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Highly polysialylated neural cell adhesion molecule positive cells increased and changed localization in rat hippocampus with exposure to repeated kindled seizures
International Congress Series 1252 (2003) 419 – 425
Highly polysialylated neural cell adhesion molecule positive cells increased and changed localization in rat hippocampus with exposure to repeated kindled seizures K. Sato *, M. Iwai, W.R. Zhang, H. Kamada, K. Ohta, I. Nagano, M. Shoji, K. Abe Department of Neurology, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1 Shikata-cho, 700-8558 Okayama, Japan Received 9 October 2002; accepted 31 January 2003
Abstract Highly polysialylated neural cell adhesion molecule (PSA-NCAM) is involved in migration of neural stem cells as well as neural plasticity. Immunoreactive PSA-NCAM expression was examined in rat with repeated exposure to amygdaloid kindled generalized seizures (GS). The number of PSA-NCAM-positive cells in bilateral dentate gyrus (DG) increased significantly at GS. Although total positive cell number was not significantly different between 3 times GS (3 GS) and 30 times GS (30 GS) groups, a greater number of positive cells were located in the outer granule cell layer (GCL), and the immunopositive dendrite greatly extended to the molecular layer in the 30 GS group. These observations indicate that increased migration of newly generated cells as well as plastic change of originally existed neural cells may occur in response to the recurrent GS, which may contribute to abnormal reconstruction of synaptic network in hippocampus and epileptogenisity in kindling. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Neural stem cell; Migration; Epilepsy; Kindling; Highly polysialylated neural cell adhesion molecule (PSA-NCAM)
* Corresponding author. Tel.: +81-86-235-7365; fax: +81-86-235-7368. E-mail address: [email protected] (K. Sato). 0531-5131/03 D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0531-5131(03)00088-8
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1. Background Repeated exposure of certain brain regions to a stimulus that triggers seizure activity results in progressively greater epileptiform responses, culminating in generalized seizures (GS), which is referred to as kindling phenomenon [1]. Kindling is an experimental model of temporal lobe epilepsy as well as neural plasticity [1,2]. Mesial temporal lobe epilepsy is the most common form of epilepsy in adults, and the seizure attacks are often intractable and accompanied by selective vulnerability in the hippocampus [3]. In kindling, the recurrent GS causes neural loss and reorganization of hippocampal circuitry, as in human epilepsy [4 –6]. These degenerative and regenerative structural changes have been demonstrated with repetition of GS, which may lead to the lowered seizure threshold culminating to spontaneous seizures [2,7,8]. Neural stem cells exist even in the adult mammalian brain especially in dentate gyrus (DG) of hippocampus [9 –12]. Neural stem cells possess two potentials such as selfrenewal and multidirectional differentiation [9 –11]. Neurogenesis is regulated by internal molecular programs and external epigenetic environments during normal development, and is also regulated under pathological conditions such as ischemia and epileptic seizures [13 – 20]. Proliferation (step 1) of neural stem cells is stimulated by prolonged seizure discharges in DG. These newborn cells migrate (step 2) and differentiate into certain neural cell species (step 3), and could make aberrant synaptic connections and neural networks [17 – 19]. Highly polysialylated neural cell adhesion molecule (PSA-NCAM) is expressed specifically at step 2 (migration) of neural stem cells development, and plays an important role for neurite outgrowth involving neural stem cells migration, neuronal circuit formation, and cell – cell interaction [21 – 23]. PSA-NCAM is involved in neural migration of neural stem cells development and also in synaptic remodeling. The present study was attempted to investigate the changes of PSA-NCAM expression in DG, and to elucidate the roles of migration of neural stem cells and synaptic plasticity on neural remodeling of hippocampus with the recurrent GS in kindling.
