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 glialmarker in commissural fibers in kindled mice

Shinji Tanakaa,b, Osamu Miyamotob, Najma A. Janjuac, Tetsuji Miyazakib,Fumio Takahashid, Ryoji Konishia, Toshifumi Itanob,∗

a Teikoku Seiyaku Co. Ltd., 567 Sanbonmatsu, Higashikagawa, Kagawa 769-2695, Japanb 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 Japand 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 2005Available online 3 October 2005

Abstract

ity. Infibersnd theial gliawhichion ofhowed. In theanteriorprogenitorgression

or

Amygdala kindling is regarded as a model of temporal lobe epilepsy in humans because of many points of similaramygdala kindling, bilateralization of epileptic seizures follows from the accumulation of stimulation and commissuralmay play a role in this process. However, new progenies of cells following amygdala kindling have not been reported aprecise nature of how bilateralization occurs is not clear. In the present study, we aim to clarify the emergence of radduring the progress of amygdala kindling in mouse brain. For this purpose, immunohistochemical staining for 3CB2,is a specific marker of radial glia, was employed. Immunoreactivity for 3CB2 was observed in the forceps minor, radiattrunk and forceps major regions at Clonus 3 and more strongly at Clonus 5. In the forceps major, the cingulate gyrus simmunopositive staining at Clonus 3, but the corpus callosum and alveus hippocampi showed staining only at Clonus 5fimbria hippocampus, the anterior commissure posterior showed staining at Clonus 5. However, the anterior commissurewas not stained at the stage progressed to Clonus 5. These findings indicate stage and region dependent expression ofcells in commissural fibers and suggest that these changes may accompany the formation of new circuits in seizure produring amygdala kindling.© 2005 Elsevier B.V. All rights reserved.

Keywords: Kindling; Radial glia; 3CB2; Commissural fibers

∗ Corresponding author. Tel.: +81 87 891 2251;fax: +81 87 891 2251.

E-mail address: [email protected] (T. Itano).

1. Introduction

Intermittent electrical stimulation of the amyg-dala results in the development of generalized motseizures (Goddard et al., 1969). Electrical kindling has

0920-1211/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.eplepsyres.2005.08.007

62 S. Tanaka et al. / Epilepsy Research 67 (2005) 61–72

been widely employed as an animal model of tem-poral lobe epilepsy in humans, the most general typeof epilepsy in adult patients (reviewed in:McNamaraet al., 1980). Following the development of kindling,morphological changes were reported with the recon-struction of neuronetworks by neurogenesis and gliosis(reviewed in:Khurgel et al., 1992; Khurgel and Ivy,1996; Morimoto et al., 2004; Parent, 2002). Thesechanges were observed mainly in the hippocampalregion. However, a recent study has shown morpho-logical changes in several regions, in addition to thehippocampus (Miyazaki et al., 2003).

Based on clinical data, the corpus callosum is knownto facilitate epileptogenic progression (Ono et al.,2002). However, morphological or molecular physio-logical changes in the commissural fibers have not beeninvestigated in detail.Condes-Lara et al. (2001)havereported stage-related propagation in the anterior com-missure in the drug-induced seizure mouse by the use ofwheat germ agglutinin-horseradish peroxidase (WGA-HRP). However, morphological changes in other com-missural fibers remain unknown (Nakagawa et al.,2004).

To investigate the relevance of kindling develop-ment in the transformation of commissural fibers, wefocused on the emergence of radial glial cells followinga or-t ntraln onali la ts seda adialg ain.

