immediate-early gene mRNAs in rat hippocampus in response to experimental seizures 7'22'24, but the identity of the late-effector gene(s) that might be responsible for the next step in the sequence is still unknown. Much attention has focussed on the gene encoding ornithine decarboxylase (ODC), the enzyme which catalyses the rate-limiting step in the synthesis of the polyamines, including putrescine, spermine and spermidine. Polyamines have long been known to play a prominent role in cell growth and differentiation16, and have more recently been shown to act as positive allosteric modulators of NMDA receptors involved in long-term potentiation 2°. These properties of the polyamines have led several investigators to suggest that the postictal surge in ODC, and ensuing changes in polyamine activity, might be the mediator of seizureelicited plastic changes in synaptic function 2'15'24. In the present study we investigated this suggestion by examining the progress of kindling in rats treated with a-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC activity4. METHOD
Subjects Male hooded rats (type PVG, Bantin & Kingman Ltd., Hull, England) weighing 250-300 g were anaesthetised with pentobarbitone and implanted with twisted bipolar stainless steel electrodes (Plastic Products Co., Roanoke, VA), aimed at a central amygdala, with coordinates A -1.0, 4.5 lateral and 8.5 mm deep relative to bregma, the incisor bar being fixed 5.0 mm above the horizontal interaural plane. Electrode coordinates were verified on photographic projections of 50/~m unstained frozen sections at the end of the experiment.
Drug treatment a-Difluoromethyl ornithine (Eflornithine, Merrell Dow) was dissolved in isotonic saline and injected subcutaneously once daily at 10.00 a.m. in a dose of 200 mg/kg and in a volume of 2.0 ml/kg. A
single such dose has previously been shown to produce rapid and prolonged depression of prostatic ODC activity, with maximal depression (>90%) after 4-6 h, and with little recovery ( 0.3, the 2-tailed 95% confidence intervals for the respective between-group differences being -0.65 to 0.98, -5.4 to 11.4, and -14.9 to 11.0). DISCUSSION Rats treated with an inhibitor of polyamine synthesis kindled as rapidly and as strongly as control rats. This was an unexpected result in view of previous findings. Firstly, as mentioned above, brain ODC shows a massive rise (by a factor of over 200) after experimental seizures2'15'21. This surge is closely associated with an enhanced expression of immediate-early genes, revealed by a transient elevation in cortical c-los mRNA 23'z4 and increased Fos and Jun immunoreactivity in putative epileptogenic sites such as the hippocampal den-
tate gyrusr; conversely, kindling is arrested by non-specific inhibitors of protein synthesis3'17, consistent with a critical role for effector gene products as typified by ODC. Secondly, the polyamine products of ODC are prominent not only in growing tissue, where they affect DNA replication and RNA expression,but also in the mature CNS, where they may be involved in long-term synaptic change 16,24. This possibility seems supported by a report that intracranial injection of polyamines may be followed, days later, by devastating and irreversible seizures 1. Despite this circumstantial evidence, the present results make it unlikely that kindling progression depends to any substantial extent on enhanced polyamine synthesis. What other explanation might there be for the apparent association between ODC activity and seizures? The striking association between post-seizure c-los expression and ODC induction is not necessarily evidence that these processes are causally related13; a more significant trigger of ODC synthesis may be provided by certain other metabolic features of brain seizure activity, in particular the sharp peri-ictal surge in extracellular adenosine. Adenosine has previously been shown to act synergistically with nerve growth factor to induce ODC synthesis in PC12 cell cultures TM, and adenosine is moreover strongly implicated in postictal processes that ordinarily prevent continued or subsequent fits5, The present findings do not exclude a role for ODC and polyamines in the short-term and long-term inhibitory processes that may be induced by seizures, and which may persist for several days after massed kindling sessions (e.g. refs. 5, 8, 18). ACKNOWLEDGEMENTS Supported by a project grant from Action Research. We thank Merrell Dow Research for a gift of DFMO.