2. Methods Tripolar electrodes were stereotaxically implanted into the left basolateral amygdala of male Sprague – Dawley rats weighing 300 –350 g under pentobarbital (50 mg/kg) anesthesia. After 14 days of recovery period, electrical stimuli (a 2-s train of 50 Hz, 1 ms rectangular waves at a current intensity of 200 AA) were given once a day to the rats. The animals developed a stage 5 GS [24] by electrical stimuli, and they were followed by additional daily stimuli to produce total 3 or 30 consecutive GS (n = 5 in each, 3 or 30 GS groups, respectively). Age-matched animals with implanted electrodes were used without stimulation as controls (n = 5). Electroencephalography was measured between the remaining poles of the tripolar electrode and the skull screw electrode, and recorded during all tests. The rat brains were rapidly removed under deep anesthesia at 8 h after the last stimulation in 3 and 30 GS groups. The Animal Committee of the Graduate School of Medicine and Dentistry, Okayama University approved this experiment.
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For the immunohistochemical staining, coronal sections at the dorsal hippocampal levels (approximately 3.3-mm posterior to bregma) were cut on a cryostat at 20 jC with a 10 Am thickness and collected on glass slides. For detection of PSA-NCAM, the sections were fixed in acetone and were first incubated in 0.3% H2O2 for 30 min and blocked with 10% normal
Fig. 1. Representative staining for immunoreactive PSA-NCAM in DG on the side of stimulation of sham control (a, d), and with 3 (b, e) and 30 generalized (c, f) seizures (3 and 30 GS, respectively). Note the stronger staining and increase of PSA-NCAM-positive cells in GCL at 3 (b, e) and 30 GS (c, f). PSA-NCAM-positive cells located in the deepest portion of GCL are shown with filled arrowheads in panels a – f, and that in outer GCL is shown with open arrowhead in panel e and f. PSA-NCAM-positive dendrites were shown with arrows in panels d – f. Scale bar: 0.5 (a – c) and 0.05 mm (d – f).
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goat serum for 30 min. Sections were then incubated overnight at 4 jC with anti-PSANCAM mouse IgM monoclonal antibody (diluted 1:2000, Seki and Arai, 1993) [22]. Then, the sections were incubated with biotinylated goat anti-mouse IgM (diluted 1:200; Vector laboratories, CA) for 1 h at room temperature. Then, all of the sections were placed in horseradish peroxidase/streptoavidin/biotin complex solution (Vectastain ABC Kit, Vector laboratories, CA) for 30 min, and were incubated for 1.5 min in a peroxidase reaction solution (0.02% diaminobenzidine, 0.02% H2O2). A set of sections was stained in a similar way without the primary antibody. The staining was examined with light microscope, and positive cells in DG were counted. The differences between them were statistically analyzed by one-way analysis of variance (ANOVA) followed by Fisher’s Protected Least Significant Difference (Fisher’s PLSD).
3. Results As for kindling characteristics, there were no differences in the mean durations of the electroencephalographical afterdischarge at the first electrical stimulation in groups with 3 and 30 GS. In rats with 3 and 30 GS, the mean duration of afterdischarge following the last
Fig. 2. Number of PSA-NCAM positive neurons in DG. Note the increased number of PSA-NCAM-positive cells both at 3 and 30 GS compared with the sham control. Mean F S.D., *: p < 0.05, **: p < 0.01, compared with control.