2

o,J sed.T darkc howa ediC ft nes-t /kgi lar

electrodes were implanted in the left side of the baso-lateral 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 stimula-tion of 1 s duration at 09:00 h and again at 15:00 h,7 days a week, with a biphasic 60 Hz square wavepulse at 100–140�A peak to peak, from two elec-tric 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 wasobtained. Based on this, the strength of the stimulatorpulse was adjusted to a sub-threshold level determinedby signs of immobility or mouth movement. In thekindled animals, the response to the successive stimula-tion was evaluated at the five kindling stages describedby Racine (1972)andRacine and Zaide (1978), withslight 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 seizuresand falling over. C3 was designated the partial stageduring kindling and C5 as the generalized stage fol-lowing kindling. In the C3 group (n = 5), stimulationw C5g ula-t n thec dw tim-u ursa ered tion( Mp mint de-h veda is-s ddedi ga xolfB edw ec-tl %e d at

mygdala kindling in mice. Radial glia plays an impant role in neurogenesis and gliogenesis in the ceervous system and sometimes result from neur

njury, such as in post-traumatic epilepsy (Campbelnd Gotz, 2002; Huttmann et al., 2003). In the presentudy, 3CB2 immunohistochemical staining was us a marker for the stage-related emergence of rlial cells in sagittal sections of epileptic mouse br

. Materials and methods

Male C57BL/6J mice (CLEA Japan, Inc., Tokyapan) weighing between 25 and 30 g were uhe animals were housed under a natural light–ycle with free access to standard laboratory cnd tap water. All animal experiments were perform

n accordance with theGuiding Principles for theare and Use of Animals approved by the Council o

he Physiological Society of Japan. Mice were ahetized with pentobarbital sodium solution (50 mg.p.) (Abbott Laboratories, North Chicago, IL). Bipo

as stopped just after reaching stage C3. In theroup (n = 5), having reached the C5 stage, stim

ion was stopped after five successive seizures. Iontrol group, C0 (n = 5), the mice were implanteith electrodes and treated as if they had been slated, but received no actual stimulation. Two hofter the final stimulation, the mice in each group weeply anesthetized with pentobarbital sodium solu60 mg/kg i.p.), then perfused transcardially with 0.1hosphate buffer saline (PBS) (pH 7.4) and, for 20

hereafter, by a fixative consisting of 4% formalyde in 0.1 M PBS (pH 7.4). The brains were remond post-fixed for 3 days in the above fixative. Tue samples were dehydrated with alcohol, emben paraffin and cut into 8�m sagittal sections usin

microtome. To stain myelin/myelinated axons, luast blue staining was carried out as follows (Kluver andarrera, 1953); paraffin sections were deparaffinizith xylene and washed in 95% ethyl alcohol. The s

ions were left in an oven overnight at 56◦C, in a 0.1%uxol fast blue solution, dissolved in 100 ml of 95thanol with 0.5 ml of 10% acetic acid, then coole

S. Tanaka et al. / Epilepsy Research 67 (2005) 61–72 63

Fig. 1. Schema (Paxinos and Watson, 1986) and luxol echt blue-stained section showing the brain regions where changes in 3CB2 immunore-activity were observed at stage C3 or C5, compared with stage C0. (A) Genu and rostrum; (B) radiation of trunk; (C) splenium; (D) fimbriahippocampus; (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 thecorpus callosum; lo, lateral olfactory tract; opt, optic tract; zo, stratum zonale. Scale bar: 1 mm.

Fs

ig. 2. Variation in 3CB2 staining during kindling in rat brain. (A) Low-taining by 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Scale bar: 1

power luxol echt blue-stained section. (B–D) Stage-related increase inmm.


room temperature and the excess staining solution wasrinsed off using 95% ethyl alcohol. After rinsing withdistilled water, the sections were separated by immer-sion in 0.05% lithium carbonate solution for 20 s andthen in 70% ethyl alcohol for 2 min. After rinsing withdistilled water, they were counterstained for 5–6 minin 0.1% cresyl violet solution dissolved in water. Theywere then immersed in 95% ethyl alcohol, dehydratedin 100% alcohol and prepared for microscopic exami-nation.

