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Changes of localization of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in rat hippocampus with exposure to repeated kindled seizures
Brain Research 946 (2002) 323–327 www.elsevier.com / locate / bres
Short communication
Changes of localization of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in rat hippocampus with exposure to repeated kindled seizures Keiko Sato*, Masanori Iwai, Isao Nagano, Mikio Shoji, Koji Abe Department of Neurology, Graduate School of Medicine and Dentistry, Okayama University, 2 -5 -1 Shikata-cho, Okayama 700 -8558, Japan Accepted 6 February 2002
Abstract Highly polysialylated neural cell adhesion molecules (PSA-NCAMs) are 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 was 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. 2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Cell differentiation and migration Keywords: Epilepsy; Kindling; Migration
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 [8]. Kindling is an experimental model of temporal lobe epilepsy as well as neural plasticity [8,16]. 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 [4]. In kindling, the recurrent GS causes the neural loss and the reorganization of hippocampal circuitry, as in human epilepsy [2,25,26]. These degenerative and re-
Abbreviations: BrdU, bromodeoxyuridine; GCL, granule cell layer; DG, dentate gyrus; GS, generalized seizures; PSA-NCAM, highly polysialylated neural cell adhesion molecule; SVZ, subventricular zone *Corresponding author. Tel.: 181-86-235-7365; fax: 181-86-2357368. E-mail address: [email protected] (K. Sato).
generative structural changes have been demonstrated with repetition of GS, which may lead to the lowered seizure threshold culminating to spontaneous seizures [13,15,16]. Neural stem cells exist even in the adult mammalian brain especially in dentate gyrus (DG) of the hippocampus [5,7,9,12]. Neural stem cells possess two potentials such as self-renewal and multidirectional differentiation [5,7,9]. 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 [1,3,10,11,17,18,21,22]. 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 [17,18,21]. Highly polysialylated neural cell adhesion molecule (PSA-NCAM) is expressed specifically at step 2 (migra-
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02616-1
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K. Sato et al. / Brain Research 946 (2002) 323 – 327
tion) of neural stem cells development, and plays an important role for neurite outgrowth involving in neural stem cells migration, neuronal circuit formation, and cell– cell interaction [20,23,24]. PSA-NCAM is involved in neural migration of neural stem cells development and also in synaptic remodeling. The present study 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. 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 mA) were given once a day to the rats. The animals developed a stage 5 GS [19] by electrical stimuli, and they were followed by additional daily stimuli to produce total 3 or 30 consecutive GS (n55 in each, 3 GS or 30 GS groups, respectively). Age-matched animals with implanted electrodes were used without stimulation as controls (n55). 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 GS and 30 GS groups. The Animal Committee of the Graduate School of Medicine and Dentistry of Okayama University approved this experiment. For the immunohistochemical staining, coronal sections at the dorsal hippocampal levels (approximately 3.3 mm posterior to bregma) were cut on a cryostat at 220 8C with a 10-mm thickness, and collected on glass slides. For detection of PSA-NCAM, the sections were fixed in acetone, and were first incubated in 0.3% H 2 O 2 for 30 min and blocked with 10% normal goat serum for 30 min. Sections were then incubated overnight at 4 8C with antiPSA-NCAM mouse IgM monoclonal antibody (diluted 1:2000, Seki and Arai [23]). Then, the sections were incubated with biotinylated goat anti-mouse IgM (diluted 1:200; Vector Labs., CA, USA) for 1 h at room temperature. Then, all of the sections were placed in horseradish peroxidase–streptoavidin–biotin complex solution (Vectastain ABC kit, Vector Labs.) for 30 min, and were incubated for 1.5 min in a peroxidase reaction solution (0.02% diaminobenzidine, 0.02% H 2 O 2 ). A set of sections was stained in a similar way without the primary antibody. The staining was examined under a light microscope, and positive cells in DG were counted. The differences between them were statistically analyzed by one-way ANOVA (analysis of variance) followed by Fisher’s protected least significant difference (Fisher’s PLSD). As for kindling characteristics, there were no differences in the mean durations of the electroencephalographical afterdischarge at the first electrical stimulation in the groups with 3 GS and 30 GS. In the rats with 3 GS and 30
