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Stage and region dependent expression of a radial glial marker in commissural fibers in kindled mice
Epilepsy Research 67 (2005) 61–72
Stage and region dependent expression of a radial glial marker in commissural fibers in kindled mice Shinji Tanaka a,b , Osamu Miyamoto b , Najma A. Janjua c , Tetsuji Miyazaki b , Fumio Takahashi d , Ryoji Konishi a , Toshifumi Itano b,∗ b
a Teikoku Seiyaku Co. Ltd., 567 Sanbonmatsu, Higashikagawa, Kagawa 769-2695, Japan Department of Neurobiology, Kagawa University, Faculty of Medicine, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan c Department of General Education, Okayama University, 2-1-1 Tsushima Naka, Okayama 700-8530 Japan d Department of Food and Nutrition, Sanyo Gakuen College, Hirai, Okayama 703-8501, Japan
Received 11 May 2005; received in revised form 23 July 2005; accepted 18 August 2005 Available online 3 October 2005
Abstract Amygdala kindling is regarded as a model of temporal lobe epilepsy in humans because of many points of similarity. In amygdala kindling, bilateralization of epileptic seizures follows from the accumulation of stimulation and commissural fibers may play a role in this process. However, new progenies of cells following amygdala kindling have not been reported and the precise nature of how bilateralization occurs is not clear. In the present study, we aim to clarify the emergence of radial glia during the progress of amygdala kindling in mouse brain. For this purpose, immunohistochemical staining for 3CB2, which is a specific marker of radial glia, was employed. Immunoreactivity for 3CB2 was observed in the forceps minor, radiation of trunk and forceps major regions at Clonus 3 and more strongly at Clonus 5. In the forceps major, the cingulate gyrus showed immunopositive staining at Clonus 3, but the corpus callosum and alveus hippocampi showed staining only at Clonus 5. In the fimbria hippocampus, the anterior commissure posterior showed staining at Clonus 5. However, the anterior commissure anterior was not stained at the stage progressed to Clonus 5. These findings indicate stage and region dependent expression of progenitor cells in commissural fibers and suggest that these changes may accompany the formation of new circuits in seizure progression during amygdala kindling. © 2005 Elsevier B.V. All rights reserved. Keywords: Kindling; Radial glia; 3CB2; Commissural fibers
1. Introduction ∗
Corresponding author. Tel.: +81 87 891 2251; fax: +81 87 891 2251. E-mail address: [email protected] (T. Itano). 0920-1211/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2005.08.007
Intermittent electrical stimulation of the amygdala results in the development of generalized motor seizures (Goddard et al., 1969). Electrical kindling has
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been widely employed as an animal model of temporal lobe epilepsy in humans, the most general type of epilepsy in adult patients (reviewed in: McNamara et al., 1980). Following the development of kindling, morphological changes were reported with the reconstruction of neuronetworks by neurogenesis and gliosis (reviewed in: Khurgel et al., 1992; Khurgel and Ivy, 1996; Morimoto et al., 2004; Parent, 2002). These changes were observed mainly in the hippocampal region. However, a recent study has shown morphological changes in several regions, in addition to the hippocampus (Miyazaki et al., 2003). Based on clinical data, the corpus callosum is known to facilitate epileptogenic progression (Ono et al., 2002). However, morphological or molecular physiological changes in the commissural fibers have not been investigated in detail. Condes-Lara et al. (2001) have reported stage-related propagation in the anterior commissure in the drug-induced seizure mouse by the use of wheat germ agglutinin-horseradish peroxidase (WGAHRP). However, morphological changes in other commissural fibers remain unknown (Nakagawa et al., 2004). To investigate the relevance of kindling development in the transformation of commissural fibers, we focused on the emergence of radial glial cells following amygdala kindling in mice. Radial glia plays an important role in neurogenesis and gliogenesis in the central nervous system and sometimes result from neuronal injury, such as in post-traumatic epilepsy (Campbell and Gotz, 2002; Huttmann et al., 2003). In the present study, 3CB2 immunohistochemical staining was used as a marker for the stage-related emergence of radial glial cells in sagittal sections of epileptic mouse brain.
2. Materials and methods Male C57BL/6J mice (CLEA Japan, Inc., Tokyo, Japan) weighing between 25 and 30 g were used. The animals were housed under a natural light–dark cycle with free access to standard laboratory chow and tap water. All animal experiments were performed in accordance with the Guiding Principles for the Care and Use of Animals approved by the Council of the Physiological Society of Japan. Mice were anesthetized with pentobarbital sodium solution (50 mg/kg i.p.) (Abbott Laboratories, North Chicago, IL). Bipolar
electrodes were implanted in the left side of the basolateral amygdaloid nuclei (A, −0.2 mm; L, 2.0 mm; V, 4.5 mm; from bregma) (Hosokawa et al., 1995). One week after surgery, the mice received a stimulation of 1 s duration at 09:00 h and again at 15:00 h, 7 days a week, with a biphasic 60 Hz square wave pulse at 100–140 A peak to peak, from two electric stimulators (NEC San-Ei, Co. Ltd., Tokyo, Japan). The stimulation strength was 100 A for four mice, 120 A for seven mice and 140 A for four mice. The strength was not altered until full kindling was obtained. Based on this, the strength of the stimulator pulse was adjusted to a sub-threshold level determined by signs of immobility or mouth movement. In the kindled animals, the response to the successive stimulation was evaluated at the five kindling stages described by Racine (1972) and Racine and Zaide (1978), with slight alterations (Hosokawa et al., 1995) as follows: C1, immobility and/or rhythmic mouth movements; C2, features of C1 plus contralateral head turning; C3, features of C2 plus contralateral forelimb clonus; C4, features of C3 plus generalized tonic-clonic seizures; C5, features of C4 plus generalized clonic seizures and falling over. C3 was designated the partial stage during kindling and C5 as the generalized stage following kindling. In the C3 group (n = 5), stimulation was stopped just after reaching stage C3. In the C5 group (n = 5), having reached the C5 stage, stimulation was stopped after five successive seizures. In the control group, C0 (n = 5), the mice were implanted with electrodes and treated as if they had been stimulated, but received no actual stimulation. Two hours after the final stimulation, the mice in each group were deeply anesthetized with pentobarbital sodium solution (60 mg/kg i.p.), then perfused transcardially with 0.1 M phosphate buffer saline (PBS) (pH 7.4) and, for 20 min thereafter, by a fixative consisting of 4% formaldehyde in 0.1 M PBS (pH 7.4). The brains were removed and post-fixed for 3 days in the above fixative. Tissue samples were dehydrated with alcohol, embedded in paraffin and cut into 8 m sagittal sections using a microtome. To stain myelin/myelinated axons, luxol fast blue staining was carried out as follows (Kluver and Barrera, 1953); paraffin sections were deparaffinized with xylene and washed in 95% ethyl alcohol. The sections were left in an oven overnight at 56 ◦ C, in a 0.1% luxol fast blue solution, dissolved in 100 ml of 95% ethanol with 0.5 ml of 10% acetic acid, then cooled at
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Fig. 1. Schema (Paxinos and Watson, 1986) and luxol echt blue-stained section showing the brain regions where changes in 3CB2 immunoreactivity were observed at stage C3 or C5, compared with stage C0. (A) Genu and rostrum; (B) radiation of trunk; (C) splenium; (D) fimbria hippocampus; (E) anterior commissure posterior; (F) thalamus; (G) olfactory bulb. Abbreviations: acp, anterior commissure, posterior part; bsc, brachium of the superior colliculus; cc, corpus callosum; cg, cingulum; fmi, forceps minor of the corpus callosum; fmj, forceps major of the corpus callosum; lo, lateral olfactory tract; opt, optic tract; zo, stratum zonale. Scale bar: 1 mm.