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electrical stimulation was 102.0 F 26.4 and 78.4 F 23.4 s, respectively (n = 5 in each, mean F S.D.). The number of electrical stimuli needed to develop first GS (stage 5) was 7.6 F 1.8 and 8.6 F 2.5 times in groups with 3 and 30 GS, respectively; and thus the 3 and 30 GS animals were sacrificed at 9.6 F 1.8 and 37.6 F 2.5 days after initial electrical stimulation, respectively. PSA-NCAM-positive cells were located in the deepest portion of granule cell layer (GCL) of the ventral part of DG in sham control brains (Fig. 1a,d, filled arrowheads). In GS rats, granule cells in both ventral (Fig. 1b,c, lower arrowheads) and dorsal (Fig. 1b,c, upper arrowheads) parts of DG became positive for PSA-NCAM. The number of PSA-NCAM positive cells significantly increased bilaterally two times in the 3 GS group (Figs. 1b,e and 2). The further increases were observed in the 30 GS group (Figs. 1c,f and 2) with no significant differences between these 3 and 30 GS groups. Although PSA-NCAM-positive cells were still mainly located in the deepest part of GCL, some positive cells were observed in the outer GCL in 3 GS animals (Fig. 1e, open arrowhead). In animals with 30 GS, the greater numbers of positive cells located in the outer GCL (Fig. 1f, open arrowheads) compared with those in 3 GS group (Fig. 1e, open arrowhead). Although PSA-NCAMpositive dendrite was minimally found in the control brain (Fig. 1d, arrow), it extended slightly in animals with 3 GS (Fig. 1e, arrows), and greatly toward the molecular layer with 30 GS (Fig. 1f, arrows).
4. Conclusions The two key findings of this study are that the numbers of PSA-NCAM-positive cells increased after GS while no further increase was observed in the 30 GS group, and that repetition of GS markedly enhances the increase of PSA-NCAM-positive cells in the outer GCL and the extension of positive dendrite. PSA-NCAM is involved in the neural migration of neural stem-cell development and also in synaptic plasticity [21,23]. Thus, the expression of PSA-NCAM should be considered from these two points of view. The first finding indicates that in the setting of kindling-induced GS, neural stem cell migration and synaptic remodeling are stimulated in DG, and may contribute to the reorganization of neural network in the hippocampus. No further increase in the number of PSA-NCAMpositive cells at 30 GS shows that the repeated GS does not lead to increment of cell number involved in the neural stem cell migration and synaptic remodeling in DG. Although the repeated GS produces cell death in DG, electrical stimuli do not facilitate neural stem cell division after GS was established [17]. It is possible that repeated GS increase the number of neurons involved in synaptic plasticity, rather than that of neural stem cells presenting migration following proliferation. Neural stem cell migration detected by the increase of PSA-NCAM-positive cells in DG was enhanced in transient brain ischemia as well as in human temporal lobe epilepsy where hippocampal neurons die [16,25,26]. The repetitive GS may not be strong enough to increase the number of neural stem cells migrating in DG where hippocampal neuronal damage is subtle. As for the second finding, the recurrent GS is considered to enhance the migration of neural stem cells from the deepest part of GCL to the outer GCL and the extension of PSANCAM-positive dendrites. It is also possible that neurons in outer GCL are involved into
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plastic change. The enhanced extension of PSA-NCAM-positive dendrites at 30 GS might represent a synaptic remodeling of both the migrating neural stem cells and granule cells existed originally. These synaptic remodeling and neural stem cell migration with repeated GS may contribute to the reorganization of neural network with abnormal synapses in hippocampus. These changes are presumed to lead to the lowered seizure threshold culminating to spontaneous seizures in epileptic brain. Although the biological significance of the enhancement of neural stem cells is not known, it occurs specifically in DG and may contribute to the optimal functioning of this region [10,13,14]. It is interesting to note that repeated GS induces significant structural changes in DG, which is shown as cell migration and synaptic plasticity. Further studies are needed to investigate the differentiation of neural stem cells and the anatomical, physiological, and chemical functions in kindling. The molecular basis underlying the facts presented in this study will offer the key to understanding the mechanisms of acquisition of epileptogenisity.
Acknowledgements We thank Dr. T. Seki for constructive criticism and technical advice for the manuscript. This work was partly supported by Grant-in-Aid for Scientific Research (B) 12470141 and (Hoga) 12877211 and National Project on Protein Structural and Functional Analyses from the Ministry of Education, Science, Culture and Sports of Japan, by grants (Itoyama Y., Kimura I., and Kuzuhara S.) from the Ministry of Health and Welfare of Japan, and by grant from Japan Epilepsy Foundation.