For immunohistological examination for 3CB2,described byShibuya et al. (2003), paraffin embeddedsections were deparaffinized with xylene, immersed inalcohol, and washed in 0.01 M PBS (pH 7.4). The sec-tions were then immersed for 1 h in 0.3% Triton X-100(Sigma, St. Louis, MO) dissolved in 0.01 M PBS. Toquench endogenous peroxidase activity, the sectionswere left for 30 min in 2% H2O2 in 0.01 M PBS. After

washing with 0.01 M PBS, non-specific reactions wereblocked with 1% albumin solution. Mab was used asthe primary antibody against the 3CB2 antigen. Thisantibody was developed by Francisco A Prada and waspurchased from the Developmental Studies HybridomaBank, maintained by the Department of Biological Sci-ences, University of Iowa (Iowa City, IA). The antibodyis known to recognize 3CB2 antigen in chick embryo,rat and chameleon (Prada et al., 1995). First, the anti-body was diluted 1:100 with a 1% albumin solution in0.01 M PBS and this was applied to the sections andleft at 4◦C overnight. Next, the sections were incu-bated for 1 h with a biotinylated secondary antibodyand for a further 1 h with an avidin–biotin peroxidasecomplex (ABC Kit; Vector Laboratories, Burlingame,CA). Peroxidase activity was visualized using a solu-tion of 3,3-diaminobenzidine in 0.03% H2O2 in 0.01 MPBS.

FoC

ig. 3. Variation in 3CB2 staining during kindling in the genu of the cof the genu and rostrum of rat brain. (B–D) Showing stage-related inc5 (D). Arrow indicates the increase in 3CB2-positive staining in the g

rpus callosum. (A) Luxol echt blue-stained section showing the locationrease in histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C);enu at stage C5. Scale bar: 500�m.


Image analysis was performed using Image-ProPlus® software (Version 4.0, Media Cybernetics, TheImaging Expert, Silver Spring, MD, USA) to assess thealtered density of the 3CB2 antigen in the immunos-tained brain regions, which included: the forceps minor(A), radiation of trunk (B), forceps major (C), fim-bria hippocampus (D), anterior commissure (E) andposterior and lateral olfactory tract (G). The immunos-tained sections were examined under a light micro-scope at×200 magnification. Photographs were takenof the areas A–E and G (Fig. 1) and used for anal-ysis. For evaluation of 3CB2 immunoreactivity, theimmunopositive density (% of analyzed area) wascalculated using Image-Pro Plus®. To evaluate dif-ferences in density between the clonus stages (0, 3and 5) within each area, a Kruskal Wallis/Tukey’s-testwas carried out. In all statistical analyses, differenceswith a P-value of <0.05 were considered statisticallysignificant.

3. Results

An illustration of sagittal section of brain (Paxinosand Watson, 1986) and Luxol echt blue-stained sec-tion from the present study are shown inFig. 1. Theareas from A–F shown inFig. 1B were investigated indetail. Fig. 3 shows the variation in 3CB2 immunos-taining, which was observed mainly in the axonalregions. These variations were stage dependent andthe strongest staining was detected at C5 (Fig. 2D) ascompared with C0 (Fig. 2B) and/or C3 (Fig. 2C). 3CB2immunopositive staining was observed for stage C3 andmore strongly for stage C5 in the regions of the majorand minor corpus callosum (Fig. 1A), in the compo-sition of the hippocampus, the anteroventral thalamicnucleus area, the bed nucleus of the stria terminalis andthe accessory olfactory bulb (Fig. 2D).

Small amounts of 3CB2-positive immunoreactivity(IR) were observed at the C0 stage in the genu and

Fo(

ig. 4. Variation in 3CB2 staining during kindling in the trunk of the corf the radiation of trunk of rat brain. (B–D) Showing the stage-relatedC); C5 (D). Arrow indicates the increase in 3CB2-positive staining in

pus callosum. (A) Luxol echt blue-stained section showing the locationincrease of histochemical staining of 3CB2. Kindling stage: C0 (B); C3the trunk at stage C5. Scale bar: 500�m.