Fig. 2. Variation in 3CB2 staining during kindling in rat brain. (A) Low-power luxol echt blue-stained section. (B–D) Stage-related increase in staining by 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Scale bar: 1 mm.
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room temperature and the excess staining solution was rinsed off using 95% ethyl alcohol. After rinsing with distilled water, the sections were separated by immersion in 0.05% lithium carbonate solution for 20 s and then in 70% ethyl alcohol for 2 min. After rinsing with distilled water, they were counterstained for 5–6 min in 0.1% cresyl violet solution dissolved in water. They were then immersed in 95% ethyl alcohol, dehydrated in 100% alcohol and prepared for microscopic examination. For immunohistological examination for 3CB2, described by Shibuya et al. (2003), paraffin embedded sections were deparaffinized with xylene, immersed in alcohol, and washed in 0.01 M PBS (pH 7.4). The sections were then immersed for 1 h in 0.3% Triton X-100 (Sigma, St. Louis, MO) dissolved in 0.01 M PBS. To quench endogenous peroxidase activity, the sections were left for 30 min in 2% H2 O2 in 0.01 M PBS. After
washing with 0.01 M PBS, non-specific reactions were blocked with 1% albumin solution. Mab was used as the primary antibody against the 3CB2 antigen. This antibody was developed by Francisco A Prada and was purchased from the Developmental Studies Hybridoma Bank, maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA). The antibody is known to recognize 3CB2 antigen in chick embryo, rat and chameleon (Prada et al., 1995). First, the antibody was diluted 1:100 with a 1% albumin solution in 0.01 M PBS and this was applied to the sections and left at 4 ◦ C overnight. Next, the sections were incubated for 1 h with a biotinylated secondary antibody and for a further 1 h with an avidin–biotin peroxidase complex (ABC Kit; Vector Laboratories, Burlingame, CA). Peroxidase activity was visualized using a solution of 3,3-diaminobenzidine in 0.03% H2 O2 in 0.01 M PBS.
Fig. 3. Variation in 3CB2 staining during kindling in the genu of the corpus callosum. (A) Luxol echt blue-stained section showing the location of the genu and rostrum of rat brain. (B–D) Showing stage-related increase in histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Arrow indicates the increase in 3CB2-positive staining in the genu at stage C5. Scale bar: 500 m.
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Image analysis was performed using Image-Pro Plus® software (Version 4.0, Media Cybernetics, The Imaging Expert, Silver Spring, MD, USA) to assess the altered density of the 3CB2 antigen in the immunostained brain regions, which included: the forceps minor (A), radiation of trunk (B), forceps major (C), fimbria hippocampus (D), anterior commissure (E) and posterior and lateral olfactory tract (G). The immunostained sections were examined under a light microscope at ×200 magnification. Photographs were taken of the areas A–E and G (Fig. 1) and used for analysis. For evaluation of 3CB2 immunoreactivity, the immunopositive density (% of analyzed area) was calculated using Image-Pro Plus® . To evaluate differences in density between the clonus stages (0, 3 and 5) within each area, a Kruskal Wallis/Tukey’s-test was carried out. In all statistical analyses, differences with a P-value of <0.05 were considered statistically significant.
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3. Results An illustration of sagittal section of brain (Paxinos and Watson, 1986) and Luxol echt blue-stained section from the present study are shown in Fig. 1. The areas from A–F shown in Fig. 1B were investigated in detail. Fig. 3 shows the variation in 3CB2 immunostaining, which was observed mainly in the axonal regions. These variations were stage dependent and the strongest staining was detected at C5 (Fig. 2D) as compared with C0 (Fig. 2B) and/or C3 (Fig. 2C). 3CB2 immunopositive staining was observed for stage C3 and more strongly for stage C5 in the regions of the major and minor corpus callosum (Fig. 1A), in the composition of the hippocampus, the anteroventral thalamic nucleus area, the bed nucleus of the stria terminalis and the accessory olfactory bulb (Fig. 2D). Small amounts of 3CB2-positive immunoreactivity (IR) were observed at the C0 stage in the genu and
Fig. 4. Variation in 3CB2 staining during kindling in the trunk of the corpus callosum. (A) Luxol echt blue-stained section showing the location of the radiation of trunk of rat brain. (B–D) Showing the stage-related increase of histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Arrow indicates the increase in 3CB2-positive staining in the trunk at stage C5. Scale bar: 500 m.
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Fig. 5. Variation in 3CB2 staining during kindling in the splenium. (A) Luxol echt blue-stained section showing the location of the splenium of rat brain. (B–D) Showing the stage-related increase in histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Arrow indicates the divergent increase in 3CB2-positive staining in the cingulate gyrus alveus hippocampi and corpus callosum at stages C3 and C5. Scale bar: 500 m.
rostrum (forceps minor), as well as on the endothelial cells (Fig. 3B). Stronger and increased 3CB2-positive dot-like IR was observed at stage C3 (Fig. 3C). At stage C5, obvious and dense 3CB2-positive processes emerged (Figs. 3D and 10). Weak 3CB2-positive IR was observed at the C0 stage in the radiation of trunk (Fig. 4B). A somewhat increased 3CB2-positive IR was observed in the corpus callosum and cingulate gyrus at stage C3 (Fig. 4C). In comparison, intense IR was clearly observed at stage C5 (Figs. 4D and 10). At stage C0, weak 3CB2-positive IR was observed in the splenium (forceps major) (Fig. 5B). There was a little 3CB2-positive staining in the region of cingulate gyrus, but the corpus callosum and alveus hippocampi had no staining (Fig. 5B). At stage C3, staining of cingulate gyrus became stronger, but little increase was observed in the corpus callosum and alveus hippocampi
(Fig. 5C). At stage C5, strong IR in the cingulate gyrus and alveus hippocampi was clearly observed (Figs. 5D and 10). However, no 3CB2-positive staining was observed in the corpus callosum (Fig. 5D). At stages C0 and C3, no 3CB2-positive staining was observed in the fimbria hippocampus (Fig. 6B and C). However, at stage C5, very strong 3CB2-positive processes were observed in this area (Figs. 6D and 10). At the anterior commissure posterior, no IR was observed at stages C0 and C3 (Fig. 7B and C). However, at stage C5, a strong IR was observed in this area (Figs. 7D and 10). IR-positive dots were observed at stages C0 and C3 on the upper side of anteroventral thalamic nucleus, in the ventrolateral and dorsomedial areas and in the bed nucleus of the stria terminalis (Fig. 8B and C). At stage C5, a strong IR was observed as compared to the C0 and C3 stages (Fig. 8D).