GS, the mean duration of afterdischarge following the last electrical stimulation was 102.0626.4 and 78.4623.4 s, respectively (n55 in each, mean6S.D.). The number of electrical stimuli needed to develop first GS (stage 5) was 7.661.8 and 8.662.5 times in the groups with 3 GS and 30 GS, respectively, and thus the 3 GS and 30 GS animals were sacrificed at 9.661.8 and 37.662.5 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 and d, filled arrowheads). In GS rats, granule cells in both ventral (Fig. 1b and c, lower arrowheads) and dorsal (Fig. 1b and c, upper arrowheads) parts of DG became positive for PSANCAM. The number of PSA-NCAM positive cells significantly increased bilaterally two times in the 3 GS group (Fig. 1b and e, Fig. 2). The further increases were observed in 30 GS group (Fig. 1c and f, Fig. 2) with no significant differences between these 3 GS and 30 GS groups. Although the PSA-NCAM positive cells were still mainly located in the deepest part of GCL, some positive cells were observed in 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-NCAM positive 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). 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 the 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 neural migration of neural stem cells development and also in synaptic plasticity [20,24]. 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 PSANCAM positive cells at 30 GS shows that the repeated GS do not lead to increment of cell number involved in the neural stem cells migration and synaptic remodeling in DG. Although the repeated GS produces cell death in DG, electrical stimuli do not facilitate neural stem cells division after GS were established [17]. It is possible that the repeated GS increase the number of neurons involved in synaptic plasticity, rather than that of neural stem cells presenting migration following proliferation. Neural stem cells 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
K. Sato et al. / Brain Research 946 (2002) 323 – 327
325
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 GS and 30 GS, respectively). Note stronger staining and increase of PSA-NCAM positive cells in GCL at 3 GS (b,e) and 30 GS (c,f). PSA-NCAM positive cells located in the deepest portion of GCL are shown with filled arrowheads in (a–f), and that in outer GCL is shown with open arrowhead in (e) and (f). PSA-NCAM positive dendrites are shown with arrows in (d–f). Scale bars: 0.5 mm (a–c), 0.05 mm (d–f).
hippocampal neurons die [6,11,14]. 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 are considered to enhance the migration of neural stem cells from the deepest part of GCL to the outer GCL and the extension of PSA-NCAM positive dendrites. It is also possible that the neurons in outer GCL are involved in 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. The synaptic remodeling and neural stem cells
migration with repeated GS may contribute to the reorganization of neural network with abnormal synapses in the 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 [1,3,7]. It is interesting to note that repeated GS induce significant structural changes in DG, which are shown as cell migration and synaptic plasticity. Further studies are needed to investigate the differentiation of neural stem cells and the anatomical, physiological and
326
K. Sato et al. / Brain Research 946 (2002) 323 – 327
Fig. 2. Number of PSA-NCAM positive neurons in DG. Note increased number of PSA-NCAM positive cells both at 3 GS and 30 GS, compared with the sham control. Mean6S.D., *, P,0.05, **, P,0.01, compared with control.
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 from the Ministry of Education, Science, Culture and Sports of Japan, by grants (Tashiro K, Itoyama Y and Tsuji S) and Comprehensive Research on Aging and Health (H11-Choju-010, No.207, Koizumi A) from the Ministry of Health and Welfare of Japan, and by Japan Epilepsy Research Foundation.
References [1] K. Abe, Therapeutic potential of neurotrophic factors and neural stem cells against ischemic brain injury, J. Cereb. Blood Flow Metab. 20 (2000) 1393–1408.