REFERENCES 1 Anderson, D.J., Crossland, J. and Shaw, G.C., The actions of spermidine and spermine on the central nervous system, Neuropharmacology, 14 (1975) 571-577. 2 Baudry, M., Lynch, G. and Gall, C., Induction of omithine decarboxylase as a possible mediator of seizure-elicited changes in genomic expression in rat hippocampus, J. Neurosci., 6 (1986) 3430-3435. 3 Cain, D.P., Corcoran, M.E. and Staines, W.A., Effects of protein synthesis inhibition on kindling in the mouse, Exp. Neurol., 68 (1980) 409-419. 4 Danzin, C., Jung, M.J., Grove, J. and Bey, P., Effect of udifluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on polyamine levels in rat tissues, Life Sci., 24 (1979) 519-524. 5 Dragunow, M., Purinergic mechanisms in epilepsy, Prog. Neurobiol., 31 (1988) 85-108. Dragunow, M., Currie, R.W., Faull, R.L.M., Robertson, H.A. and Jansen, K., Immediate-early genes, kindling and long-term potentiation, Neurosci. Biobehav. Rev., 13 (1989) 301-313. 7 Dragunow, M. and Robertson, H.A., Kindling stimulation induces c-los protein(s) in granule cells in the rat dentate nucleus, Nature, 329 (1987) 442-442. 8 Engel, J: and Ackermann, R.F., Interictal EEG spikes correlate with decreased, rather than increased, epileptogenicity in amygdaloid kindled rats, Brain Res., 190 (1980) 543-548. 9 Ferchmin, P.A. and Eterovi~, V.A., Role of pelyamines in experience-dependent brain plasticity, Pharmacol. Biochem. Behav., 26 (1987) 341-349. 10 Ferchmin, P.A. and Eterovi~, V.A., Experience affects cortical but not subcortical polyamines, Pharmacol. Biochem. Behav., 35 (1990) 355-358. 11 Goddard, G.V., McIntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. 12 Goelet, P., Castelucci, V.F., Schacher, S. and Kandel, E.R., The long and the short of long-term memory - - a molecular framework, Nature, 322 (1986) 419-422.
13 Guerrero, I., Pellicer, A. and Burstein, D.E., Dissociation of c-fos from ODC expression in a PC12 subline stably transfected with an inducible N-ras oncogene, Biochem. Biophys. Res. Commun., 150 (1988) 1185-1192. 14 Guroff, G., Dickens, G., End, D. and Londos, C., The action of adenosine analogs on PC12 cells, J. Neurochem., 37 (1981) 1431-1439. 15 Hayashi, Y., Hattori, Y., Moriwaki, A., Saeki, K. and Hori, Y., Changes in polyamine concentration in arnygdaloid-kindled rats, I. Neurochem., 53 (1989) 986-988. 16 Heby, O., Role of polyamines in the control of cell proliferation and differentiation, Differentiation, 19 (1981) 1-20. 17 Jonec, V. and Wasterlaln, C.G., Effect of inhibition of protein synthesis on the development of kindled seizures in rats, Exp. Neurol., 66 (1979) 524-532. 18 Mucha, R.F. and Pinel, J.P.J., Postseizure inhibition of kindled seizures, Exp. Neurol., 54 (1977) 266-282. 19 Racine, R.J., Modification of seizure activity by electrical stimulation. II. Motor seizure, EEG Clin. Neurophysiol., 32 (1972) 281-294. 20 Ransom, R.W. and Stec, N.L., Cooperative modulation of [3H]MK-801 binding to the N-methyl-D-aspartate receptorion channel complex by L-glutamate, glycine, and polyamines, J. Neurochem., 51 (1988) 830-836. 21 Reed, L.J. and de Belleroehe, J.S., Induction of ornithine decarboxylase in cerebral cortex by excitotoxin lesion of nucleus basalis: association with postsynaptic responsiveness and N-methyl-D-aspartate activation, J. Neurochem., 55 (1990) 780-787. 22 Saffen, D.W., Cole, A.J., Worley, P.F., Christy, B.A., Ryder, K. and Baraban, J.M., Convulsant-induced increase in transcription factor messenger RNAs in rat brain, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 7795-7799. 23 Wood, H. and de Belleroche, J.S., Induction of c-los mRNA in cerebral cortex by excitotoxin stimulation of cortical inputs: involvement of N-methyl-D-aspartate receptors, Brain Res., (1991) in press. 24 Zawia, N.H. and Bondy, S.C., Electrically stimulated rapid gene expression in the brain: ornithine decarboxylase and c-los, Mol. Brain Res., 7 (1990) 243-247.