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Fig. 6. Variation in 3CB2 staining during kindling in the anterior commissure. (A) Luxol echt blue-stained section showing the location of the anterior commissure posterior of rat brain. (B–D) Showing the stage-related increase of histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Arrow indicates the increase in 3CB2-positive staining in the anterior commissure posterior at stage C5. Scale bar: 500 m.
Obvious staining at stage C0 was seen in the nucleus of the optic tract, cerebral peduncle and longitudinal fasciculus pons, but there was no change at stages C3 and C5 (Fig. 8). A strong IR was observed at stage C5 in the following regions: the reticular thalamic nucleus, zona limitans, bed nucleus of the stria terminalis, medial posteromedial part, stria terminals, internal capsule, fields of forel, medial lemniscus, parafascicular thalamic nucleus, ethmoid thalamic nucleus, sub-parafascicul thalamic nucleus, parvo, lateral posterior thalamic nucleus, substantia nigra and pars compacta (Fig. 8D). At C0, the olfactory bulb, lateral olfactory tract and glomerular layer were lightly stained (Fig. 9B). At stage C3, in the glomerular layer, the light staining increased (Fig. 9C). At stage C5, the lateral olfactory tract and glomerular layer showed a considerably increased reaction compared with stage C3 (Figs. 9D and 10).
Quantification of the densities at each of the kindling stages in immunostained brain areas is shown in Fig. 10.
4. Discussion The results of the present study make clear a relationship between the rearrangement of commissural fibers, as indicated by radial glial cell expression, and the progressive stages of kindling. Since radial glia has been regarded as multiple-purpose precursors of neurons and glia (Campbell and Gotz, 2002), the expression of radial glial marker protein may be related to neurogenesis and/or gliogenesis. However, no expression in neuronal pericarya was observed in the present study. Therefore, this may be expressed in glial cells. Glial fibrillary acidic protein and vimentin expressing cells have been associated with human temporal lobe
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Fig. 7. Variation in 3CB2 staining during kindling in the fimbria hippocampus. (A) Luxol echt blue-stained section showing the location of the fimbria hippocampus of rat brain. (B–D) Showing the stage-related increase in histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Arrow indicates the increase in 3CB2-positive staining in the fimbria hippocampus at stage C5. Scale bar: 200 m.
epilepsy (Crespel et al., 2002). Since vimentin is also one of the marker proteins of radial glia (Nakagawa et al., 2004), we suggest that 3CB2 IR may be related to possible gliosis. In the genu, rostrum, trunk and splenium, IR was observed with kindling stage progression. Since the propagation of discharges is transmitted through the commissural fibers of corpus callosum, some alteration may occur in the connections between the hemispheres. Previously, we have shown that the radial glial cell marker, vimentin, is increased in these regions (Nakagawa et al., 2004). The importance of the commissural connection was not discussed in that study. However, in the present study, the analysis was focused on commissural fibers in explanation of the former data as well. It was for this purpose, that sagittal sections were used. The IR of 3CB2 expanded from the rostrum to the truncus in the corpus callosum from an early stage of
kindling. Changes in the corpus callosum began in the frontal area and spread to the occipital region. Another study has reported on the evaluation and cortical projection of temporal seizures in the cat, following an asymmetrical projection (Fernandez-Mas et al., 1992). A bisectional study has suggested that the anterior area in the corpus callosum contributes to inter-hemispheric synchrony (Usuki et al., 1992) and these results support the findings of the present study. During clinical callosotomy, the anterior two-third of the corpus callosum is often removed without any associated deficiency (Purves et al., 1988; Sakas and Phillips, 1996). The results of the present study also support the significance of the extent of resection, in which the IR of the forceps major was observed to differ from that of the cingulum, corpus callosum and alveus hippocampi. Furthermore, although the cingulum showed a strong IR at stage C3, the alveus hippocampi was an area where IR was observed at the C5 stage. On the other hand, the corpus
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Fig. 8. Variation in 3CB2 staining during kindling in the thalamus. (A) Luxol echt blue-stained section showing the location of the optic thalamus of rat brain. (B–D) Showing the stage-related increase of histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Scale bar: 1 mm.
callosum exhibited no IR at any stage. These results are also in agreement with the above-mentioned findings, which suggest that the splenium corpus callosum is not directly related to generalized seizures. In the anterior commissure posterior, 3CB2 IR was only observed at the C5 stage, whereas no staining was observed in the anterior commissure anterior and intrabulber at any stage. McIntyre (1975) reported that the combined sectioning of the anterior corpus callosum and the anterior commissure blocks the propagation of after-discharge in the inter-amygdala. Furthermore, chemical labeling using wheat germ agglutinin-horseradish peroxidase has indicated stagerelated staining in the anterior commissure (CondesLara et al., 2001), also supporting our data. In the present study, IR at stage C5 was observed not only in commissural fibers, but also in the cingulum, alveus hippocampi, fimbria hippocampus, brachium of superior colliculus and thalamic areas. Vimentin
immunoreactivity was also observed in the cingulum with stage elevation (Nakagawa et al., 2004), and c-fos expression in amygdala kindling was reported at early stages (Sato et al., 1998). Following systemic kainic acid administration, about 100 times more c-fos expression was observed in the cingulum in both pre- and postseizure states as compared to controls (Willoughby et al., 1997). The alveus hippocampi and fimbria hippocampus are efferent, elongated regions projecting from the hippocampus to the fornix. Bilateral transection of the fimbria/fornix produces a decrease in the after-discharge threshold, clonus and the rise of seizure duration (Mohapel et al., 1997). Combined with these observations our findings suggest that morphological changes in these areas at a latter stage may be introduced by seizure reinforcement and development. The thalamus has been thought to be one of the important areas in limbic epileptogenesis (Mraovitch
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Fig. 9. Variation in 3CB2 staining during kindling in the olfactory bulb. (A) Luxol echt blue-stained section showing the location of the lateral olfactory tract and glomerular layer in the olfactory bulb of rat brain. (B–D) Showing the stage-related increase in histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Arrows indicate the increase in 3CB2-positive staining in the lateral olfactory tract and glomerular layer. Scale bar: 1 mm.
and Calando, 1999). Increased WGA-HRP labeling was found at the medial and lateral bed nuclei stria terminalis (Condes-Lara et al., 2001). Moreover, following carbachol injection into the thalamus, c-fos expression was observed as generalized convulsive seizures progressed in the basal ganglia circuit (Mraovitch and Calando, 1999). C-fos expression was then observed first in the thalamus and was later detected in the anteroventral, laterodorsal and ventromedial hypothalamic nuclei. Finally, its expression has been observed in the superior colliculus, in the ventral posteromedial thalamic nucleus and in the ventrolateral nucleus of the reticular nucleus (Mraovitch and Calando, 1999). These data coincide with the 3CB2 staining data of the present study. Since the zona incerta is one of the important areas for carbachol-induced generalized seizures, 3CB2 expression in this region would suggest significant seizure generalization.
