Reorganization of CA3 area of the mouse ... - Wiley Online Library

Journal of Neuroscience Research 83:318–331 (2006)

Reorganization of CA3 Area of the Mouse Hippocampus After Pilocarpine Induced Temporal Lobe Epilepsy With Special Reference to the CA3-Septum Pathway Dong Liang Ma,1,6 Yong Cheng Tang,1 Peng Min Chen,1,7 Shwn Chin Chia,1 Feng Li Jiang,1 Jean-Marc Burgunder,5 Wei Ling Lee,2 and Feng Ru Tang1,3,4* 1

Epilepsy Research Lab, National Neuroscience Institute, Singapore Department of Neurology, National Neuroscience Institute, Singapore 3 Department of Anatomy, National University of Singapore, Singapore 4 Department of Medicine, National University of Singapore, Singapore 5 Department of Neurology, University of Berne, Switzerland 6 Neuroscience Research Center, School of Medicine, Xi’an Jiaotong University, Xi’an, People’s Republic of China 7 Department of Biochemistry, Institute of Clinical Research, Sino-Japanese Friendship Hospital, Beijing, People’s Republic of China 2

We showed that when CA3 pyramidal neurons in the caudal 80% of the dorsal hippocampus had almost disappeared completely, the efferent pathway of CA3 was rarely detectable. We used the mouse pilocarpine model of temporal lobe epilepsy (TLE), and injected iontophoretically the anterograde tracer phaseolus vulgaris leucoagglutinin (PHA-L) into gliotic CA3, medial septum and the nucleus of diagonal band of Broca, median raphe, and lateral supramammillary nuclei, or the retrograde tracer cholera toxin B subunit (CTB) into gliotic CA3 area of hippocampus. In the afferent pathway, the number of neurons projecting to CA3 from medial septum and the nucleus of diagonal band of Broca, median raphe, and lateral supramammillary nuclei increased significantly. In the hippocampus, where CA3 pyramidal neurons were partially lost, calbindin, calretinin, parvalbumin immunopositive back-projection neurons from CA1–CA3 area were observed. Sprouting of Schaffer collaterals with increased number of large boutons in both sides of CA1 area, particularly in the stratum pyramidale, was found. When CA3 pyramidal neurons in caudal 80% of the dorsal hippocampus have almost disappeared completely, surviving CA3 neurons in the rostral 20% of the dorsal hippocampus may play an important role in transmitting hyperactivity of granule cells to surviving CA1 neurons or to dorsal part of the lateral septum. We concluded that reorganization of CA3 area with its downstream or upstream nuclei may be involved in the occurrence of epilepsy. VC 2005 Wiley-Liss, Inc.

ganization in CA3 and CA1 areas with sprouting of axons from CA1 pyramidal neurons to other CA1 pyramidal neurons (Perez et al., 1996; Esclapez et al., 1999; Lehmann et al., 2000, 2001) or to CA3 neurons (Lehmann et al., 2001) and to neurons in the dentate gyrus (Bausch and McNamara, 2004), from the subiculum to CA1 and to the hilus of the dentate gyrus (Lehmann et al., 2001) has also been found to be related to epileptic activity. Limitation of in vitro studies using hippocampal slices makes it impossible, however, to show the abnormal connections between hippocampus and other brain regions such as medial and lateral septum, supramammillary, and median raphe nuclei. In previous anterograde tracing studies in the rat pilocarpine model (Lehmann et al., 2001), most of CA1 and CA3 pyramidal neurons were intact, therefore, the lack of replication of profound loss of CA1 and CA3 pyramidal neurons did not allow further characterization of the mechanism involved. In patients with TLE, trisynaptic neural pathway in the hippocampus might be interrupted, however, gliotic hippocampus still plays an important role for the occur-

Key words: reorganization; CA3 area; hippocampus; epilepsy; mouse

*Correspondence to: Dr. Feng-Ru Tang, Epilepsy Research Lab, National Neuroscience Institute, 11 Jalan Tan Tock Seng, Singapore 308433. E-mail: [email protected]

Mossy fiber sprouting in the dentate gyrus is well known to be involved in epileptogenesis in temporal lobe epilepsy (TLE). In hippocampal slices, axon reor' 2005 Wiley-Liss, Inc.

Contract grant sponsor: National Medical Research Council of Singapore; Contract grant number: NMRC/0670/2002, NMRC/0777/2003, NMRC/0731/2003, NMRC/0768/2003, NMRC/0960/2005.

Received 9 August 2005; Revised 4 October 2005; Accepted 20 October 2005 Published online 29 December 2005 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.20731