Fig. 5. Variation in 3CB2 staining during kindling in the splenium. (A) Luxol echt blue-stained section showing the location of the splenium ofrat brain. (B–D) Showing the stage-related increase in histochemical staining of 3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Arrow indicatesthe 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 endothelialcells (Fig. 3B). Stronger and increased 3CB2-positivedot-like IR was observed at stage C3 (Fig. 3C). Atstage C5, obvious and dense 3CB2-positive processesemerged (Figs.3D and 10). Weak 3CB2-positive IRwas observed at the C0 stage in the radiation of trunk(Fig. 4B). A somewhat increased 3CB2-positive IRwas observed in the corpus callosum and cingulategyrus at stage C3 (Fig. 4C). In comparison, intenseIR was clearly observed at stage C5 (Figs.4D and10).

At stage C0, weak 3CB2-positive IR was observedin the splenium (forceps major) (Fig. 5B). There was alittle 3CB2-positive staining in the region of cingulategyrus, but the corpus callosum and alveus hippocampihad no staining (Fig. 5B). At stage C3, staining of cin-gulate gyrus became stronger, but little increase wasobserved in the corpus callosum and alveus hippocampi

(Fig. 5C). At stage C5, strong IR in the cingulate gyrusand alveus hippocampi was clearly observed (Figs.5D and10). However, no 3CB2-positive staining wasobserved in the corpus callosum (Fig. 5D).

At stages C0 and C3, no 3CB2-positive staining wasobserved in the fimbria hippocampus (Fig. 6B and C).However, at stage C5, very strong 3CB2-positive pro-cesses were observed in this area (Figs.6D and10).

At the anterior commissure posterior, no IR wasobserved at stages C0 and C3 (Fig. 7B and C). How-ever, at stage C5, a strong IR was observed in this area(Figs.7D and10).

IR-positive dots were observed at stages C0 and C3on the upper side of anteroventral thalamic nucleus, inthe ventrolateral and dorsomedial areas and in the bednucleus of the stria terminalis (Fig. 8B and C). At stageC5, a strong IR was observed as compared to the C0and C3 stages (Fig. 8D).


Fig. 6. Variation in 3CB2 staining during kindling in the anterior commissure. (A) Luxol echt blue-stained section showing the location of theanterior 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 nucleusof the optic tract, cerebral peduncle and longitudinalfasciculus pons, but there was no change at stages C3and C5 (Fig. 8). A strong IR was observed at stage C5in the following regions: the reticular thalamic nucleus,zona limitans, bed nucleus of the stria terminalis,medial posteromedial part, stria terminals, internalcapsule, fields of forel, medial lemniscus, parafas-cicular thalamic nucleus, ethmoid thalamic nucleus,sub-parafascicul thalamic nucleus, parvo, lateral pos-terior thalamic nucleus, substantia nigra and pars com-pacta (Fig. 8D).

At C0, the olfactory bulb, lateral olfactory tract andglomerular layer were lightly stained (Fig. 9B). Atstage C3, in the glomerular layer, the light stainingincreased (Fig. 9C). At stage C5, the lateral olfac-tory tract and glomerular layer showed a considerablyincreased reaction compared with stage C3 (Figs.9Dand10).

Quantification of the densities at each of the kin-dling stages in immunostained brain areas is shown inFig. 10.

4. Discussion

The results of the present study make clear a rela-tionship between the rearrangement of commissuralfibers, as indicated by radial glial cell expression, andthe progressive stages of kindling. Since radial glia hasbeen regarded as multiple-purpose precursors of neu-rons and glia (Campbell and Gotz, 2002), the expres-sion of radial glial marker protein may be related toneurogenesis and/or gliogenesis. However, no expres-sion in neuronal pericarya was observed in the presentstudy. Therefore, this may be expressed in glial cells.Glial fibrillary acidic protein and vimentin expressingcells have been associated with human temporal lobe


Fig. 7. Variation in 3CB2 staining during kindling in the fimbria hippocampus. (A) Luxol echt blue-stained section showing the location of thefimbria 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 alsoone of the marker proteins of radial glia (Nakagawa etal., 2004), we suggest that 3CB2 IR may be related topossible gliosis.