Neuronal progenitor cell migration, from the subventricular zone to the olfactory bulb, has been reported in the rostral migratory stream (Winner et al., 2002; Yamada et al., 2004). The regeneration expanded from the rostral migratory stream to the granule cell and glomerular layers, respectively. Pilocarpininduced seizures produce proliferating neuroblasts in this region in rat (Parent et al., 2002). However, in the present study, IR in the olfactory bulb was observed in the lateral olfactory tract and glomerular layer. These results suggest that gliosis in the olfactory bulb may not always be linked with neurogenesis. The reason why we selected stage 3 seizures and refer to them as partial seizures is as flows. At stage C3, the forelimb clonus was expanded to the contra-lateral side. However, the animals show no clear generalized tonic clonic seizures. Hence, the stage C3 is thought to be one of the proceedings for kindling development.
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Fig. 10. Density values according to clonus stage in six immunostained brain regions in amygdala-kindled mice. Density was measured by Image-Pro Plus® software. Results are expressed as mean ± S.E.M. (n = 5). * P < 0.05; ** P < 0.01 vs. Clonus 0. # P < 0.05; ## P < 0.01 vs. Clonus 3. The areas in which density was measured included the forceps minor (A), radiation of trunk (B), forceps major (C), fimbria hippocampus (D), anterior commissure posterior (E) and lateral olfactory tract (G). A–G represent corresponding areas in Fig. 1.
This is also one reason why we call partial seizure at C3. In previous study of ours (Nakagawa et al., 2004), vimentin immunoreactivity was found to be highest at Clonus 3 decreasing at Clonus 5 in the hippocampal formation, regions around third ventricle, caudate putamen and lateral habenular nucleus. In contrast, vimentin immunoreactivity consistently increased with progression of kindling in the cingulum and parietal cortex (Nakagawa et al., 2004). This may suggest that different staining proteins have different expressions depending on the kindling stage. We have examined the morphological changes during and after kindling process since 1995 (Hosokawa et al., 1995; Umeoka et al., 2000; Miyazaki et al., 2003; Nakagawa et al., 2004). In order to be able to compare these studies with each other and each steps in the procedure must be same. This was one of the most important reasons why we selected the 2 h period for the sacrifice after final stimulation. The preliminary experiment showed no clear differences between 2 h after and one day after the final stimulation (data not shown). Another concern that may be raised is that radial glial marker expression might be the same as “permanent” change as kindling. Since radial glial immunoreac-
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tivities were increased after spinal cord injury and decreased after 12 weeks, these results suggest the expression of 3CB2 might be temporary (Shibuya et al., 2003). As 3CB2 is the antigen for the intermediate filament and non-enzyme, the changes might not be reflected in the enzymatic reaction. Further studies must be done to clarify these possibilities. In conclusion, the results of the present study reaffirm that the commissural fibers connecting the cerebral hemispheres may be necessary for kindling development. In addition, regional and quantitative changes suggest that specific transmission may be occurring in the commissures during the kindling process. These findings contribute to the understanding of generalized seizures and development of remedies for epilepsy. Acknowledgement This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. References Campbell, K., Gotz, M., 2002. Radial glia: multi-purpose cells for vertebrate brain development. Trends Neurosci. 25, 235–238. Condes-Lara, M., Talavera-Cuevas, E., Larriva-Sahd, J., MartinezLorenzana, G., 2001. Different wheat germ agglutininhorseradish peroxidase labeling in structures related to the development of amygdaline kindling in the rat. Neurosci. Lett. 299, 13–16. Crespel, A., Coubes, P., Rousset, M.C., Alonso, G., Bockaert, J., Baldy-Moulinier, M., Lerner-Natoli, M., 2002. Immature-like astrocytes are associated with dentate granule cell migration in human temporal lobe epilepsy. Neurosci. Lett. 330, 114–118. Fernandez-Mas, R., Martinez, A., Gutierrez, R., FernandezGuardiola, A., 1992. EEG frequency and time domain mapping study of the cortical projections of temporal lobe amygdala afterdischarge during kindling in the cat. Epilepsy Res. 1, 23–34. Goddard, G.V., McIntyre, D., Leech, C., 1969. A permanent change in brain function resulting from daily electrical stimulation. Exp. Neurol. 25, 295–330. Hosokawa, J., Itano, T., Usuki, T., Tokuda, M., Matsui, H., Janjua, N.A., Suwaki, H., Okada, Y., Negi, T., Murakami, T.H., Konishi, R., Hatase, O., 1995. Morphological changes in the hippocampus in amygdaloid kindled mouse. Epilepsy Res. 20, 11–20. Huttmann, K., Sadgrove, M., Wallraff, A., Hinterkeuser, S., Kirchhoff, F., Steinhauser, C., Gray, W.P., 2003. Seizures preferentially stimulate proliferation of radial glia-like astrocytes in the adult dentate gyrus: functional and immunocytochemical analysis. Eur. J. Neurosci. 18, 2769–2778.