[2] 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. USA 94 (1997) 10432–10437. [3] 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. [4] I. Blumcke, H. Beck, A.A. Lie, O.D. Wiestler, Molecular neuropathology of human mesial temporal lobe epilepsy, Epilepsy Res. 36 (1999) 205–223. [5] 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. [6] 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. [7] F.H. Gage, Mammalian neural stem cells, Science 287 (2000) 1433–1438. [8] 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. [9] E. Gould, A.J. Reeves, M.S. Graziano, C.G. Gross, Neurogenesis in the neocortex of adult primates, Science 286 (1999) 548–552. [10] W.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. [11] 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. [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] 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. [14] 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. [15] 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. [16] I. Mody, Synaptic plasticity in kindling, Adv. Neurol. 79 (1999) 631–643. [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] R.J. Racine, Modification of seizure activity by electrical stimulation: II. Motor seizure, Electroenceph. Clin. Neurophysiol. 32 (1972) 281–294. [20] 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. [21] H.E. Scharfman, J.H. Goodman, A.L. Sollas, Granule-like neurons
K. Sato et al. / Brain Research 946 (2002) 323 – 327 at the hilar / CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: functional implications of seizureinduced neurogenesis, J. Neurosci. 20 (2000) 6144–6158. [22] 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. [23] 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. [24] T. Seki, Y. Arai, Distribution and possible roles of the highly
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Short communication
Changes of localization of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in rat hippocampus with exposure to repeated kindled seizures Keiko Sato*, Masanori Iwai, Isao Nagano, Mikio Shoji, Koji Abe Department of Neurology, Graduate School of Medicine and Dentistry, Okayama University, 2 -5 -1 Shikata-cho, Okayama 700 -8558, Japan Accepted 6 February 2002
Abstract Highly polysialylated neural cell adhesion molecules (PSA-NCAMs) are 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 was 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. 2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Cell differentiation and migration Keywords: Epilepsy; Kindling; Migration
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 [8]. Kindling is an experimental model of temporal lobe epilepsy as well as neural plasticity [8,16]. 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 [4]. In kindling, the recurrent GS causes the neural loss and the reorganization of hippocampal circuitry, as in human epilepsy [2,25,26]. These degenerative and re-
Abbreviations: BrdU, bromodeoxyuridine; GCL, granule cell layer; DG, dentate gyrus; GS, generalized seizures; PSA-NCAM, highly polysialylated neural cell adhesion molecule; SVZ, subventricular zone *Corresponding author. Tel.: 181-86-235-7365; fax: 181-86-2357368. E-mail address: [email protected] (K. Sato).
generative structural changes have been demonstrated with repetition of GS, which may lead to the lowered seizure threshold culminating to spontaneous seizures [13,15,16]. Neural stem cells exist even in the adult mammalian brain especially in dentate gyrus (DG) of the hippocampus [5,7,9,12]. Neural stem cells possess two potentials such as self-renewal and multidirectional differentiation [5,7,9]. 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 [1,3,10,11,17,18,21,22]. 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 [17,18,21]. Highly polysialylated neural cell adhesion molecule (PSA-NCAM) is expressed specifically at step 2 (migra-
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02616-1
324
K. Sato et al. / Brain Research 946 (2002) 323 – 327
tion) of neural stem cells development, and plays an important role for neurite outgrowth involving in neural stem cells migration, neuronal circuit formation, and cell– cell interaction [20,23,24]. PSA-NCAM is involved in neural migration of neural stem cells development and also in synaptic remodeling. The present study 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. 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 mA) were given once a day to the rats. The animals developed a stage 5 GS [19] by electrical stimuli, and they were followed by additional daily stimuli to produce total 3 or 30 consecutive GS (n55 in each, 3 GS or 30 GS groups, respectively). Age-matched animals with implanted electrodes were used without stimulation as controls (n55). 