References [1] G.V.D. Goddard, C. McIntyre, C.K. Leech, A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol. 25 (1969) 295 – 330. [2] I. Mody, Synaptic plasticity in kindling, Adv. Neurol. 79 (1999) 631 – 643. [3] I. Blumcke, H. Beck, A.A. Lie, O.D. Wiestler, Molecular neuropathology of human mesial temporal lobe epilepsy, Epilepsy Res. 36 (1999) 205 – 223. [4] J. Bengzon, Z. Kokaia, E. Elmer, A. Nanobashvili, M. Kokaia, O. Lindvall, Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 10432 – 10437. [5] T. Sutula, X.X. He, J. Cavazos, G. Scott, Synaptic reorganization in the hippocampus induced by abnormal functional activity, Science 239 (1988) 1147 – 1150. [6] T. Sutula, G. Cascino, J. Cavazos, I. Parada, L. Ramirez, Mossy fiber synaptic reorganization in the epileptic human temporal lobe, Ann. Neurol. 26 (1989) 321 – 330. [7] M. Michalakis, D. Holsinger, C. Ikeda-Douglas, S. Cammisuli, J. Ferbinteanu, C. DeSouza, S. DeSouza, J. Fecteau, R.J. Racine, N.W. Milgram, Development of spontaneous seizures over extended electrical kindling. I. Electrographic, behavioral, and transfer kindling correlates, Brain Res. 793 (1998) 197 – 211. [8] N.W. Milgram, M. Michael, S. Cammisuli, E. Head, J. Ferbinteanu, C. Reid, M.P. Murphy, R.J. Racine, Development of spontaneous seizures over extended electrical kindling. II. Persistence of dentate inhibitory suppression, Brain Res. 670 (1995) 112 – 120. [9] P.S. Eriksson, E. Perfilieva, T. Bjork-Eriksson, A.M. Alborn, C. Nordborg, D.A. Peterson, F.H. Gage, Neurogenesis in the adult human hippocampus, Nat. Med. 4 (1998) 1313 – 1317.
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[10] F.H. Gage, Mammalian neural stem cells, Science 287 (2000) 1433 – 1438. [11] E. Gould, A.J. Reeves, M.S. Graziano, C.G. Gross, Neurogenesis in the neocortex of adult primates, Science 286 (1999) 548 – 552. [12] C.B. Johansson, S. Momma, D.L. Clarke, M. Risling, U. Lendahl, J. Frisen, Identification of a neural stem cell in the adult mammalian central nervous system, Cell 96 (1999) 25 – 34. [13] K. Abe, Therapeutic potential of neurotrophic factors and neural stem cells against ischemic brain injury, J. Cereb. Blood Flow Metab. 20 (2000) 1393 – 1408. [14] R. Bernabeu, F.R. Sharp, NMDA and AMPA/kainate glutamate receptors modulate dentate neurogenesis and CA3 synapsin I in normal and ischemic hippocampus, J. Cereb. Blood Flow Metab. 20 (2000) 1669 – 1680. [15] E.P. Gray, L.E. Sundstrom, Kainic acid increases the proliferation of granule cell progenitors in the dentate gyrus of the adult rat, Brain Res. 790 (1998) 52 – 59. [16] M. Iwai, T. Hayashi, W.R. Zhang, K. Sato, Y. Manabe, K. Abe, Induction of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in postischemic gerbil hippocampus mainly dissociated with neural stem cell proliferation, Brain Res. 902 (2001) 288 – 293. [17] E. Nakagawa, Y. Aimi, O. Yasuhara, I. Tooyama, M. Shimada, P.L. McGeer, H. Kimura, Enhancement of progenitor cell division in the dentate gyrus triggered by initial limbic seizures in rat models of epilepsy, Epilepsia 41 (2000) 10 – 18. [18] J.M. Parent, T.W. Yu, R.T. Leibowitz, D.H. Geschwind, R.S. Sloviter, D.H. Lowenstein, Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult hippocampus, J. Neurosci. 17 (1997) 3727 – 3738. [19] H.E. Scharfman, J.H. Goodman, A.L. Sollas, Granule-like neurons at the hilar/CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: functional implications of seizure-induced neurogenesis, J. Neurosci. 20 (2000) 6144 – 6158. [20] B.W. Scott, S. Wang, W.M. Burnham, U. De Boni, J.M. Wojtowicz, Kindling-induced neurogenesis in the dentate gyrus of the rat, Neurosci. Lett. 248 (1998) 73 – 76. [21] U. Rutishauser, L. Landmesser, Polysialic acid in the vertebrate nervous system: a promoter of plasticity in cell – cell interactions, Trends Neurosci. 