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Kainic acid-induced seizures increase c-fos-like protein in the hippocampus
European Journal of Pharmacology, 150 (1988) 405-406
405
Elsevier EJP 0129R
Rapid communication
Kainic acid-induced seizures increase c-los-like protein in the hippocampus Th. Popovici, G. B a r b i n a n d Y. Ben Ari * I N S E R M U. 29, H3pital de Port-Royal, 123 Bd. de Port-Royal, 75674 Paris Cedex 14, France
Received 10 May 1988, accepted 11 May 1988
The levels of the proto-oncogene c-fos in PC 12 cells increase following sustained opening of voltage-dependent calcium channels (Morgan and Curran, 1986). A similar increase in rodent brain is observed after seizure activity (Dragunow and Robertson, 1987; Morgan et al., 1987). Kainic acid, a powerful excitatory analogue of glutamate induces seizures and a brain damage syndrome involving the hippocampus and limbic regions. This syndrome has some relevance to human temporal lobe epilepsy (Ben Ari, 1985). We now report on a sequential increase in c-fos protein within the the hippocampal formation of the rat following i.p. administration of kainate. Adult male Wistar rats received kainic acid (8 m g / k g i.p.) and were killed at various time intervals. Immunoreactivity was detected as previously described, with minor modifications (Dragunow et al., 1987). The rats were perfused with 1% paraformaldehyde, their brains were immersed in 15% sucrose and cut with a cryostat. Sections (30 /~m thick) were incubated in 5% normal rabbit serum. The primary c-los antibody (C.R.B., England) was used at a dilution of 1/1600. A biotinylated donkey anti-sheep antibody (1/100) and a streptavidin-biotin peroxidase complex (1/400), both from Amersham, were applied successively before visualization with diaminobenzidine. In keeping with earlier observations (Ben Ari, 1985), a limbic motor seizure developed 1 h after parenteral kainate administration. Whereas the
* To whom all correspondenceshould be addressed.
control brains showed no immunoreactivity, a strong nuclear staining was exclusively restricted to the granule neurons of the dentate gyms, 3 h after treatment (fig. 1A). At 6 h the nuclear immunoreaction extended to the pyramidal cells and the polymorphic interneurons located in the stratum orients, stratum radiatum and hilus (fig. 1B). The labeling disappeared progressively in the same order as it had appeared (data not shown). Granule cell staining was dramatically reduced 12 h after the injection and disappeared at 24 h whereas the decline was delayed in the the pyramidal cells. The immunostaining in granule and pyramidal cells was predominant within the nucleus and very faint in the cytoplasm. Axons and dendrites as well as sections incubated without the c-fos antibody were devoid of labeling. In conclusion, kainic acid produced a seizure syndrome associated with a transient rise in the c-los protein within the hippocampus. The time course of this increase, which affected the granule neurons before the pyramidal ones, was in agreement with the results of electrographic and metabolic studies. Together, these data suggest that the area dendata is first activated by the paroxysmal discharge through the perforant pathway; the entire hippocampal formation and related structures of the limbic system are involved subsequently (Popovici et al., in preparation), c-fos protein induction as shown here may be produced either through specific neurotransmitter activation or through seizure discharges and the associated calcium influx (Morgan and Curran, 1986). Since the c-los protein may be involved in the regulation
0014-2999/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
406
Fig. 1. c-los protein immunoreactivity within the hippocampus 3 h (A) and 6 h (B) following kainate administration.
o f g e n e e x p r e s s i o n its s e q u e n t i a l a c t i v a t i o n b y k a i n a t e m a y b e a r s o m e r e l e v a n c e to t h e p e r s i s t e n t c h a n g e s w h i c h o c c u r d u r i n g epilepsy.
References Ben Ari, Y., 1985, Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy, Neuroscience 14, 375.
Dragunow, M., M.R. Peterson and H.A. Robertson, 1987, Presence of c-fos like immunoreactivity in the adult rat brain, European J. Pharmacol. 135, 113. Dragunow, M. and H.A. Robertson, 1987, Kindling stimulation induces c-fos protein(s) in granule cells of the rat dentate gyms, Nature 329, 441. Morgan, J.I. and T. Curran, 1986, Role of ion flux in the control of c-los expression, Nature 322, 552. Morgan J.I., D.R. Cohen, J.L. Hempstead and T. Curran, 1987, Mapping patterns of c-fos expression in the central nervous system after seizure, Science 237, 192.