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Purves, S.J., Wada, J.A., Woodhurst, W.B., Moyes, P.D., Strauss, E., Kosaka, B., Li, D., 1988. Results of anterior corpus callosum section in 24 patients with medically intractable seizures. Neurology 38, 1194–1201. Racine, R.J., 1972. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr. Clin. Neurophysiol. 32, 281–294. Racine, R.J., Zaide, J., 1978. A further investigation into the mechanisms of the kindling phenomenon. In: Livibingston, K., Horynkiewicz, O. (Eds.), Limbic Mechanisms: the Continuing Evaluation of the Limbic System Concept. Plenum Press, New York, pp. 457–493. Sakas, D.E., Phillips, J., 1996. Anterior callosotomy in the management of intractable epileptic seizures: significance of the extent of resection. Acta Neurochir. (Wien) 138, 700– 707. Sato, T., Yamada, N., Morimoto, K., Uemura, S., Kuroda, S., 1998. A behavioral and immunohistochemical study on the development of perirhinal cortical kindling: a comparison with other types of limbic kindling. Brain Res. 811, 122–132. Shibuya, S., Miyamoto, O., Itano, T., Mori, S., Norimatsu, H., 2003. Temporal progressive antigen expression in radial glia after contusive spinal cord injury in adult rats. Glia 42, 172– 183. Umeoka, S., Miyamoto, O., Janjua, N.A., Nagao, S., Itano, T., 2000. Appearance and alteration of TUNEL positive cells through epileptogenesis in amygdaloid kindled rat. Epilepsy Res. 42, 97–103. Usuki, T., Iwahashi, K., Tanaka, K., Murakami, T., Kugoh, T., Hosokawa, K., 1992. Bilateral interhemispheric synchrony and amygdaloid kindling in congenitally acallosal and corpus callosum bisected mice. Jpn. J. Psychiatry Neurol. 46, 498–500. Willoughby, J.O., Mackenzie, L., Medvedev, A., Hiscock, J.J., 1997. Fos induction following systemic kainic acid: early expression in hippocampus and later widespread expression correlated with seizure. Neuroscience 77, 379–392. Winner, B., Cooper-Kuhn, C.M., Aigner, R., Winkler, J., Kuhn, H.G., 2002. Long-term survival and cell death of newly generated neurons in the adult rat olfactory bulb. Eur. J. Neurosci. 16, 1681–1689. Yamada, M., Onodera, M., Mizuno, Y., Mochizuki, H., 2004. Neurogenesis in olfactory bulb identified by retroviral labeling in normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated adult mice. Neuroscience 124, 173–181.
Stage and region dependent expression of a radial glial marker in commissural fibers in kindled mice Shinji Tanaka a,b , Osamu Miyamoto b , Najma A. Janjua c , Tetsuji Miyazaki b , Fumio Takahashi d , Ryoji Konishi a , Toshifumi Itano b,∗ b
a Teikoku Seiyaku Co. Ltd., 567 Sanbonmatsu, Higashikagawa, Kagawa 769-2695, Japan Department of Neurobiology, Kagawa University, Faculty of Medicine, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan c Department of General Education, Okayama University, 2-1-1 Tsushima Naka, Okayama 700-8530 Japan d Department of Food and Nutrition, Sanyo Gakuen College, Hirai, Okayama 703-8501, Japan
Received 11 May 2005; received in revised form 23 July 2005; accepted 18 August 2005 Available online 3 October 2005
Abstract Amygdala kindling is regarded as a model of temporal lobe epilepsy in humans because of many points of similarity. In amygdala kindling, bilateralization of epileptic seizures follows from the accumulation of stimulation and commissural fibers may play a role in this process. However, new progenies of cells following amygdala kindling have not been reported and the precise nature of how bilateralization occurs is not clear. In the present study, we aim to clarify the emergence of radial glia during the progress of amygdala kindling in mouse brain. For this purpose, immunohistochemical staining for 3CB2, which is a specific marker of radial glia, was employed. Immunoreactivity for 3CB2 was observed in the forceps minor, radiation of trunk and forceps major regions at Clonus 3 and more strongly at Clonus 5. In the forceps major, the cingulate gyrus showed immunopositive staining at Clonus 3, but the corpus callosum and alveus hippocampi showed staining only at Clonus 5. In the fimbria hippocampus, the anterior commissure posterior showed staining at Clonus 5. However, the anterior commissure anterior was not stained at the stage progressed to Clonus 5. These findings indicate stage and region dependent expression of progenitor cells in commissural fibers and suggest that these changes may accompany the formation of new circuits in seizure progression during amygdala kindling. © 2005 Elsevier B.V. All rights reserved. Keywords: Kindling; Radial glia; 3CB2; Commissural fibers
1. Introduction ∗
Corresponding author. Tel.: +81 87 891 2251; fax: +81 87 891 2251. E-mail address: [email protected] (T. Itano). 0920-1211/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2005.08.007
Intermittent electrical stimulation of the amygdala results in the development of generalized motor seizures (Goddard et al., 1969). Electrical kindling has
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been widely employed as an animal model of temporal lobe epilepsy in humans, the most general type of epilepsy in adult patients (reviewed in: McNamara et al., 1980). Following the development of kindling, morphological changes were reported with the reconstruction of neuronetworks by neurogenesis and gliosis (reviewed in: Khurgel et al., 1992; Khurgel and Ivy, 1996; Morimoto et al., 2004; Parent, 2002). These changes were observed mainly in the hippocampal region. However, a recent study has shown morphological changes in several regions, in addition to the hippocampus (Miyazaki et al., 2003). Based on clinical data, the corpus callosum is known to facilitate epileptogenic progression (Ono et al., 2002). However, morphological or molecular physiological changes in the commissural fibers have not been investigated in detail. Condes-Lara et al. (2001) have reported stage-related propagation in the anterior commissure in the drug-induced seizure mouse by the use of wheat germ agglutinin-horseradish peroxidase (WGAHRP). However, morphological changes in other commissural fibers remain unknown (Nakagawa et al., 2004). To investigate the relevance of kindling development in the transformation of commissural fibers, we focused on the emergence of radial glial cells following amygdala kindling in mice. Radial glia plays an important role in neurogenesis and gliogenesis in the central nervous system and sometimes result from neuronal injury, such as in post-traumatic epilepsy (Campbell and Gotz, 2002; Huttmann et al., 2003). In the present study, 3CB2 immunohistochemical staining was used as a marker for the stage-related emergence of radial glial cells in sagittal sections of epileptic mouse brain.