Rewiring of Epileptic Mouse Hippocampus

rence of spontaneously recurrent seizures (Tang and Lee, 2001; Tang et al., 2001c). Although Lehmann et al. (2000) was unable to demonstrate any aberrant connection in human hippocampus with severe neuronal loss in CA1 and CA3 area, stating difficulties to ‘‘imagine that such a ruined piece of network is still capable of contributing to seizure genesis and propagation,’’ surgical removal of such a hippocampus clearly reduced or abolished seizures in TLE due to mesial temporal sclerosis. A shifting of primary seizure foci of CA3 and CA1 areas to a secondary epileptogenic centre in the subiculum has been demonstrated (Cohen et al., 2002; Knopp et al., 2005). However, the role of surviving neurons in primary seizure foci of CA3 and CA1 still remains to be elucidated. We have shown recently the reinnervation of surviving CA1 neurons by sprouted mossy fibers, suggesting that surviving neurons in gliotic CA1 may act as a bridge between dentate gyrus and subiculum contributing toward epileptogenesis (Tang et al., 2005). In the present in vivo study in the mouse pilocarpine model of TLE, we show the reorganization of the afferent and efferent pathways of surviving CA3 neurons by iontophoretic injection of the anterograde tracer phaseolus vulgaris leucoagglutinin (PHA-L) into gliotic CA3, medial septum and the nucleus of diagonal band of Broca (MS-nDBB complex), median raphe, and lateral supramammillary nuclei, or the retrograde tracer cholera toxin B subunit (CTB) into gliotic CA3 area of hippocampus. By combined PHA-L or CTB with calbindin (CB), calretinin (CR), parvalbumin (PV), or choline acetyltransferase (ChAT) double immunostaining, we hoped to demonstrate chemical anatomy of preand post-synaptic neurons projecting to or receiving input from CA3 area in mice after pilocarpine-induced status epilepticus. The results may provide some clues for understanding epileptogenesis at both intra- and extra-hippocampal sites. MATERIALS AND METHODS Pilocarpine Treatment Male Swiss mice weighing 25–30 g were used for the study according to our established procedures (Tang et al., 2001a,b, 2004a,b, 2005). Mice were given a single subcutaneous injection of methyl-scopolamine nitrate (1 mg/kg) 30 min before the injection of either saline in the control or pilocarpine in the experimental group. In the latter group, the mice received a single intraperitoneal injection of 300 mg/kg pilocarpine and experienced acute status epilepticus (SE). All experiments were approved by the Tan Tock Seng Hospital, National Neuroscience Institute Institutional Animal Care and Use Committee. In the handling and care of all animals, the guidelines for animal research of NIH were strictly followed. Efforts were made throughout the study to minimize animal suffering and to use the minimum number of animals. These mice were used for iontophoretic injection of PHA-L or CTB at 60 days after pilocarpineinduced status epilepticus. Journal of Neuroscience Research DOI 10.1002/jnr

319

PHA-L or CTB Immunocytochemistry Iontophoretic injection of PHA-L or CTB into CA3 area of the hippocampus. In experimental mice at 60 days after pilocarpine-induced status epilepticus, the loss of CA3 pyramidal neurons in the hippocampus made it impossible to distinguish three parts of CA3 area, (i.e., CA3a, b, and c) (Tang et al., 2005). We viewed CA3 as a single entity in both the control and experimental mice although in the control mice we specifically injected the CA3b area. Forty-nine mice were used, 22 control (11 for PHA-L, 11 for CTB groups) and 27 experimental mice at 60 days after pilocarpine-induced status epilepticus (15 for PHA-L, 12 for CTB groups) were used. Mice were anesthetized with chloral hydrate (40 mg/kg) and fixed in a Stoelting stereotaxic apparatus. A small hole was drilled in the skull above the intended injection sites and a glass micropipette (diameter ¼ 20–30 lm) containing a 2.5% solution of PHA-L (Vector Laboratories, Burlingame, CA) in 0.1 M phosphate buffered saline (pH 7.4) or 10% CTB (List Biological Laboratories, Campbell, CA) in distilled water was lowered into CA3b area of the hippocampus in the control mice at 0.21 mm posterior to bregma, 0.26 mm lateral to midline, 0.18 mm ventral to dura or in the experimental mice at 0.20 mm posterior to bregma, 0.24 mm lateral to midline, 0.15 mm ventral to dura according to the atlas of Paxinos and Franklin (2001). PHA-L or CTB was delivered iontophoretically with positive current (5 lA: 7 sec on, 7 sec off) for 10–15 min. Seven days after PHA-L delivery, animals were deeply anesthetized, and perfused transcardially with 10 ml of saline, followed by 100 ml of 4% paraformaldehyde and 0.2% picric acid in 0.1 M PB (pH 7.4) for 30 min. Frozen coronal sections at 40-lm thickness were cut in a cryostat. Serial sections were transferred to different wells of a 24-well tissue culture dish for control, NeuN, PHA-L single labeling, PHA-L and calbindin (CB), calretinin (CR), parvalbumin (PV), choline acetyltransferase (ChAT), NeuN and CB double immunostaining in PHA-L injected mice, or for NeuN, CTB single labeling, CTB and CB, CR, PV, or ChAT double immunostaining in CTB injected mice. Iontophoretic injection of PHA-L into the medial septum and the nucleus of diagonal band of Broca (MS-nDBB complex), median raphe, and lateral supramammillary nuclei. Fifty-five mice, 31 control (10 MSnDBB complex, 11 median raphe nucleus, 10 lateral supramammillary nucleus) and 24 SE (10 MS-nDBB complex, 6 median raphe nucleus, 8 lateral supramammillary nucleus) were used for this study. PHA-L injection and immunostaining were done according to the protocols mentioned above. For tracer injection, glass micropipettes were lowered into MS-nDBB complex at 1.0 mm anterior to bregma, 0.5 mm lateral to midline with the angle of 88, 4.0 mm ventral to dura, into lateral supramammillary nucleus at 2.7 mm posterior to bregma, 0.8 mm lateral to midline, 4.8 mm ventral to dura, and into median raphe nucleus at 4.1 mm posterior to bregma, 1.0 mm lateral to midline, 4.2 mm ventral to dura according to the atlas of Paxinos and Franklin (2001). NeuN, PHA-L, PHA-L, and CB, CR, PV, ChAT, or NeuN, and CB Immunocytochemical Studies For NeuN or PHA-L immunocytochemical study, freely floating sections were washed in 0.1 M Tris buffered

320

Ma et al.