3
Elsevier EPIRES 00448
Ornithine decarboxylase induction and polyamine synthesis in the kindling of seizures: the effect of a-difluoromethylornithine L.J. Herberg a, I.C. Rose a, J.S. de B e l l e r o c h e b and M. Mintz c aExperimental Psychology Laboratory, Instituteof Neurology, London, bDepartmentof Biochemistry, CharingCross Westminster Medical School, London (UK) and CDepartmentof Psychology, Universityof Tel A viv (Israel) (Received 17 July 1991; revision received 28 October 1991; accepted 4 November 1991
Key words: Amygdala; a-Difluoromethylomithine; Immediate-early genes; Kindling; Ornithine decarboxylase; Polyamines; Seizure
It has been suggested that the kindling of seizures may depend on the induction of genes encoding enzymes involved in neurotransmission. Experimental seizures are followed by an especially rapid and massive induction of brain omithine decarboxylase (ODC), an enzyme which catalyses the rate-limiting step in the synthesis of polyamines. The latter compounds have been shown to act as positive allosteric modulators of the NMDA receptor, and also to play an important role in cell growth and differentiation. The induction of ODC by seizures has accordingly been suggested to play a pivotal role in the changes in synaptic structure and function that underlie kindling. In the present study we examined the progress of kindling during treatment with a-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. We found that progressive increase in the duration and severity of kindled seizures and in the duration of local afterdischarges was unaffected by daily injections of DFMO in doses previously shown to cause substantial depression of brain ODC activity. Treatment with DFMO also failed to produce significant anticonvulsant or proconvulsant effects. Progressive increase in seizure activity during kindling is therefore unlikely to depend to any appreciable extent on enhanced synthesis of polyamines by ODC.
INTRODUCTION Recent studies suggest that long-term synaptic changes underlying learning and memory may be brought about by activation of immediate-early genes such as the proto-oncogenes c-los and c-jun. These genes encode transcription factors which regulate the activity of other, 'effector' genes, whose translated products are thought to produce
Correspondence to: Jacob Herberg MBBCh, DPhil, Experimental Psychology Lab., Institute of Neurology, Queen Square, London WC1N 3BG, UK.
long-term change in synaptic function 12,22. The kindling of seizures may depend on similar mechanisms. In the kindling procedure, daily administration of a brief electrical stimulus, initially without overt effect, leads to progressively longer local afterdischarges, culminating in generalised tonic-clonic convulsions at every trial n. It has been suggested that kindling involves a sequential activation of immediate-early competence genes, such as c-los and c-]un, encoding transcription factors that regulate the activity of delayed-early or late-effector genes (reviewed by Dragunow and colleaguest). Products of effector genes would be directly or indirectly responsible for the perma-
0920-1211/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved
nent changes in synaptic structure underlying the kindling process. Recent studies have in fact demonstrated sharp increases in c-los, c-jun and other immediate-early gene mRNAs in rat hippocampus in response to experimental seizures 7'22'24, but the identity of the late-effector gene(s) that might be responsible for the next step in the sequence is still unknown. Much attention has focussed on the gene encoding ornithine decarboxylase (ODC), the enzyme which catalyses the rate-limiting step in the synthesis of the polyamines, including putrescine, spermine and spermidine. Polyamines have long been known to play a prominent role in cell growth and differentiation16, and have more recently been shown to act as positive allosteric modulators of NMDA receptors involved in long-term potentiation 2°. These properties of the polyamines have led several investigators to suggest that the postictal surge in ODC, and ensuing changes in polyamine activity, might be the mediator of seizureelicited plastic changes in synaptic function 2'15'24. In the present study we investigated this suggestion by examining the progress of kindling in rats treated with a-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC activity4. METHOD
Subjects Male hooded rats (type PVG, Bantin & Kingman Ltd., Hull, England) weighing 250-300 g were anaesthetised with pentobarbitone and implanted with twisted bipolar stainless steel electrodes (Plastic Products Co., Roanoke, VA), aimed at a central amygdala, with coordinates A -1.0, 4.5 lateral and 8.5 mm deep relative to bregma, the incisor bar being fixed 5.0 mm above the horizontal interaural plane. Electrode coordinates were verified on photographic projections of 50/~m unstained frozen sections at the end of the experiment.