2. Materials and methods Male C57BL/6J mice (CLEA Japan, Inc., Tokyo, Japan) weighing between 25 and 30 g were used. The animals were housed under a natural light–dark cycle with free access to standard laboratory chow and tap water. All animal experiments were performed in accordance with the Guiding Principles for the Care and Use of Animals approved by the Council of the Physiological Society of Japan. Mice were anesthetized with pentobarbital sodium solution (50 mg/kg i.p.) (Abbott Laboratories, North Chicago, IL). Bipolar
electrodes were implanted in the left side of the basolateral amygdaloid nuclei (A, −0.2 mm; L, 2.0 mm; V, 4.5 mm; from bregma) (Hosokawa et al., 1995). One week after surgery, the mice received a stimulation of 1 s duration at 09:00 h and again at 15:00 h, 7 days a week, with a biphasic 60 Hz square wave pulse at 100–140 A peak to peak, from two electric stimulators (NEC San-Ei, Co. Ltd., Tokyo, Japan). The stimulation strength was 100 A for four mice, 120 A for seven mice and 140 A for four mice. The strength was not altered until full kindling was obtained. Based on this, the strength of the stimulator pulse was adjusted to a sub-threshold level determined by signs of immobility or mouth movement. In the kindled animals, the response to the successive stimulation was evaluated at the five kindling stages described by Racine (1972) and Racine and Zaide (1978), with slight alterations (Hosokawa et al., 1995) as follows: C1, immobility and/or rhythmic mouth movements; C2, features of C1 plus contralateral head turning; C3, features of C2 plus contralateral forelimb clonus; C4, features of C3 plus generalized tonic-clonic seizures; C5, features of C4 plus generalized clonic seizures and falling over. C3 was designated the partial stage during kindling and C5 as the generalized stage following kindling. In the C3 group (n = 5), stimulation was stopped just after reaching stage C3. In the C5 group (n = 5), having reached the C5 stage, stimulation was stopped after five successive seizures. In the control group, C0 (n = 5), the mice were implanted with electrodes and treated as if they had been stimulated, but received no actual stimulation. Two hours after the final stimulation, the mice in each group were deeply anesthetized with pentobarbital sodium solution (60 mg/kg i.p.), then perfused transcardially with 0.1 M phosphate buffer saline (PBS) (pH 7.4) and, for 20 min thereafter, by a fixative consisting of 4% formaldehyde in 0.1 M PBS (pH 7.4). The brains were removed and post-fixed for 3 days in the above fixative. Tissue samples were dehydrated with alcohol, embedded in paraffin and cut into 8 m sagittal sections using a microtome. To stain myelin/myelinated axons, luxol fast blue staining was carried out as follows (Kluver and Barrera, 1953); paraffin sections were deparaffinized with xylene and washed in 95% ethyl alcohol. The sections were left in an oven overnight at 56 ◦ C, in a 0.1% luxol fast blue solution, dissolved in 100 ml of 95% ethanol with 0.5 ml of 10% acetic acid, then cooled at
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Fig. 1. Schema (Paxinos and Watson, 1986) and luxol echt blue-stained section showing the brain regions where changes in 3CB2 immunoreactivity were observed at stage C3 or C5, compared with stage C0. (A) Genu and rostrum; (B) radiation of trunk; (C) splenium; (D) fimbria hippocampus; (E) anterior commissure posterior; (F) thalamus; (G) olfactory bulb. Abbreviations: acp, anterior commissure, posterior part; bsc, brachium of the superior colliculus; cc, corpus callosum; cg, cingulum; fmi, forceps minor of the corpus callosum; fmj, forceps major of the corpus callosum; lo, lateral olfactory tract; opt, optic tract; zo, stratum zonale. Scale bar: 1 mm.
Fig. 2. Variation in 3CB2 staining during kindling in rat brain. (A) Low-power luxol echt blue-stained section. (B–D) Stage-related increase in staining by 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Scale bar: 1 mm.
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room temperature and the excess staining solution was rinsed off using 95% ethyl alcohol. After rinsing with distilled water, the sections were separated by immersion in 0.05% lithium carbonate solution for 20 s and then in 70% ethyl alcohol for 2 min. After rinsing with distilled water, they were counterstained for 5–6 min in 0.1% cresyl violet solution dissolved in water. They were then immersed in 95% ethyl alcohol, dehydrated in 100% alcohol and prepared for microscopic examination. For immunohistological examination for 3CB2, described by Shibuya et al. (2003), paraffin embedded sections were deparaffinized with xylene, immersed in alcohol, and washed in 0.01 M PBS (pH 7.4). The sections were then immersed for 1 h in 0.3% Triton X-100 (Sigma, St. Louis, MO) dissolved in 0.01 M PBS. To quench endogenous peroxidase activity, the sections were left for 30 min in 2% H2 O2 in 0.01 M PBS. After
washing with 0.01 M PBS, non-specific reactions were blocked with 1% albumin solution. Mab was used as the primary antibody against the 3CB2 antigen. This antibody was developed by Francisco A Prada and was purchased from the Developmental Studies Hybridoma Bank, maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA). The antibody is known to recognize 3CB2 antigen in chick embryo, rat and chameleon (Prada et al., 1995). First, the antibody was diluted 1:100 with a 1% albumin solution in 0.01 M PBS and this was applied to the sections and left at 4 ◦ C overnight. Next, the sections were incubated for 1 h with a biotinylated secondary antibody and for a further 1 h with an avidin–biotin peroxidase complex (ABC Kit; Vector Laboratories, Burlingame, CA). Peroxidase activity was visualized using a solution of 3,3-diaminobenzidine in 0.03% H2 O2 in 0.01 M PBS.
Fig. 3. Variation in 3CB2 staining during kindling in the genu of the corpus callosum. (A) Luxol echt blue-stained section showing the location of the genu and rostrum of rat brain. (B–D) Showing stage-related increase in histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Arrow indicates the increase in 3CB2-positive staining in the genu at stage C5. Scale bar: 500 m.
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Image analysis was performed using Image-Pro Plus® software (Version 4.0, Media Cybernetics, The Imaging Expert, Silver Spring, MD, USA) to assess the altered density of the 3CB2 antigen in the immunostained brain regions, which included: the forceps minor (A), radiation of trunk (B), forceps major (C), fimbria hippocampus (D), anterior commissure (E) and posterior and lateral olfactory tract (G). The immunostained sections were examined under a light microscope at ×200 magnification. Photographs were taken of the areas A–E and G (Fig. 1) and used for analysis. For evaluation of 3CB2 immunoreactivity, the immunopositive density (% of analyzed area) was calculated using Image-Pro Plus® . To evaluate differences in density between the clonus stages (0, 3 and 5) within each area, a Kruskal Wallis/Tukey’s-test was carried out. In all statistical analyses, differences with a P-value of <0.05 were considered statistically significant.
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3. Results An illustration of sagittal section of brain (Paxinos and Watson, 1986) and Luxol echt blue-stained section from the present study are shown in Fig. 1. The areas from A–F shown in Fig. 1B were investigated in detail. Fig. 3 shows the variation in 3CB2 immunostaining, which was observed mainly in the axonal regions. These variations were stage dependent and the strongest staining was detected at C5 (Fig. 2D) as compared with C0 (Fig. 2B) and/or C3 (Fig. 2C). 3CB2 immunopositive staining was observed for stage C3 and more strongly for stage C5 in the regions of the major and minor corpus callosum (Fig. 1A), in the composition of the hippocampus, the anteroventral thalamic nucleus area, the bed nucleus of the stria terminalis and the accessory olfactory bulb (Fig. 2D). Small amounts of 3CB2-positive immunoreactivity (IR) were observed at the C0 stage in the genu and
Fig. 4. Variation in 3CB2 staining during kindling in the trunk of the corpus callosum. (A) Luxol echt blue-stained section showing the location of the radiation of trunk of rat brain. (B–D) Showing the stage-related increase of histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Arrow indicates the increase in 3CB2-positive staining in the trunk at stage C5. Scale bar: 500 m.