saline (TBS) containing 0.1% Triton-X 100 and placed overnight in primary goat antibody for PHA-L (1:5,000) (Vector Laboratories, Burlingame, CA), mouse antibody for NeuN (1:1,000) (Chemicon International, Inc., Temecula, CA), and for 2 hr in biotinylated horse anti-goat IgG for PHA-L or goat anti-mouse IgG for NeuN diluted 1:500 in TBS/Triton X-100. Sections were then incubated in avidin-biotin complex (ABC) reagent in TBS/Triton X-100 for 2 hr, and washed in Tris buffered (TB) and reacted in a solution of 0.12% H2O2 and 0.05% 3,30 -diaminobenzidine (DAB) (Sigma-Aldrich, St. Louis, MO) in TB for 20 min and mounted. Alternative PHA-L stained sections were counterstained with cresyl fast violet (CFV), coverslipped, and photographed by using image analysis system. For double labeling of PHA-L and CB, CR, PV, ChAT, sections were incubated in primary goat antibody for PHA-L (1:1,000) (Vector Laboratories) and rabbit antibodies for CB (1:200), CR (1:150), PV (1:150), ChAT (1:100) (Chemicon International, Inc.) for 48 hr, washed in TBS/Triton X-100, and placed in biotinylated horse anti-goat IgG (1:500) and swain anti-rabbit IgG (1:100) for 4 hr, incubated in ABC solution for 2 hr and reacted in DAB-Nickel solution for 20 min. Sections were then incubated in rabbit peroxidase anti-peroxidase (PAP) (1:100) solution overnight, and developed with DAB alone. For double labeling of NeuN and CB to outline intermediate part of the lateral septum (LSI) (most of neurons in this part are CB immunopositive), so as to show dorsal part of the lateral septum (LSD) for neuronal counting, sections were incubated overnight in primary mouse antibody for NeuN (1:100) and rabbit antibodies for CB (1:200) (Chemicon International, Inc.), then washed in PBS and placed for 1 hr in fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG against NeuN and Cy3 conjugated goat anti-rabbit IgG for CB, The sections were then mounted, dried, and coverslipped by using FluorSave Reagent (Calbiochem-Novabiochem, La Jolla, CA) to retard fading. In control sections, one or both primary antibodies were omitted. The tissue preparations were examined by using an Olympus FLUOVIEW FV500 Confocal Laser Scanning Biological Microscope.

NeuN, CTB, CTB, and CB, CR, PV, ChAT immunocytochemistry NeuN or CTB immunocytochemical study was done according to the similar protocol for NeuN or PHA-L immunostaining as stated as the above. Primary goat anti-CTB (List Biological Laboratories, Campbell, CA) instead of anti-PHA-L antibody (1:5,000) was used. For CTB and CB, CR, PV, ChAT immunocytochemistry to characterize the retrogradely labeled neurons in the MS-nDBB, lateral supramammillary or median raphe nucleus, sections were incubated overnight in primary goat antibody for CTB (1:1,000) and rabbit antibodies for CB (1:200), CR (1:150), PV (1:150), ChAT (1:100), then washed in PBS and placed for 1 hr in FITC-conjugated goat anti-rabbit IgG against CB, CR, PV, ChAT, and Cy3 conjugated goat anti-goat IgG for CTB, The sections were then processed for NeuN and CB double immunostaining.

Data Analysis In the present study, the identification of different brain regions was made according to the stereotaxic coordinates of Paxinos and Franklin (2001). Quantitative analysis was done by using Imaging-Pro Plus (MediaCybernetics, Silver Spring, MD). Immunostained neuronal profiles in different brain regions such as the rostral part of CA3 area of the hippocampus, lateral septum, medial septum, and MS-nDBB complex, lateral supramammillary or median raphe nucleus were counted and indicated as a number per square millimeter (No/mm2) in mean value 6 standard deviation (SD). In the rostral part of CA3 area of the hippocampus, neuronal loss was indicated as a percentage. In MS-nDBB complex, lateral supramammillary or median raphe nuclei, the percentages of the number of CTB and CB, CR, PV, ChAT double labeled neurons to all the CTB labeled neurons was obtained. Neuronal profile counting was done by an investigator who was blind to the experimental conditions to which the mice had been subjected. As comparative studies were done among different groups of mice, the number of neuronal profiles was not calibrated. The data obtained were then subjected to statistical analysis by t-test for control and experimental groups. A P-value of 0.05). PHA-L Immunocytochemistry PHA-L injection sites in CA3 area of the hippocampus. Eleven control and fifteen SE mice were used for PHA-L injection. Four control and three SE mice for PHA-L were excluded from data analysis due to incorrect injection sites. PHA-L immunostaining demonstrated that the injection sites were mainly located in CA3b area in the control mice. In SE mice, due to the shrinkage of CA3 area after gliosis, adjacent granule cells in the upper blade of dentate gyrus were also labeled occasionally. Quantitative study showed that in the transverse section, the diameter of PHA-L injection Journal of Neuroscience Research DOI 10.1002/jnr


321

Fig. 1. NeuN immunocytochemistry shows partial neuronal lost in CA3 and CA1 in caudal (A, in the control; B, in SE, arrowheads) and rostral (E, in SE; D, in the control) parts of dorsal hippocampus. C: CA3 and CA1 neurons have almost disappeared completely in caudal part of dorsal hippocampus. In rostral part of CA3 area of the same hippocampus, only partial neuronal loss occurs (F). NeuN (green) and CB (red) double labeling shows the boundary between

the intermediate part of the lateral septum (LSI) and dorsal part of the lateral septum (LSD) (white stippled line in G,H). In the LSD, only a few CB immunopositive neurons are demonstrated (G,H). In LSD of SE mouse, NeuN immunopositive neurons appear reduced (H). Scale bar ¼ 150 lm (A); 100 lm (B,C); 75 lm (D–F); 50 lm (G,H).