19 (1996) 422 – 427. [22] T. Seki, Y. Arai, Highly polysialylated neural cell adhesion molecule (NCAM-H) is expressed by newly generated granule cells in the dentate gyrus of the adult rat, J. Neurosci. 13 (1993) 2351 – 2358. [23] T. Seki, Y. Arai, Distribution and possible roles of the highly polysialylated neural cell adhesion molecule (NCAM-H) in the developing and adult central nervous system, Neurosci. Res. 17 (1993) 265 – 290. [24] R.J. Racine, Modification of seizure activity by electrical stimulation: II, Motor seizure, Electroencephalogr. Clin. Neurophysiol. 32 (1972) 281 – 294. [25] G.B. Fox, C. Kjoller, K.J. Murphy, C.M. Regan, The modulations of NCAM polysialylation state that follow transient global ischemia are brief on neurons but enduring on glia, J. Neuropathol. Exp. Neurol. 60 (2001) 132 – 140. [26] M. Mikkonen, H. Soininen, R. Kalvianen, T. Tapiola, A. Ylinen, M. Vapalahti, L. Paljarvi, A. Pitkanen, Remodeling of neuronal circuitries in human temporal lobe epilepsy: increased expression of highly polysialylated neural cell adhesion molecule in the hippocampus and the entorhinal cortex, Ann. Neurol. 44 (1998) 923 – 934.
Highly polysialylated neural cell adhesion molecule positive cells increased and changed localization in rat hippocampus with exposure to repeated kindled seizures K. Sato *, M. Iwai, W.R. Zhang, H. Kamada, K. Ohta, I. Nagano, M. Shoji, K. Abe Department of Neurology, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1 Shikata-cho, 700-8558 Okayama, Japan Received 9 October 2002; accepted 31 January 2003
Abstract Highly polysialylated neural cell adhesion molecule (PSA-NCAM) is involved in migration of neural stem cells as well as neural plasticity. Immunoreactive PSA-NCAM expression was examined in rat with repeated exposure to amygdaloid kindled generalized seizures (GS). The number of PSA-NCAM-positive cells in bilateral dentate gyrus (DG) increased significantly at GS. Although total positive cell number was not significantly different between 3 times GS (3 GS) and 30 times GS (30 GS) groups, a greater number of positive cells were located in the outer granule cell layer (GCL), and the immunopositive dendrite greatly extended to the molecular layer in the 30 GS group. These observations indicate that increased migration of newly generated cells as well as plastic change of originally existed neural cells may occur in response to the recurrent GS, which may contribute to abnormal reconstruction of synaptic network in hippocampus and epileptogenisity in kindling. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Neural stem cell; Migration; Epilepsy; Kindling; Highly polysialylated neural cell adhesion molecule (PSA-NCAM)
* Corresponding author. Tel.: +81-86-235-7365; fax: +81-86-235-7368. E-mail address: [email protected] (K. Sato). 0531-5131/03 D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0531-5131(03)00088-8
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1. Background Repeated exposure of certain brain regions to a stimulus that triggers seizure activity results in progressively greater epileptiform responses, culminating in generalized seizures (GS), which is referred to as kindling phenomenon [1]. Kindling is an experimental model of temporal lobe epilepsy as well as neural plasticity [1,2]. Mesial temporal lobe epilepsy is the most common form of epilepsy in adults, and the seizure attacks are often intractable and accompanied by selective vulnerability in the hippocampus [3]. In kindling, the recurrent GS causes neural