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Fig. 5. Variation in 3CB2 staining during kindling in the splenium. (A) Luxol echt blue-stained section showing the location of the splenium of rat brain. (B–D) Showing the stage-related increase in histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Arrow indicates the divergent increase in 3CB2-positive staining in the cingulate gyrus alveus hippocampi and corpus callosum at stages C3 and C5. Scale bar: 500 m.
rostrum (forceps minor), as well as on the endothelial cells (Fig. 3B). Stronger and increased 3CB2-positive dot-like IR was observed at stage C3 (Fig. 3C). At stage C5, obvious and dense 3CB2-positive processes emerged (Figs. 3D and 10). Weak 3CB2-positive IR was observed at the C0 stage in the radiation of trunk (Fig. 4B). A somewhat increased 3CB2-positive IR was observed in the corpus callosum and cingulate gyrus at stage C3 (Fig. 4C). In comparison, intense IR was clearly observed at stage C5 (Figs. 4D and 10). At stage C0, weak 3CB2-positive IR was observed in the splenium (forceps major) (Fig. 5B). There was a little 3CB2-positive staining in the region of cingulate gyrus, but the corpus callosum and alveus hippocampi had no staining (Fig. 5B). At stage C3, staining of cingulate gyrus became stronger, but little increase was observed in the corpus callosum and alveus hippocampi
(Fig. 5C). At stage C5, strong IR in the cingulate gyrus and alveus hippocampi was clearly observed (Figs. 5D and 10). However, no 3CB2-positive staining was observed in the corpus callosum (Fig. 5D). At stages C0 and C3, no 3CB2-positive staining was observed in the fimbria hippocampus (Fig. 6B and C). However, at stage C5, very strong 3CB2-positive processes were observed in this area (Figs. 6D and 10). At the anterior commissure posterior, no IR was observed at stages C0 and C3 (Fig. 7B and C). However, at stage C5, a strong IR was observed in this area (Figs. 7D and 10). IR-positive dots were observed at stages C0 and C3 on the upper side of anteroventral thalamic nucleus, in the ventrolateral and dorsomedial areas and in the bed nucleus of the stria terminalis (Fig. 8B and C). At stage C5, a strong IR was observed as compared to the C0 and C3 stages (Fig. 8D).
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Fig. 6. Variation in 3CB2 staining during kindling in the anterior commissure. (A) Luxol echt blue-stained section showing the location of the anterior commissure posterior of rat brain. (B–D) Showing the stage-related increase of histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Arrow indicates the increase in 3CB2-positive staining in the anterior commissure posterior at stage C5. Scale bar: 500 m.
Obvious staining at stage C0 was seen in the nucleus of the optic tract, cerebral peduncle and longitudinal fasciculus pons, but there was no change at stages C3 and C5 (Fig. 8). A strong IR was observed at stage C5 in the following regions: the reticular thalamic nucleus, zona limitans, bed nucleus of the stria terminalis, medial posteromedial part, stria terminals, internal capsule, fields of forel, medial lemniscus, parafascicular thalamic nucleus, ethmoid thalamic nucleus, sub-parafascicul thalamic nucleus, parvo, lateral posterior thalamic nucleus, substantia nigra and pars compacta (Fig. 8D). At C0, the olfactory bulb, lateral olfactory tract and glomerular layer were lightly stained (Fig. 9B). At stage C3, in the glomerular layer, the light staining increased (Fig. 9C). At stage C5, the lateral olfactory tract and glomerular layer showed a considerably increased reaction compared with stage C3 (Figs. 9D and 10).
Quantification of the densities at each of the kindling stages in immunostained brain areas is shown in Fig. 10.
4. Discussion The results of the present study make clear a relationship between the rearrangement of commissural fibers, as indicated by radial glial cell expression, and the progressive stages of kindling. Since radial glia has been regarded as multiple-purpose precursors of neurons and glia (Campbell and Gotz, 2002), the expression of radial glial marker protein may be related to neurogenesis and/or gliogenesis. However, no expression in neuronal pericarya was observed in the present study. Therefore, this may be expressed in glial cells. Glial fibrillary acidic protein and vimentin expressing cells have been associated with human temporal lobe
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Fig. 7. Variation in 3CB2 staining during kindling in the fimbria hippocampus. (A) Luxol echt blue-stained section showing the location of the fimbria hippocampus of rat brain. (B–D) Showing the stage-related increase in histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Arrow indicates the increase in 3CB2-positive staining in the fimbria hippocampus at stage C5. Scale bar: 200 m.
epilepsy (Crespel et al., 2002). Since vimentin is also one of the marker proteins of radial glia (Nakagawa et al., 2004), we suggest that 3CB2 IR may be related to possible gliosis. In the genu, rostrum, trunk and splenium, IR was observed with kindling stage progression. Since the propagation of discharges is transmitted through the commissural fibers of corpus callosum, some alteration may occur in the connections between the hemispheres. Previously, we have shown that the radial glial cell marker, vimentin, is increased in these regions (Nakagawa et al., 2004). The importance of the commissural connection was not discussed in that study. However, in the present study, the analysis was focused on commissural fibers in explanation of the former data as well. It was for this purpose, that sagittal sections were used. The IR of 3CB2 expanded from the rostrum to the truncus in the corpus callosum from an early stage of
kindling. Changes in the corpus callosum began in the frontal area and spread to the occipital region. Another study has reported on the evaluation and cortical projection of temporal seizures in the cat, following an asymmetrical projection (Fernandez-Mas et al., 1992). A bisectional study has suggested that the anterior area in the corpus callosum contributes to inter-hemispheric synchrony (Usuki et al., 1992) and these results support the findings of the present study. During clinical callosotomy, the anterior two-third of the corpus callosum is often removed without any associated deficiency (Purves et al., 1988; Sakas and Phillips, 1996). The results of the present study also support the significance of the extent of resection, in which the IR of the forceps major was observed to differ from that of the cingulum, corpus callosum and alveus hippocampi. Furthermore, although the cingulum showed a strong IR at stage C3, the alveus hippocampi was an area where IR was observed at the C5 stage. On the other hand, the corpus
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Fig. 8. Variation in 3CB2 staining during kindling in the thalamus. (A) Luxol echt blue-stained section showing the location of the optic thalamus of rat brain. (B–D) Showing the stage-related increase of histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Scale bar: 1 mm.