site was 387.7 6 27.15 lm in the control and 388.8 6 22.89 lm in SE mice. No significant difference was shown between the two groups of mice by unpaired ttest (P > 0.05). The septo temporal extent of the PHAL injection site was 626.7 6 24.59 lm in the control and 613.3 6 26.67 lm in the SE mice respectively. No significant difference was shown between the two groups of mice by unpaired t-test (P > 0.05). PHA-L immunostaining in the LSD and MSnDBB complex. When PHA-L was iontophoretically

injected into CA3b area of the hippocampus of the control mice, PHA-L immunopositive axons and terminals were demonstrated mainly in the ipsi- and contra-lateral LSD (Fig. 2A), and in MS-nDBB complex. In the bilateral LSD, dense PHA-L immunopositive axons and terminals were shown. In MS-nDBB complex, however, the labeled axons and terminals were sparsely distributed. In SE mice at 60 days after pilocarpine-induced status epilepticus, when CA3 neurons had almost completely disappeared, PHA-L immunopositive axons and termi-

Journal of Neuroscience Research DOI 10.1002/jnr

Fig. 2. PHA-L immunopositive staining shows dense PHA-L immunopositive axons and terminals in the ipsi- and contra-lateral dorsal part of the lateral septum (LSD) (A), and moderate staining in the medial septum (MS) (A) after iontophoretic injection of PHA-L into CA3b area of the hippocampus in the control mouse. In SE mouse, however, when CA3 neurons have almost disappeared completely in the injection site, PHA-L immunopositive axons and terminals are rarely seen in LSD and MS (B). In the ipsilateral hippocampus of the control mouse, the densest PHA-L immunopositive staining in CA1 and CA3 area was demonstrated in the deep part of stratum radiatum in the ipsilateral, however, in the contralateral site, the densest PHAL immunopositive staining in CA1 and CA3 area is demonstrated in the stratum oriens (so) (C). Moderate PHA-L immunopositive axons

and terminals were shown in the stratum radiatum (sr) (C). In the stratum pyramidale, they are rarely seen. In SE mouse, when CA3 and CA1 neurons have almost disappeared completely, PHA-L immunopositive axons and terminals are almost undetectable in the above area (D). When CA3 and CA1 neurons are partially lost (F,H,I,J in SE compared to E,G in the control mouse), sprouted axons and terminals are found not only in the strata radiatum (sr) and oriens (so), but also in the stratum pyramidal (sp) (Fig. F,I). More boutons in PHA-L immunopositive axons are shown in CA1 area in SE mouse when compared to the control (H in SE; G in the control mouse). Boutons in SE mouse also become larger (H–J). Scale bar ¼ 100 lm (A–D); 25 lm (E,F); 10 lm (G–J). Figure can be viewed in color online via www.interscience.wiley.com.

Fig. 3. Double immunostaining shows the contact between PHA-L immunopositive boutons and terminals (black, indicated by arrowheads) and PV (A), CB (B), and CR (C) immunopositive neurons (brown) in CA1 areas in the control mouse. In SE mouse, sprouted Schaffer collaterals with larger boutons and terminals are found (D,E). Surviving CR (D), CB (F), or PV (G) immunopositive neurons are also contacted by PHA-L immunopositive boutons and terminals (arrowheads). In the hilus of the dentate gyrus of the control

mouse, the contacts between PHA-L immunopositive boutons or terminals with CR (H) or CB (I) immunopositive neurons are demonstrated. These contacts have almost completely disappeared in SE mouse. In the septum of the control mouse, PHA-L immunopositive boutons or terminals contact with CB immunopositive neurons in LSD (J) and MS-nDBB complex (K), with CR (L), ChAT (M), and PV (N) immunopositive neurons in MS-nDBB complex. Scale bar ¼ 10 lm (A–N).

324

Ma et al.

Fig. 4. CTB immunopositive staining shows CTB injection site in the control (A) and SE (B) mice. White circle indicates the centre of CTB injection site. Note that stronger background staining in (B) is caused by no-specific staining of reactive astrocytes in gliotic hippocampus. Scale bar ¼ 100 lm. Figure can be viewed in color online via www.interscience.wiley.com.

nals were rarely detected in LSD and MS-nDBB complex (Fig. 2B). When CA3 neurons were partially lost, reduced PHA-L immunopositive axons and terminals were shown in the two areas. PHA-L immunostaining in the hippocampus. When PHA-L was iontophoretically injected into CA3b area, the densest PHA-L immunopositive staining in CA1 and CA3 area was demonstrated in the deep part of stratum radiatum in the ipsilateral, and in the stratum oriens in the contralateral (Fig. 2C) hippocampus in the control mice. Moderate PHA-L immunopositive axons and terminals were shown in the stratum oriens of the ipsilateral and stratum radiatum of the contralateral CA3 and CA1 area (Fig. 2C). In the stratum pyramidale, they were seen occasionally. In SE mice, when CA3 and CA1 neurons had almost completely disappeared, few PHA-L immunopositive axons and terminals were detected in the above area (Fig. 2D). When CA3 and CA1 neurons were only partially lost, however, sprouting axons and terminals were found not only in the strata radiatum and oriens, but also in the stratum pyramidal (Fig. 2E,G in the control and 2F,H–J in SE mice). High magnification quantitative assessment showed that the number of boutons in PHA-L immunopositive axons in the stratum radiatum of CA1 area increased significantly in SE mice (Fig. 2H) (the density of boutons was 1/per 4.98 6 0.47 lm in the control and 1/per 3.68 6 0.19 lm in SE mice, P < 0.05). Boutons in SE mice were also larger (Fig. 2H–J) (the area range of boutons was from 0.45–2.10 lm2 in the control, and from 1.04– 19.15 lm2 in SE mice). PHA-L and CB, CR, PV, ChAT double labeling. Double immunostaining showed that PHA-L immunopositive boutons and terminals contacted with PV (Fig. 3A), CB (Fig. 3B), and CR (Fig. 3C) immunopositive neurons in CA1 areas in the control. In SE mice, the surviving CR (Fig. 3D, E), CB (Fig. 3F), or PV (Fig. 3G) immunopositive neurons were also contacted by PHA-L immunopositive boutons and terminals. In the hilus of the dentate gyrus of the control mice, the contacts between PHA-L immunopositive