loss and reorganization of hippocampal circuitry, as in human epilepsy [4 –6]. These degenerative and regenerative structural changes have been demonstrated with repetition of GS, which may lead to the lowered seizure threshold culminating to spontaneous seizures [2,7,8]. Neural stem cells exist even in the adult mammalian brain especially in dentate gyrus (DG) of hippocampus [9 –12]. Neural stem cells possess two potentials such as selfrenewal and multidirectional differentiation [9 –11]. Neurogenesis is regulated by internal molecular programs and external epigenetic environments during normal development, and is also regulated under pathological conditions such as ischemia and epileptic seizures [13 – 20]. Proliferation (step 1) of neural stem cells is stimulated by prolonged seizure discharges in DG. These newborn cells migrate (step 2) and differentiate into certain neural cell species (step 3), and could make aberrant synaptic connections and neural networks [17 – 19]. Highly polysialylated neural cell adhesion molecule (PSA-NCAM) is expressed specifically at step 2 (migration) of neural stem cells development, and plays an important role for neurite outgrowth involving neural stem cells migration, neuronal circuit formation, and cell – cell interaction [21 – 23]. PSA-NCAM is involved in neural migration of neural stem cells development and also in synaptic remodeling. The present study was attempted to investigate the changes of PSA-NCAM expression in DG, and to elucidate the roles of migration of neural stem cells and synaptic plasticity on neural remodeling of hippocampus with the recurrent GS in kindling.
2. Methods Tripolar electrodes were stereotaxically implanted into the left basolateral amygdala of male Sprague – Dawley rats weighing 300 –350 g under pentobarbital (50 mg/kg) anesthesia. After 14 days of recovery period, electrical stimuli (a 2-s train of 50 Hz, 1 ms rectangular waves at a current intensity of 200 AA) were given once a day to the rats. The animals developed a stage 5 GS [24] by electrical stimuli, and they were followed by additional daily stimuli to produce total 3 or 30 consecutive GS (n = 5 in each, 3 or 30 GS groups, respectively). Age-matched animals with implanted electrodes were used without stimulation as controls (n = 5). Electroencephalography was measured between the remaining poles of the tripolar electrode and the skull screw electrode, and recorded during all tests. The rat brains were rapidly removed under deep anesthesia at 8 h after the last stimulation in 3 and 30 GS groups. The Animal Committee of the Graduate School of Medicine and Dentistry, Okayama University approved this experiment.
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For the immunohistochemical staining, coronal sections at the dorsal hippocampal levels (approximately 3.3-mm posterior to bregma) were cut on a cryostat at 20 jC with a 10 Am thickness and collected on glass slides. For detection of PSA-NCAM, the sections were fixed in acetone and were first incubated in 0.3% H2O2 for 30 min and blocked with 10% normal
Fig. 1. Representative staining for immunoreactive PSA-NCAM in DG on the side of stimulation of sham control (a, d), and with 3 (b, e) and 30 generalized (c, f) seizures (3 and 30 GS, respectively). Note the stronger staining and increase of PSA-NCAM-positive cells in GCL at 3 (b, e) and 30 GS (c, f). PSA-NCAM-positive cells located in the deepest portion of GCL are shown with filled arrowheads in panels a – f, and that in outer GCL is shown with open arrowhead in panel e and f. PSA-NCAM-positive dendrites were shown with arrows in panels d – f. Scale bar: 0.5 (a – c) and 0.05 mm (d – f).