In the genu, rostrum, trunk and splenium, IR wasobserved with kindling stage progression. Since thepropagation of discharges is transmitted through thecommissural fibers of corpus callosum, some alter-ation may occur in the connections between the hemi-spheres. Previously, we have shown that the radial glialcell marker, vimentin, is increased in these regions(Nakagawa et al., 2004). The importance of the com-missural connection was not discussed in that study.However, in the present study, the analysis was focusedon commissural fibers in explanation of the former dataas well. It was for this purpose, that sagittal sectionswere used.

The IR of 3CB2 expanded from the rostrum to thetruncus in the corpus callosum from an early stage of

kindling. Changes in the corpus callosum began in thefrontal area and spread to the occipital region. Anotherstudy has reported on the evaluation and cortical pro-jection of temporal seizures in the cat, following anasymmetrical projection (Fernandez-Mas et al., 1992).A bisectional study has suggested that the anterior areain the corpus callosum contributes to inter-hemisphericsynchrony (Usuki et al., 1992) and these results sup-port the findings of the present study. During clinicalcallosotomy, the anterior two-third of the corpus callo-sum is often removed without any associated deficiency(Purves et al., 1988; Sakas and Phillips, 1996). Theresults of the present study also support the significanceof the extent of resection, in which the IR of the forcepsmajor was observed to differ from that of the cingulum,corpus callosum and alveus hippocampi. Furthermore,although the cingulum showed a strong IR at stageC3, the alveus hippocampi was an area where IR wasobserved at the C5 stage. On the other hand, the corpus


Fig. 8. Variation in 3CB2 staining during kindling in the thalamus. (A) Luxol echt blue-stained section showing the location of the optic thalamusof 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 arealso in agreement with the above-mentioned findings,which suggest that the splenium corpus callosum is notdirectly related to generalized seizures.

In the anterior commissure posterior, 3CB2 IR wasonly observed at the C5 stage, whereas no stainingwas observed in the anterior commissure anterior andintrabulber at any stage.McIntyre (1975) reportedthat the combined sectioning of the anterior cor-pus callosum and the anterior commissure blocks thepropagation of after-discharge in the inter-amygdala.Furthermore, chemical labeling using wheat germagglutinin-horseradish peroxidase has indicated stage-related staining in the anterior commissure (Condes-Lara et al., 2001), also supporting our data.

In the present study, IR at stage C5 was observed notonly in commissural fibers, but also in the cingulum,alveus hippocampi, fimbria hippocampus, brachiumof superior colliculus and thalamic areas. Vimentin

immunoreactivity was also observed in the cingulumwith stage elevation (Nakagawa et al., 2004), and c-fosexpression in amygdala kindling was reported at earlystages (Sato et al., 1998). Following systemic kainicacid administration, about 100 times more c-fos expres-sion was observed in the cingulum in both pre- and post-seizure states as compared to controls (Willoughbyet al., 1997). The alveus hippocampi and fimbriahippocampus are efferent, elongated regions project-ing from the hippocampus to the fornix. Bilateraltransection of the fimbria/fornix produces a decreasein the after-discharge threshold, clonus and the riseof seizure duration (Mohapel et al., 1997). Com-bined with these observations our findings suggestthat morphological changes in these areas at a latterstage may be introduced by seizure reinforcement anddevelopment.

The thalamus has been thought to be one of theimportant areas in limbic epileptogenesis (Mraovitch


Fig. 9. Variation in 3CB2 staining during kindling in the olfactory bulb. (A) Luxol echt blue-stained section showing the location of the lateralolfactory tract and glomerular layer in the olfactory bulb of rat brain. (B–D) Showing the stage-related increase in histochemical staining of3CB2. Kindling stage: C0 (B); C3 (C); C5 (D). Arrows indicate the increase in 3CB2-positive staining in the lateral olfactory tract and glomerularlayer. Scale bar: 1 mm.