boutons or terminals with CR (Fig. 3H), CB (Fig. 3I) immunopositive neurons were found. These contacts were rarely seen in SE mice. In the septum of the control mice, PHA-L immunopositive boutons or terminals had contacts with CB immunopositive neurons in LSD (Fig. 3J) and MS-nDBB complex (Fig. 3K), with CR (Fig. 3L), ChAT (Fig. 3M), and PV (Fig. 3N) immunopositive neurons in MS-nDBB complex. Such contacts were reduced in SE mice. CTB Immunocytochemistry CTB injection sites in CA3 area of the hippocampus. Eleven controls and twelve SE mice were used for CTB injection. Five controls and three SE for CTB injection were excluded from data analysis due to incorrect injection sites. CTB immunostaining demonstrated that the injection sites were mainly located in CA3b area in the control (Fig. 4A) and SE (Fig. 4B) mice. In SE mice, no specific background staining on reactive astrocytes was obvious when compared to the control. Quantitative study showed that in the transverse section, the diameter of CTB injection site was 392.4 6 22.13 lm in the control and 348.2 6 8.57 lm in SE mice. No significant difference was shown between the two groups of mice by unpaired t-test (P > 0.05). The septo temporal extent of the CTB injection site was 608.0 6 34.41 lm in the control and 616.0 6 53.07 lm in the SE mice. No significant difference was shown between the two groups of mice by unpaired t-test (P > 0.05). When CTB was injected into CA3 area of the control mice, retrogradely labeled cells were observed in the ipsilateral stratum granulosum of the dentate gyrus, in the ipsilateral and contralateral rostral or caudal part of CA3 region (Fig. 5A), particularly in the former. A few labeled neurons were also found in the hilus of the dentate gyrus of both sides (Fig. 5A). No labeled granule cell was observed in the contralateral dentate gyrus. In both sides of CA1 and CA3 areas, anterogradely labeled CTB immunopositive axon and terminals were also demonstrated. In SE mice, when CA3 pyramidal neuJournal of Neuroscience Research DOI 10.1002/jnr

Fig. 5. CTB immunopositive staining shows retrogradely labeled pyramidal neurons in the contralateral caudal (A, counterstained with cresyl fast violet) or rostral (C) part of CA3 area after CTB injection into the CA3 area of the control mouse. A few labeled neurons are also found in the hilus (h) of the dentate gyrus of the both sides (A, arrowhead). In both sides of CA1and CA3 areas, anterogradely labeled CTB immunopositive axon and terminals are demonstrated (A), which is similar to PHA-L immunostaining in (C). In SE mouse, when CA3 pyramidal neurons are partially lost, only a few CTB labeled neurons could be found in caudal part of both side of CA3 area of hippocampus (B). No CTB immunopositive axons or immu-

nopositive cells were detected in the contralateral caudal part of CA3 and CA1 areas when CA3 and CA1 pyramidal neurons have almost disappeared completely. Granule cells in the ipsilateral dentate gyrus and CA3 neurons in the rostral part of both sides of the dorsal hippocampus (C,D) are still labeled despite loss of almost all CA3 pyramidal neurons in the injection site. In the ipsilateral lateral supramammillary nucleus (E in the control; F in SE mouse), MS-nDBB complex (G in the control; H in SE mouse), and median raphe nucleus (I in the control; J in SE mouse), CTB labeled neurons seem increased. Scale bar ¼100 lm (A–H); 50 lm (I,J). Figure can be viewed in color online via www.interscience.wiley.com.

326

Ma et al.

TABLE I. CTB Labeled Neurons in Different Parts of the Brain After Injection Into CA3 Area of the Hippocampus*

Control SE P-value

MS-nDBB

SUM

MR

Rostral CA3

34.5 6 2.3 129.2 6 21.6 0.05) and in the lateral supramammillary nucleus, 14.45 6 1.27% and 14.44 6 0.74% of them were CR immunopositive in the control and SE mice respectively (Fig. 6I,J) (P > 0.05). In the gliotic CA1 area, some CTB immunopositive neurons were also found. They were CB (Fig. 6K), CR (Fig. 6L), or PV (Fig. 6M) immunopositive. In the hilus of the dentate gyrus of the control mice, retrogradely labeled CTB immunopositive neurons were CR immunopositive (Fig. 6N, N0 ). Iontophoretic Injection of PHA-L Into MS-nDBB Complex, Median Raphe, and Lateral Supramammillary Nuclei When PHA-L was iontophoretically injected into MS-nDBB complex , median raphe, and lateral supramammillary nuclei, PHA-L immunopositive axons with boutons and terminals were found in CA3 area in the control (MS-nDBB complex, Fig. 7A; median raphe nucleus, Fig. 7D; lateral supramammillary nucleus, Fig. 7G) and SE mice at 60 days after pilocarpine-in-