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 GS and 30 GS groups. The Animal Committee of the Graduate School of Medicine and Dentistry of Okayama University approved this experiment. For the immunohistochemical staining, coronal sections at the dorsal hippocampal levels (approximately 3.3 mm posterior to bregma) were cut on a cryostat at 220 8C with a 10-mm thickness, and collected on glass slides. For detection of PSA-NCAM, the sections were fixed in acetone, and were first incubated in 0.3% H 2 O 2 for 30 min and blocked with 10% normal goat serum for 30 min. Sections were then incubated overnight at 4 8C with antiPSA-NCAM mouse IgM monoclonal antibody (diluted 1:2000, Seki and Arai [23]). Then, the sections were incubated with biotinylated goat anti-mouse IgM (diluted 1:200; Vector Labs., CA, USA) for 1 h at room temperature. Then, all of the sections were placed in horseradish peroxidase–streptoavidin–biotin complex solution (Vectastain ABC kit, Vector Labs.) for 30 min, and were incubated for 1.5 min in a peroxidase reaction solution (0.02% diaminobenzidine, 0.02% H 2 O 2 ). A set of sections was stained in a similar way without the primary antibody. The staining was examined under a light microscope, and positive cells in DG were counted. The differences between them were statistically analyzed by one-way ANOVA (analysis of variance) followed by Fisher’s protected least significant difference (Fisher’s PLSD). As for kindling characteristics, there were no differences in the mean durations of the electroencephalographical afterdischarge at the first electrical stimulation in the groups with 3 GS and 30 GS. In the rats with 3 GS and 30
GS, the mean duration of afterdischarge following the last electrical stimulation was 102.0626.4 and 78.4623.4 s, respectively (n55 in each, mean6S.D.). The number of electrical stimuli needed to develop first GS (stage 5) was 7.661.8 and 8.662.5 times in the groups with 3 GS and 30 GS, respectively, and thus the 3 GS and 30 GS animals were sacrificed at 9.661.8 and 37.662.5 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 and d, filled arrowheads). In GS rats, granule cells in both ventral (Fig. 1b and c, lower arrowheads) and dorsal (Fig. 1b and c, upper arrowheads) parts of DG became positive for PSANCAM. The number of PSA-NCAM positive cells significantly increased bilaterally two times in the 3 GS group (Fig. 1b and e, Fig. 2). The further increases were observed in 30 GS group (Fig. 1c and f, Fig. 2) with no significant differences between these 3 GS and 30 GS groups. Although the PSA-NCAM positive cells were still mainly located in the deepest part of GCL, some positive cells were observed in 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-NCAM positive 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). 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 the 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 neural migration of neural stem cells development and also in synaptic plasticity [20,24]. 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 PSANCAM positive cells at 30 GS shows that the repeated GS do not lead to increment of cell number involved in the neural stem cells migration and synaptic remodeling in DG. Although the repeated GS produces cell death in DG, electrical stimuli do not facilitate neural stem cells division after GS were established [17]. It is possible that the repeated GS increase the number of neurons involved in synaptic plasticity, rather than that of neural stem cells presenting migration following proliferation. Neural stem cells 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
K. Sato et al. / Brain Research 946 (2002) 323 – 327
325
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 GS and 30 GS, respectively). Note stronger staining and increase of PSA-NCAM positive cells in GCL at 3 GS (b,e) and 30 GS (c,f). PSA-NCAM positive cells located in the deepest portion of GCL are shown with filled arrowheads in (a–f), and that in outer GCL is shown with open arrowhead in (e) and (f). PSA-NCAM positive dendrites are shown with arrows in (d–f). Scale bars: 0.5 mm (a–c), 0.05 mm (d–f).
hippocampal neurons die [6,11,14]. 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 are considered to enhance the migration of neural stem cells from the deepest part of GCL to the outer GCL and the extension of PSA-NCAM positive dendrites. It is also possible that the neurons in outer GCL are involved in 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. The synaptic remodeling and neural stem cells
migration with repeated GS may contribute to the reorganization of neural network with abnormal synapses in the 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 [1,3,7]. It is interesting to note that repeated GS induce significant structural changes in DG, which are shown as cell migration and synaptic plasticity. Further studies are needed to investigate the differentiation of neural stem cells and the anatomical, physiological and
326
K. Sato et al. / Brain Research 946 (2002) 323 – 327
Fig. 2. Number of PSA-NCAM positive neurons in DG. Note increased number of PSA-NCAM positive cells both at 3 GS and 30 GS, compared with the sham control. Mean6S.D., *, P,0.05, **, P,0.01, compared with control.
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 from the Ministry of Education, Science, Culture and Sports of Japan, by grants (Tashiro K, Itoyama Y and Tsuji S) and Comprehensive Research on Aging and Health (H11-Choju-010, No.207, Koizumi A) from the Ministry of Health and Welfare of Japan, and by Japan Epilepsy Research Foundation.
References [1] K. Abe, Therapeutic potential of neurotrophic factors and neural stem cells against ischemic brain injury, J. Cereb. Blood Flow Metab. 20 (2000) 1393–1408.
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