callosum exhibited no IR at any stage. These results are also in agreement with the above-mentioned findings, which suggest that the splenium corpus callosum is not directly related to generalized seizures. In the anterior commissure posterior, 3CB2 IR was only observed at the C5 stage, whereas no staining was observed in the anterior commissure anterior and intrabulber at any stage. McIntyre (1975) reported that the combined sectioning of the anterior corpus callosum and the anterior commissure blocks the propagation of after-discharge in the inter-amygdala. Furthermore, chemical labeling using wheat germ agglutinin-horseradish peroxidase has indicated stagerelated staining in the anterior commissure (CondesLara et al., 2001), also supporting our data. In the present study, IR at stage C5 was observed not only in commissural fibers, but also in the cingulum, alveus hippocampi, fimbria hippocampus, brachium of superior colliculus and thalamic areas. Vimentin
immunoreactivity was also observed in the cingulum with stage elevation (Nakagawa et al., 2004), and c-fos expression in amygdala kindling was reported at early stages (Sato et al., 1998). Following systemic kainic acid administration, about 100 times more c-fos expression was observed in the cingulum in both pre- and postseizure states as compared to controls (Willoughby et al., 1997). The alveus hippocampi and fimbria hippocampus are efferent, elongated regions projecting from the hippocampus to the fornix. Bilateral transection of the fimbria/fornix produces a decrease in the after-discharge threshold, clonus and the rise of seizure duration (Mohapel et al., 1997). Combined with these observations our findings suggest that morphological changes in these areas at a latter stage may be introduced by seizure reinforcement and development. The thalamus has been thought to be one of the important areas in limbic epileptogenesis (Mraovitch
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Fig. 9. Variation in 3CB2 staining during kindling in the olfactory bulb. (A) Luxol echt blue-stained section showing the location of the lateral olfactory tract and glomerular layer in the olfactory bulb of rat brain. (B–D) Showing the stage-related increase in histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Arrows indicate the increase in 3CB2-positive staining in the lateral olfactory tract and glomerular layer. Scale bar: 1 mm.
and Calando, 1999). Increased WGA-HRP labeling was found at the medial and lateral bed nuclei stria terminalis (Condes-Lara et al., 2001). Moreover, following carbachol injection into the thalamus, c-fos expression was observed as generalized convulsive seizures progressed in the basal ganglia circuit (Mraovitch and Calando, 1999). C-fos expression was then observed first in the thalamus and was later detected in the anteroventral, laterodorsal and ventromedial hypothalamic nuclei. Finally, its expression has been observed in the superior colliculus, in the ventral posteromedial thalamic nucleus and in the ventrolateral nucleus of the reticular nucleus (Mraovitch and Calando, 1999). These data coincide with the 3CB2 staining data of the present study. Since the zona incerta is one of the important areas for carbachol-induced generalized seizures, 3CB2 expression in this region would suggest significant seizure generalization.
Neuronal progenitor cell migration, from the subventricular zone to the olfactory bulb, has been reported in the rostral migratory stream (Winner et al., 2002; Yamada et al., 2004). The regeneration expanded from the rostral migratory stream to the granule cell and glomerular layers, respectively. Pilocarpininduced seizures produce proliferating neuroblasts in this region in rat (Parent et al., 2002). However, in the present study, IR in the olfactory bulb was observed in the lateral olfactory tract and glomerular layer. These results suggest that gliosis in the olfactory bulb may not always be linked with neurogenesis. The reason why we selected stage 3 seizures and refer to them as partial seizures is as flows. At stage C3, the forelimb clonus was expanded to the contra-lateral side. However, the animals show no clear generalized tonic clonic seizures. Hence, the stage C3 is thought to be one of the proceedings for kindling development.
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Fig. 10. Density values according to clonus stage in six immunostained brain regions in amygdala-kindled mice. Density was measured by Image-Pro Plus® software. Results are expressed as mean ± S.E.M. (n = 5). * P < 0.05; ** P < 0.01 vs. Clonus 0. # P < 0.05; ## P < 0.01 vs. Clonus 3. The areas in which density was measured included the forceps minor (A), radiation of trunk (B), forceps major (C), fimbria hippocampus (D), anterior commissure posterior (E) and lateral olfactory tract (G). A–G represent corresponding areas in Fig. 1.
This is also one reason why we call partial seizure at C3. In previous study of ours (Nakagawa et al., 2004), vimentin immunoreactivity was found to be highest at Clonus 3 decreasing at Clonus 5 in the hippocampal formation, regions around third ventricle, caudate putamen and lateral habenular nucleus. In contrast, vimentin immunoreactivity consistently increased with progression of kindling in the cingulum and parietal cortex (Nakagawa et al., 2004). This may suggest that different staining proteins have different expressions depending on the kindling stage. We have examined the morphological changes during and after kindling process since 1995 (Hosokawa et al., 1995; Umeoka et al., 2000; Miyazaki et al., 2003; Nakagawa et al., 2004). In order to be able to compare these studies with each other and each steps in the procedure must be same. This was one of the most important reasons why we selected the 2 h period for the sacrifice after final stimulation. The preliminary experiment showed no clear differences between 2 h after and one day after the final stimulation (data not shown). Another concern that may be raised is that radial glial marker expression might be the same as “permanent” change as kindling. Since radial glial immunoreac-
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tivities were increased after spinal cord injury and decreased after 12 weeks, these results suggest the expression of 3CB2 might be temporary (Shibuya et al., 2003). As 3CB2 is the antigen for the intermediate filament and non-enzyme, the changes might not be reflected in the enzymatic reaction. Further studies must be done to clarify these possibilities. In conclusion, the results of the present study reaffirm that the commissural fibers connecting the cerebral hemispheres may be necessary for kindling development. In addition, regional and quantitative changes suggest that specific transmission may be occurring in the commissures during the kindling process. These findings contribute to the understanding of generalized seizures and development of remedies for epilepsy. Acknowledgement This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. References Campbell, K., Gotz, M., 2002. Radial glia: multi-purpose cells for vertebrate brain development. Trends Neurosci. 25, 235–238. Condes-Lara, M., Talavera-Cuevas, E., Larriva-Sahd, J., MartinezLorenzana, G., 2001. Different wheat germ agglutininhorseradish peroxidase labeling in structures related to the development of amygdaline kindling in the rat. Neurosci. Lett. 299, 13–16. Crespel, A., Coubes, P., Rousset, M.C., Alonso, G., Bockaert, J., Baldy-Moulinier, M., Lerner-Natoli, M., 2002. Immature-like astrocytes are associated with dentate granule cell migration in human temporal lobe epilepsy. Neurosci. Lett. 330, 114–118. Fernandez-Mas, R., Martinez, A., Gutierrez, R., FernandezGuardiola, A., 1992. EEG frequency and time domain mapping study of the cortical projections of temporal lobe amygdala afterdischarge during kindling in the cat. Epilepsy Res. 1, 23–34. Goddard, G.V., McIntyre, D., Leech, C., 1969. A permanent change in brain function resulting from daily electrical stimulation. Exp. Neurol. 25, 295–330. Hosokawa, J., Itano, T., Usuki, T., Tokuda, M., Matsui, H., Janjua, N.A., Suwaki, H., Okada, Y., Negi, T., Murakami, T.H., Konishi, R., Hatase, O., 1995. Morphological changes in the hippocampus in amygdaloid kindled mouse. Epilepsy Res. 20, 11–20. Huttmann, K., Sadgrove, M., Wallraff, A., Hinterkeuser, S., Kirchhoff, F., Steinhauser, C., Gray, W.P., 2003. Seizures preferentially stimulate proliferation of radial glia-like astrocytes in the adult dentate gyrus: functional and immunocytochemical analysis. Eur. J. Neurosci. 18, 2769–2778.
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