and Calando, 1999). Increased WGA-HRP labelingwas found at the medial and lateral bed nuclei stria ter-minalis (Condes-Lara et al., 2001). Moreover, follow-ing carbachol injection into the thalamus, c-fos expres-sion was observed as generalized convulsive seizuresprogressed in the basal ganglia circuit (Mraovitch andCalando, 1999). C-fos expression was then observedfirst in the thalamus and was later detected in theanteroventral, laterodorsal and ventromedial hypotha-lamic nuclei. Finally, its expression has been observedin the superior colliculus, in the ventral posteromedialthalamic nucleus and in the ventrolateral nucleus ofthe reticular nucleus (Mraovitch and Calando, 1999).These data coincide with the 3CB2 staining data of thepresent study. Since the zona incerta is one of the impor-tant areas for carbachol-induced generalized seizures,3CB2 expression in this region would suggest signifi-cant seizure generalization.

Neuronal progenitor cell migration, from the sub-ventricular zone to the olfactory bulb, has been reportedin the rostral migratory stream (Winner et al., 2002;Yamada et al., 2004). The regeneration expandedfrom the rostral migratory stream to the granulecell and glomerular layers, respectively. Pilocarpin-induced seizures produce proliferating neuroblasts inthis region in rat (Parent et al., 2002). However, in thepresent study, IR in the olfactory bulb was observed inthe lateral olfactory tract and glomerular layer. Theseresults suggest that gliosis in the olfactory bulb maynot always be linked with neurogenesis.

The reason why we selected stage 3 seizures andrefer to them as partial seizures is as flows. At stage C3,the forelimb clonus was expanded to the contra-lateralside. However, the animals show no clear generalizedtonic clonic seizures. Hence, the stage C3 is thoughtto be one of the proceedings for kindling development.


Fig. 10. Density values according to clonus stage in six immunos-tained brain regions in amygdala-kindled mice. Density was mea-sured by Image-Pro Plus® software. Results are expressed asmean± 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 measuredincluded 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 inFig. 1.

This is also one reason why we call partial seizure atC3. In previous study of ours (Nakagawa et al., 2004),vimentin immunoreactivity was found to be highestat Clonus 3 decreasing at Clonus 5 in the hippocam-pal formation, regions around third ventricle, caudateputamen and lateral habenular nucleus. In contrast,vimentin immunoreactivity consistently increased withprogression of kindling in the cingulum and parietalcortex (Nakagawa et al., 2004). This may suggest thatdifferent staining proteins have different expressionsdepending on the kindling stage.

We have examined the morphological changes dur-ing and after kindling process since 1995 (Hosokawa etal., 1995; Umeoka et al., 2000; Miyazaki et al., 2003;Nakagawa et al., 2004). In order to be able to com-pare these studies with each other and each steps inthe procedure must be same. This was one of the mostimportant reasons why we selected the 2 h period for thesacrifice after final stimulation. The preliminary exper-iment showed no clear differences between 2 h after andone day after the final stimulation (data not shown).Another concern that may be raised is that radial glialmarker expression might be the same as “permanent”change as kindling. Since radial glial immunoreac-

tivities were increased after spinal cord injury anddecreased after 12 weeks, these results suggest theexpression of 3CB2 might be temporary (Shibuya etal., 2003). As 3CB2 is the antigen for the intermedi-ate filament and non-enzyme, the changes might notbe reflected in the enzymatic reaction. Further studiesmust be done to clarify these possibilities.

In conclusion, the results of the present study reaf-firm that the commissural fibers connecting the cerebralhemispheres may be necessary for kindling develop-ment. In addition, regional and quantitative changessuggest that specific transmission may be occurring inthe commissures during the kindling process. Thesefindings contribute to the understanding of generalizedseizures and development of remedies for epilepsy.

Acknowledgement

This work was supported by a Grant-in-Aid forScientific Research from the Ministry of Education,Science and Culture of Japan.

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Stage and region dependent expression of a radial glial marker in commissural fibers in kindled mice