duced status epilepticus (MS-nDBB complex, Fig. 7B,C; median raphe nucleus, Fig. 7E,F; lateral supramammillary nucleus, Fig. 7H,I). In gliotic CA3 area, large (with diameter >5 lm) PHA-L immunopositive bouton- or terminal-like structures (Fig. 7B,C,E,F) or axon expansion (Fig. 7H,I) were demonstrated, and some of them aggregated to form grape-bundle-like structures (Fig. 7B, C,F). In the control mice, the area of axon terminals or boutons in CA3 area from MS-nDBB complex, median raphe, and lateral supramammillary nuclei ranged from 1.17–2.67 lm2 (Fig. 7A,D,G). In SE mice it ranged from 1.28–47.92 lm2 (Fig. 7B,C,E,F,H,I). DISCUSSION Methodologic Consideration In the present study, although there was no significant difference in the dimensions of PHA-L or CTB injection sites, the upper blade of the dentate gyrus near CA3 area was always stained in SE mice due to shrinkage of CA3 area. This diffusion of PHA-L may not affect efferent pathway of gliotic CA3 area due to the limited projecting site of granule cells. The afferent pathway may be amplified, however, because CTB could also label some neurons in MS-nDBB complex, median raphe, and lateral supramammillary nuclei that project to the dentate gyrus. As PHA-L or CTB was injected mainly into CA3 area, it was impossible for us to do a control study by injecting just the two tracers into a very small portion of the upper blade of the dentate gyrus near CA3 area, to exclude retrogradely labeled dentate gyrus projecting neurons from MS-nDBB complex, median raphe, and lateral supramammillary nuclei in the quantitative study. Due to the staining of a very small portion of dentate gyrus, more retrogradely labeled neurons in these areas might be found, but it should not affect our quantitative analysis significantly, as we noticed that diffused CTB far away from the injected point would not produce any retrograde labeling. CA3 Area and Epileptogenesis Neuronal loss in CA3 area of the hippocampus from patients with TLE is one of the characteristic neuropathologic changes. Systemic or local injection of kainic acid, pilocarpine, or other substances have been used to produce temporal lobe epilepsy in animal models (Nitecka et al., 1984; Tremblay et al., 1984; Dube et al., 1998; Covolan and Mello, 2000; Magloczky and Freund, 1993, 1995; Tang et al., 2004a,b). Few studies have been done, however, to explain why recurrent seizures occur spontaneously after loss of neurons in CA3 area or interruption of trisynaptic neural pathway in hippocampus. Previous calbindin immunostaining in our group has shown the existence of calbindin immunopositive mossy fibers and terminals in gliotic CA3 area (Chen et al., 2004; Zhang et al., 2004). This finding was further supported by our recent study showing many PHA-L immunopositive mossy fibers and terminals in gliotic Journal of Neuroscience Research DOI 10.1002/jnr


Fig. 6. CTB (red) and CB, CR, PV, ChAT (green) double labeling shows that in MSnDBB complex, some CTB immunopositive neurons are CB (A,A0 ,B,B0 ), ChAT (C,D), and PV (E,F) immunopositive in the control and SE mice respectively. In the median raphe (G,H) and lateral supramammillary nuclei, some CTB immunopositive neurons are CR immunopositive (I,J). In the gliotic CA1 area, some CTB immunopositive neurons are CB (K), CR (L), or PV (M) immunopositive. In the hilus of the dentate gyrus of the control mouse, retrogradely labeled CTB immunopositive neurons are CR immunopositive (N,N0 ). Scale bar ¼ 100 lm (A,B); 25 lm (C–H); 50 lm (K–N); 10 lm (A0 ,B0 , I,J,N0 ).


327

328

Ma et al.

Fig. 7. PHA-L immunopositive staining shows MS-nDBB– CA3 (A in the control; B,C in SE mouse), MS–CA3 (D in the control; E,F in SE mouse) and supramammillary–CA3 (G in the control; H,I in SE mouse) projecting axons with boutons and terminals in CA3 area. Note that in gliotic CA3 area, large (with diameter >5 lm) PHA-L immunopositive bouton- or terminal-like structures (arrowheads in B,C,E) or axon expansions (arrowheads in H,I) are demonstrated. Some of axons with boutons and terminals aggregate to form grape-bundle-like structures (F). Scale bar ¼ 10 lm (A–I). Figure can be viewed in color online via www.interscience.wiley.com.

CA3 area after iontophoretic injection of PHA-L into the granule layer of the dentate gyrus (Tang et al., 2005). This suggests that synaptic activity still exist in the gliotic CA3 area. In the present study, when CA3 pyramidal neurons had almost disappeared completely, the efferent of CA3 was rarely detected in both sides of CA1, CA3, and LSD. The number of CA3 projecting neurons from the MS-nDBB complex, median raphe, and lateral supramammillary nuclei increased signifi-

cantly, however, suggesting the reorganization of CA3– LSD, MS-nDBB–CA3, median raphe–CA3, or lateral supramammillary–CA3 pathways. In the hippocampus, when CA3 pyramidal neurons were partially lost, CB, CR, or PV immunopositive back-projection neurons from CA1–CA3 area were found. PHA-L anterograde labeling showed sprouting of Schaffer collaterals with increased number of larger boutons in both sides of CA1 area. Journal of Neuroscience Research DOI 10.1002/jnr


Intra-Hippocampal Connection of CA3 Areas in SE Mice In the hippocampal slices of the rat pilocarpine model, Lehmann et al. (2001) found back-projection of CA1 neurons to CA3 area. It was confirmed by the present in vivo CTB retrograde labeling in the mouse pilocarpine model. We showed that some back-projection neurons in CA1 area were CB, CR, or PV immunopositive, suggesting that they were interneurons. Furthermore, our PHA-L anterograde labeling has shown sprouting of Schaffer collaterals with increased number of larger boutons in both sides of CA1 area. The sprouted boutons or axon terminals have established perisomatic contact with neurons in the stratum pyramidale in SE mice, which was rarely seen in controls, suggesting that such a perisomatic innervation may be related to the hyperactivity of CA1 pyramidal neurons and epileptogenesis. The demonstration of the contacts between PHA-L immunopositive axon terminals and boutons with CB, CR, and PV immunopositive neurons suggests the existence of the forward inhibition in CA1 area in epilepsy. Previous in vivo study showed that the dentate gyrus initially restricted the entry of seizures from entorhinal cortex into the rest of hippocampus. When this was overcome, there was rapid bilateral spread through the hippocampal formation (Collins et al., 1983). In vitro study showed that the epileptogenic injury reduced inhibition of layer II neurons in the entorhinal cortex and resulted in excessive synaptic input to the dentate gyrus (Kobayashi et al., 2003). In the present NeuN immunostaining, CA1 and CA3 pyramidal neurons in caudal 80% of the dorsal hippocampus had almost disappeared completely in 16 of 27 SE mice, however, most of CA3 pyramidal neurons in the rostral 20% of the dorsal hippocampus still existed. This suggests that this part of CA3 may play an important role in transmitting hyperactivity of granule cells to surviving CA1 neurons in the rostral portion of the dorsal hippocampus or to LSD. CA3–LSD Pathway When muscarinic agonist bethanechol chloride was injected into CA3 subfield of the dorsal hippocampus in unrestrained rats, EEG showed spiking activity of high frequency in the injected hippocampus, with rapid propagation to the lateral septum. Subsequent neuropathologic alterations were observed in this area (Turski et al., 1983). Intense electrographic seizures were also induced by microinjection of naloxone into the lateral septum (Calder et al., 1982). In weanling rats with genetic absence epilepsy, metabolic activity is increased in the lateral septum before the occurrence of spike-and-wave discharges (Nehlig et al., 1998). Lateral septum may play some roles in the development of epilepsy when combined with the prominent loss of neurons in the lateral septum of the rat kainic acid and pilocarpine models of epilepsy (Nitecka et al., 1984; Tremblay et al., 1984; Dube et al., 1998; Covolan and Mello 2000). In our Journal of Neuroscience Research DOI 10.1002/jnr