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goat serum for 30 min. Sections were then incubated overnight at 4 jC with anti-PSANCAM mouse IgM monoclonal antibody (diluted 1:2000, Seki and Arai, 1993) [22]. Then, the sections were incubated with biotinylated goat anti-mouse IgM (diluted 1:200; Vector laboratories, CA) for 1 h at room temperature. Then, all of the sections were placed in horseradish peroxidase/streptoavidin/biotin complex solution (Vectastain ABC Kit, Vector laboratories, CA) for 30 min, and were incubated for 1.5 min in a peroxidase reaction solution (0.02% diaminobenzidine, 0.02% H2O2). A set of sections was stained in a similar way without the primary antibody. The staining was examined with light microscope, and positive cells in DG were counted. The differences between them were statistically analyzed by one-way analysis of variance (ANOVA) followed by Fisher’s Protected Least Significant Difference (Fisher’s PLSD).
3. Results As for kindling characteristics, there were no differences in the mean durations of the electroencephalographical afterdischarge at the first electrical stimulation in groups with 3 and 30 GS. In rats with 3 and 30 GS, the mean duration of afterdischarge following the last
Fig. 2. Number of PSA-NCAM positive neurons in DG. Note the increased number of PSA-NCAM-positive cells both at 3 and 30 GS compared with the sham control. Mean F S.D., *: p < 0.05, **: p < 0.01, compared with control.
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electrical stimulation was 102.0 F 26.4 and 78.4 F 23.4 s, respectively (n = 5 in each, mean F S.D.). The number of electrical stimuli needed to develop first GS (stage 5) was 7.6 F 1.8 and 8.6 F 2.5 times in groups with 3 and 30 GS, respectively; and thus the 3 and 30 GS animals were sacrificed at 9.6 F 1.8 and 37.6 F 2.5 days after initial electrical stimulation, respectively. PSA-NCAM-positive cells were located in the deepest portion of granule cell layer (GCL) of the ventral part of DG in sham control brains (Fig. 1a,d, filled arrowheads). In GS rats, granule cells in both ventral (Fig. 1b,c, lower arrowheads) and dorsal (Fig. 1b,c, upper arrowheads) parts of DG became positive for PSA-NCAM. The number of PSA-NCAM positive cells significantly increased bilaterally two times in the 3 GS group (Figs. 1b,e and 2). The further increases were observed in the 30 GS group (Figs. 1c,f and 2) with no significant differences between these 3 and 30 GS groups. Although PSA-NCAM-positive cells were still mainly located in the deepest part of GCL, some positive cells were observed in the outer GCL in 3 GS animals (Fig. 1e, open arrowhead). In animals with 30 GS, the greater numbers of positive cells located in the outer GCL (Fig. 1f, open arrowheads) compared with those in 3 GS group (Fig. 1e, open arrowhead). Although PSA-NCAMpositive dendrite was minimally found in the control brain (Fig. 1d, arrow), it extended slightly in animals with 3 GS (Fig. 1e, arrows), and greatly toward the molecular layer with 30 GS (Fig. 1f, arrows).