Drug treatment a-Difluoromethyl ornithine (Eflornithine, Merrell Dow) was dissolved in isotonic saline and injected subcutaneously once daily at 10.00 a.m. in a dose of 200 mg/kg and in a volume of 2.0 ml/kg. A
single such dose has previously been shown to produce rapid and prolonged depression of prostatic ODC activity, with maximal depression (>90%) after 4-6 h, and with little recovery ( 0.3, the 2-tailed 95% confidence intervals for the respective between-group differences being -0.65 to 0.98, -5.4 to 11.4, and -14.9 to 11.0). DISCUSSION Rats treated with an inhibitor of polyamine synthesis kindled as rapidly and as strongly as control rats. This was an unexpected result in view of previous findings. Firstly, as mentioned above, brain ODC shows a massive rise (by a factor of over 200) after experimental seizures2'15'21. This surge is closely associated with an enhanced expression of immediate-early genes, revealed by a transient elevation in cortical c-los mRNA 23'z4 and increased Fos and Jun immunoreactivity in putative epileptogenic sites such as the hippocampal den-
tate gyrusr; conversely, kindling is arrested by non-specific inhibitors of protein synthesis3'17, consistent with a critical role for effector gene products as typified by ODC. Secondly, the polyamine products of ODC are prominent not only in growing tissue, where they affect DNA replication and RNA expression,but also in the mature CNS, where they may be involved in long-term synaptic change 16,24. This possibility seems supported by a report that intracranial injection of polyamines may be followed, days later, by devastating and irreversible seizures 1. Despite this circumstantial evidence, the present results make it unlikely that kindling progression depends to any substantial extent on enhanced polyamine synthesis. What other explanation might there be for the apparent association between ODC activity and seizures? The striking association between post-seizure c-los expression and ODC induction is not necessarily evidence that these processes are causally related13; a more significant trigger of ODC synthesis may be provided by certain other metabolic features of brain seizure activity, in particular the sharp peri-ictal surge in extracellular adenosine. Adenosine has previously been shown to act synergistically with nerve growth factor to induce ODC synthesis in PC12 cell cultures TM, and adenosine is moreover strongly implicated in postictal processes that ordinarily prevent continued or subsequent fits5, The present findings do not exclude a role for ODC and polyamines in the short-term and long-term inhibitory processes that may be induced by seizures, and which may persist for several days after massed kindling sessions (e.g. refs. 5, 8, 18). ACKNOWLEDGEMENTS Supported by a project grant from Action Research. We thank Merrell Dow Research for a gift of DFMO.