329

present study, significant loss of neurons in LSD was also observed in experimental mice. When PHA-L was injected into CA3 area of SE mice, loss of its immunopositive axons and terminals in LSD may suggest the deactivation of surviving neurons, leading to a hypoactive or even ‘‘dormant’’ LSD. It is known that lateral septal nucleus projects to the supramammillary nucleus, which projects back to the hippocampus and median septum (Risold and Swanson, 1997). We speculate, therefore, that hypoactive LSD may be indirectly involved in epileptic activity. MS-nDBB Complex–, Lateral Supramammillary–, and Median Raphe–CA3 Pathways MS-nDBB complex–hippocampal neurons are responsible for the anti-epileptogenic effect of the cholinergic system in amygdala or hippocampal kindling (Miller et al., 1994; Ferencz et al., 2000, 2001). Medial septal carbachol injections during electrically kindled limbic status epilepticus stopped ictal behavior and reduced EEG spike rate (Miller et al., 1994). Microinjection of kainic acid into the medial septum produced seizures with a longer latency (Ben-Ari et al., 1980). Electrolytic medial septal lesions abolished hippocampal u activity and lowered myoclonic and facial–forelimb pentylenetetrazol (PTZ) seizure thresholds. Medial septal electrical stimulation at 4–8 Hz during PTZ induced facial–forelimb seizures, stopped behavioral seizures, and EEG spiking. As u rhythm has a cholinergic component (Whishaw et al., 1978; Brazhnik et al., 1986), it is possible that medial septum cholinergic activation of hippocampal interneurons may play a role in anti-epileptogenesis. A recent study has also shown that a complex interplay between glutamatergic afferents from the entorhinal cortex and cholinergic afferents from the medial septum could be involved in the normal regulation of granule cell function, and any change of their relationships may result in the hyperactivity of granular cells, leading to temporal lobe epilepsy (Frazier et al., 2003). Supramammillary nucleus, particularly the lateral part, innervates the hippocampal formation with excitatory neurotransmitter (Vertes, 1992; Kiss et al., 2000). Most postsynaptic targets of its projection are principal cells in the dentate gyrus and in the CA2–CA3a subfields, suggesting a mechanism other than disinhibition is responsible for the facilitatory effect of this pathway on hippocampal evoked activity (Magloczky et al., 1994). Acute interruption of the facilitatory hypothalamic afferents by intra supramammillary injection of muscimol may deprive the hippocampal intensification mechanism produced by supramammillary–hippocampal pathway and block the genesis and spread of limbic seizures in the hippocampus. It may, therefore, not be related to the inactivation of the disinhibition mechanism as suggested by Saji et al. (2000). A qualitative study by Magloczky et al. (1994) demonstrated that in the rat supramammillary nucleus, majority of retrogradely labeled neurons was CR immu-

330

Ma et al.

nopositive. In the present study, only14.45 6 1.27% and 14.44 6 0.74% of CTB immunopositive neurons were CR immunopositive in the control and SE mice, respectively. The number of CTB immunopositive neurons increased significantly in SE mice, however, the proportion of CR immunopositive neurons in all CTB labeled neurons did not change significantly in the control and SE mice. The lower percentage of CR immunopositive CTB labeled neurons in the present study may suggest the existence of species difference for CR projecting neurons from supramammillary nucleus to the hippocampus between the rat and mouse. Direct projection from median raphe nucleus to hippocampus has been reported to mainly innervate hippocampal interneurons (Freund et al., 1990; Halasy et al., 1992). Neurophysiologic study showed that electrical stimulation of the raphe nucleus induced inhibition of the penicillin-induced seizure activity. It was supported by neuropharmacologic approaches showing that adrenoblocking agents facilitate the effects of raphe nucleus on the seizure activity (Khanbabian et al., 1986). In the epileptogenic hippocampus and amygdala, a reduced 5-HT1A receptor binding potential was demonstrated by PET imaging (Savic et al., 2004). Neurosurgical destruction of serotoninergic terminals in the epileptic focus (olfactory bulb or amygdala) facilitated the initial development of kindling obtained by stimulations of these structures (Lerner-Natoli, 1987). Transplantation of fetal raphe tissue promotes lasting reductions in increased seizure severity resulting from depletion of serotonin in the brain of genetically epilepsy-prone rats (GEPR-3s) (Clough et al., 1996). It suggests that median raphe 5-HT system may involve in the desynchronization of the hippocampal EEG. In our present study, CTB immunopositive neurons in the MS-nDBB complex, lateral supramammillary, and median raphe nuclei increased significantly after CA3 injection in SE mice when compared to the control. This may suggest the sprouting of adjacent CA1 or dentate gyrus projecting fibers from all three areas. Borhegyi et al. (1998) showed that at least 25% of the supramammillary–hippocampal cells also projected to MS-nDBB complex. As the number of CTB labeled neurons increased in the supramammillary nucleus and MS-nDBB complex, it is possible that supramammillary nucleus may strongly facilitate epileptogenicity through supramammillary–hippocampal and supramammillary– MS-nDBB complex hippocampal pathways. Our previous study has demonstrated newly sprouted dendrites with growth cone-like dendritic spines in CA3 area (Tang et al., 2005), they may form the postsynaptic elements of the sprouted fibers from median raphe nucleus, MS-nDBB complex, or supramammillary nucleus. The enlargement of axon terminals and boutons or probably degenerating axon expansion in CA3 areas from MSnDBB complex, median raphe, and lateral supramammillary nuclei may also increase the uptake of CTB from the injection site, leading to retrograde labeling of more neurons.