405
Elsevier EJP 0129R
Rapid communication
Kainic acid-induced seizures increase c-los-like protein in the hippocampus Th. Popovici, G. B a r b i n a n d Y. Ben Ari * I N S E R M U. 29, H3pital de Port-Royal, 123 Bd. de Port-Royal, 75674 Paris Cedex 14, France
Received 10 May 1988, accepted 11 May 1988
The levels of the proto-oncogene c-fos in PC 12 cells increase following sustained opening of voltage-dependent calcium channels (Morgan and Curran, 1986). A similar increase in rodent brain is observed after seizure activity (Dragunow and Robertson, 1987; Morgan et al., 1987). Kainic acid, a powerful excitatory analogue of glutamate induces seizures and a brain damage syndrome involving the hippocampus and limbic regions. This syndrome has some relevance to human temporal lobe epilepsy (Ben Ari, 1985). We now report on a sequential increase in c-fos protein within the the hippocampal formation of the rat following i.p. administration of kainate. Adult male Wistar rats received kainic acid (8 m g / k g i.p.) and were killed at various time intervals. Immunoreactivity was detected as previously described, with minor modifications (Dragunow et al., 1987). The rats were perfused with 1% paraformaldehyde, their brains were immersed in 15% sucrose and cut with a cryostat. Sections (30 /~m thick) were incubated in 5% normal rabbit serum. The primary c-los antibody (C.R.B., England) was used at a dilution of 1/1600. A biotinylated donkey anti-sheep antibody (1/100) and a streptavidin-biotin peroxidase complex (1/400), both from Amersham, were applied successively before visualization with diaminobenzidine. In keeping with earlier observations (Ben Ari, 1985), a limbic motor seizure developed 1 h after parenteral kainate administration. Whereas the
* To whom all correspondenceshould be addressed.
control brains showed no immunoreactivity, a strong nuclear staining was exclusively restricted to the granule neurons of the dentate gyms, 3 h after treatment (fig. 1A). At 6 h the nuclear immunoreaction extended to the pyramidal cells and the polymorphic interneurons located in the stratum orients, stratum radiatum and hilus (fig. 1B). The labeling disappeared progressively in the same order as it had appeared (data not shown). Granule cell staining was dramatically reduced 12 h after the injection and disappeared at 24 h whereas the decline was delayed in the the pyramidal cells. The immunostaining in granule and pyramidal cells was predominant within the nucleus and very faint in the cytoplasm. Axons and dendrites as well as sections incubated without the c-fos antibody were devoid of labeling. In conclusion, kainic acid produced a seizure syndrome associated with a transient rise in the c-los protein within the hippocampus. The time course of this increase, which affected the granule neurons before the pyramidal ones, was in agreement with the results of electrographic and metabolic studies. Together, these data suggest that the area dendata is first activated by the paroxysmal discharge through the perforant pathway; the entire hippocampal formation and related structures of the limbic system are involved subsequently (Popovici et al., in preparation), c-fos protein induction as shown here may be produced either through specific neurotransmitter activation or through seizure discharges and the associated calcium influx (Morgan and Curran, 1986). Since the c-los protein may be involved in the regulation
0014-2999/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
406
Fig. 1. c-los protein immunoreactivity within the hippocampus 3 h (A) and 6 h (B) following kainate administration.
o f g e n e e x p r e s s i o n its s e q u e n t i a l a c t i v a t i o n b y k a i n a t e m a y b e a r s o m e r e l e v a n c e to t h e p e r s i s t e n t c h a n g e s w h i c h o c c u r d u r i n g epilepsy.
References Ben Ari, Y., 1985, Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy, Neuroscience 14, 375.
Dragunow, M., M.R. Peterson and H.A. Robertson, 1987, Presence of c-fos like immunoreactivity in the adult rat brain, European J. Pharmacol. 135, 113. Dragunow, M. and H.A. Robertson, 1987, Kindling stimulation induces c-fos protein(s) in granule cells of the rat dentate gyms, Nature 329, 441. Morgan, J.I. and T. Curran, 1986, Role of ion flux in the control of c-los expression, Nature 322, 552. Morgan J.I., D.R. Cohen, J.L. Hempstead and T. Curran, 1987, Mapping patterns of c-fos expression in the central nervous system after seizure, Science 237, 192.