REFERENCES 1 Anderson, D.J., Crossland, J. and Shaw, G.C., The actions of spermidine and spermine on the central nervous system, Neuropharmacology, 14 (1975) 571-577. 2 Baudry, M., Lynch, G. and Gall, C., Induction of omithine decarboxylase as a possible mediator of seizure-elicited changes in genomic expression in rat hippocampus, J. Neurosci., 6 (1986) 3430-3435. 3 Cain, D.P., Corcoran, M.E. and Staines, W.A., Effects of protein synthesis inhibition on kindling in the mouse, Exp. Neurol., 68 (1980) 409-419. 4 Danzin, C., Jung, M.J., Grove, J. and Bey, P., Effect of udifluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on polyamine levels in rat tissues, Life Sci., 24 (1979) 519-524. 5 Dragunow, M., Purinergic mechanisms in epilepsy, Prog. Neurobiol., 31 (1988) 85-108. Dragunow, M., Currie, R.W., Faull, R.L.M., Robertson, H.A. and Jansen, K., Immediate-early genes, kindling and long-term potentiation, Neurosci. Biobehav. Rev., 13 (1989) 301-313. 7 Dragunow, M. and Robertson, H.A., Kindling stimulation induces c-los protein(s) in granule cells in the rat dentate nucleus, Nature, 329 (1987) 442-442. 8 Engel, J: and Ackermann, R.F., Interictal EEG spikes correlate with decreased, rather than increased, epileptogenicity in amygdaloid kindled rats, Brain Res., 190 (1980) 543-548. 9 Ferchmin, P.A. and Eterovi~, V.A., Role of pelyamines in experience-dependent brain plasticity, Pharmacol. Biochem. Behav., 26 (1987) 341-349. 10 Ferchmin, P.A. and Eterovi~, V.A., Experience affects cortical but not subcortical polyamines, Pharmacol. Biochem. Behav., 35 (1990) 355-358. 11 Goddard, G.V., McIntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. 12 Goelet, P., Castelucci, V.F., Schacher, S. and Kandel, E.R., The long and the short of long-term memory - - a molecular framework, Nature, 322 (1986) 419-422.
13 Guerrero, I., Pellicer, A. and Burstein, D.E., Dissociation of c-fos from ODC expression in a PC12 subline stably transfected with an inducible N-ras oncogene, Biochem. Biophys. Res. Commun., 150 (1988) 1185-1192. 14 Guroff, G., Dickens, G., End, D. and Londos, C., The action of adenosine analogs on PC12 cells, J. Neurochem., 37 (1981) 1431-1439. 15 Hayashi, Y., Hattori, Y., Moriwaki, A., Saeki, K. and Hori, Y., Changes in polyamine concentration in arnygdaloid-kindled rats, I. Neurochem., 53 (1989) 986-988. 16 Heby, O., Role of polyamines in the control of cell proliferation and differentiation, Differentiation, 19 (1981) 1-20. 17 Jonec, V. and Wasterlaln, C.G., Effect of inhibition of protein synthesis on the development of kindled seizures in rats, Exp. Neurol., 66 (1979) 524-532. 18 Mucha, R.F. and Pinel, J.P.J., Postseizure inhibition of kindled seizures, Exp. Neurol., 54 (1977) 266-282. 19 Racine, R.J., Modification of seizure activity by electrical stimulation. II. Motor seizure, EEG Clin. Neurophysiol., 32 (1972) 281-294. 20 Ransom, R.W. and Stec, N.L., Cooperative modulation of [3H]MK-801 binding to the N-methyl-D-aspartate receptorion channel complex by L-glutamate, glycine, and polyamines, J. Neurochem., 51 (1988) 830-836. 21 Reed, L.J. and de Belleroehe, J.S., Induction of ornithine decarboxylase in cerebral cortex by excitotoxin lesion of nucleus basalis: association with postsynaptic responsiveness and N-methyl-D-aspartate activation, J. Neurochem., 55 (1990) 780-787. 22 Saffen, D.W., Cole, A.J., Worley, P.F., Christy, B.A., Ryder, K. and Baraban, J.M., Convulsant-induced increase in transcription factor messenger RNAs in rat brain, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 7795-7799. 23 Wood, H. and de Belleroche, J.S., Induction of c-los mRNA in cerebral cortex by excitotoxin stimulation of cortical inputs: involvement of N-methyl-D-aspartate receptors, Brain Res., (1991) in press. 24 Zawia, N.H. and Bondy, S.C., Electrically stimulated rapid gene expression in the brain: ornithine decarboxylase and c-los, Mol. Brain Res., 7 (1990) 243-247.