4. Conclusions The two key findings of this study are that the numbers of PSA-NCAM-positive cells increased after GS while no further increase was observed in the 30 GS group, and that repetition of GS markedly enhances the increase of PSA-NCAM-positive cells in the outer GCL and the extension of positive dendrite. PSA-NCAM is involved in the neural migration of neural stem-cell development and also in synaptic plasticity [21,23]. Thus, the expression of PSA-NCAM should be considered from these two points of view. The first finding indicates that in the setting of kindling-induced GS, neural stem cell migration and synaptic remodeling are stimulated in DG, and may contribute to the reorganization of neural network in the hippocampus. No further increase in the number of PSA-NCAMpositive cells at 30 GS shows that the repeated GS does not lead to increment of cell number involved in the neural stem cell migration and synaptic remodeling in DG. Although the repeated GS produces cell death in DG, electrical stimuli do not facilitate neural stem cell division after GS was established [17]. It is possible that repeated GS increase the number of neurons involved in synaptic plasticity, rather than that of neural stem cells presenting migration following proliferation. Neural stem cell migration detected by the increase of PSA-NCAM-positive cells in DG was enhanced in transient brain ischemia as well as in human temporal lobe epilepsy where hippocampal neurons die [16,25,26]. The repetitive GS may not be strong enough to increase the number of neural stem cells migrating in DG where hippocampal neuronal damage is subtle. As for the second finding, the recurrent GS is considered to enhance the migration of neural stem cells from the deepest part of GCL to the outer GCL and the extension of PSANCAM-positive dendrites. It is also possible that neurons in outer GCL are involved into
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plastic change. The enhanced extension of PSA-NCAM-positive dendrites at 30 GS might represent a synaptic remodeling of both the migrating neural stem cells and granule cells existed originally. These synaptic remodeling and neural stem cell migration with repeated GS may contribute to the reorganization of neural network with abnormal synapses in hippocampus. These changes are presumed to lead to the lowered seizure threshold culminating to spontaneous seizures in epileptic brain. Although the biological significance of the enhancement of neural stem cells is not known, it occurs specifically in DG and may contribute to the optimal functioning of this region [10,13,14]. It is interesting to note that repeated GS induces significant structural changes in DG, which is shown as cell migration and synaptic plasticity. Further studies are needed to investigate the differentiation of neural stem cells and the anatomical, physiological, and chemical functions in kindling. The molecular basis underlying the facts presented in this study will offer the key to understanding the mechanisms of acquisition of epileptogenisity.
Acknowledgements We thank Dr. T. Seki for constructive criticism and technical advice for the manuscript. This work was partly supported by Grant-in-Aid for Scientific Research (B) 12470141 and (Hoga) 12877211 and National Project on Protein Structural and Functional Analyses from the Ministry of Education, Science, Culture and Sports of Japan, by grants (Itoyama Y., Kimura I., and Kuzuhara S.) from the Ministry of Health and Welfare of Japan, and by grant from Japan Epilepsy Foundation.
References [1] G.V.D. Goddard, C. McIntyre, C.K. Leech, A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol. 25 (1969) 295 – 330. [2] I. Mody, Synaptic plasticity in kindling, Adv. Neurol. 79 (1999) 631 – 643. [3] I. Blumcke, H. Beck, A.A. Lie, O.D. Wiestler, Molecular neuropathology of human mesial temporal lobe epilepsy, Epilepsy Res. 36 (1999) 205 – 223. [4] J. Bengzon, Z. Kokaia, E. Elmer, A. Nanobashvili, M. Kokaia, O. Lindvall, Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 10432 – 10437. [5] T. Sutula, X.X. He, J. Cavazos, G. Scott, Synaptic reorganization in the hippocampus induced by abnormal functional activity, Science 239 (1988) 1147 – 1150. [6] T. Sutula, G. Cascino, J. Cavazos, I. Parada, L. Ramirez, Mossy fiber synaptic reorganization in the epileptic human temporal lobe, Ann. Neurol. 26 (1989) 321 – 330. [7] M. Michalakis, D. Holsinger, C. Ikeda-Douglas, S. Cammisuli, J. Ferbinteanu, C. DeSouza, S. DeSouza, J. Fecteau, R.J. Racine, N.W. Milgram, Development of spontaneous seizures over extended electrical kindling. I. Electrographic, behavioral, and transfer kindling correlates, Brain Res. 793 (1998) 197 – 211. [8] N.W. Milgram, M. Michael, S. Cammisuli, E. Head, J. Ferbinteanu, C. Reid, M.P. Murphy, R.J. Racine, Development of spontaneous seizures over extended electrical kindling. II. Persistence of dentate inhibitory suppression, Brain Res. 670 (1995) 112 – 120. [9] P.S. Eriksson, E. Perfilieva, T. Bjork-Eriksson, A.M. Alborn, C. Nordborg, D.A. Peterson, F.H. Gage, Neurogenesis in the adult human hippocampus, Nat. Med. 4 (1998) 1313 – 1317.
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