ACKNOWLEDGMENT This study was supported by research grants from the National Medical Research Council of Singapore to F.R.T. REFERENCES Bausch SB, McNamara JO. 2004. Contributions of mossy fiber and CA1 pyramidal cell sprouting to dentate granule cell hyperexcitability in kainic acid-treated hippocampal slice cultures. J Neurophysiol 92:3582– 3595. Ben-Ari Y, Tremblay E, Ottersen OP, Meldrum BS. 1980. The role of epileptic activity in hippocampal and ‘‘remote’’ cerebral lesions induced by kainic acid. Brain Res 191:79–97. Borhegyi Z, Magloczky Z, Acsady L, Freund TF. 1998. The supramammillary nucleus innervates cholinergic and GABAergic neurons in the medial septum-diagonal band of Broca complex. Neuroscience 82: 1053–1065. Brazhnik ES, Vinogradova OS. 1986. Control of the neuronal rhythmic bursts in the septal pacemaker of theta-rhythm: effects of anesthetic and anticholinergic drugs. Brain Res 380:94–106. Calder LD, Snyder EW, Dustman RE. 1982. Naloxone-induced epileptogenesis has brain-site specificity in rats. Neuropharmacology 21:1001– 1004. Chen PM, Chia SC, Lee WL, Tang FR. 2004. Calcium-binding protein (CBP) immunopositive neurons in the gliotic mouse hippocampus after pilocarpine induced temporal lobe epilepsy. Proceedings of International Biomedical Science Conference, Kunmin, China. Clough R, Statnick M, Maring-Smith M, Wang C, Eells J, Browning R, Dailey J, Jobe P. 1996. Fetal raphe transplants reduce seizure severity in serotonin-depleted GEPRs. Neuroreport 8:341–346. Cohen I, Navarro V, Clemenceau S, Baulac M, Miles R. 2002. On the origin of interictal activity in human temporal lobe epilepsy in vitro. Science 298:1418–1421. Collins RC, Tearse RG, Lothman EW. 1983. Functional anatomy of limbic seizures: focal discharges from medial entorhinal cortex in rat. Brain Res 280:25–40. Covolan L, Mello LE. 2000. Temporal profile of neuronal injury following pilocarpine or kainic acid-induced status epilepticus. Epilepsy Res 39:133–152. Dube C, Andre V, Covolan L, Ferrandon A, Marescaux C, Nehlig A. 1998. C-Fos, Jun D and HSP72 immunoreactivity, and neuronal injury following lithium-pilocarpine-induced status epilepticus in immature and adult rats. Brain Res Mol Brain Res 63:139–154. Esclapez M, Hirsch JC, Ben-Ari Y, Bernard C. 1999. Newly formed excitatory pathways provide a substrate for hyperexcitability in experimental temporal lobe epilepsy. J Comp Neurol 408:449–460. Ferencz I, Leanza G, Nanobashvili A, Kokaia M, Lindvall O. 2000. Basal forebrain neurons suppress amygdala kindling via cortical but not hippocampal cholinergic projections in rats. Eur J Neurosci 12:2107– 2116. Ferencz I, Leanza G, Nanobashvili A, Kokaia Z, Kokaia M, Lindvall O. 2001. Septal cholinergic neurons suppress seizure development in hippocampal kindling in rats: comparison with noradrenergic neurons. Neuroscience 102:819–832. Frazier CJ, Strowbridge BW, Papke RL. 2003. Nicotinic receptors on local circuit neurons in dentate gyrus: a potential role in regulation of granule cell excitability. J Neurophysiol 89:3018–3028. Freund TF, Gulyas AI, Acsady L, Gorcs T, Toth K. 1990. Serotonergic control of the hippocampus via local inhibitory interneurons. Proc Natl Acad Sci USA 87:8501–8505. Halasy K, Miettinen R, Szabat E, Freund TF. 1992. GABAergic interneurons are the major postsynaptic targets of median raphe afferents in the rat dentate gyrus. Eur J Neurosci 4:144–153. Journal of Neuroscience Research DOI 10.1002/jnr

Rewiring of Epileptic Mouse Hippocampus Khanbabian MV, Sarkisian RSh, Sarkisian LV. 1986. Interaction of adrenergic and serotoninergic mechanisms in modulation of seizure activity of the brain. Fiziol Zh SSSR Im I M Sechenova 72:306–310. Kiss J, Csaki A, Bokor H, Shanabrough M, Leranth C. 2000. The supramammillo-hippocampal and supramammillo-septal glutamatergic/aspartatergic projections in the rat: a combined [3H]D-aspartate autoradiographic and immunohistochemical study. Neuroscience 97:657–669. Knopp A, Kivi A, Wozny C, Heinemann U, Behr J. 2005. Cellular and network properties of the subiculum in the pilocarpine model of temporal lobe epilepsy. J Comp Neurol 483:476–488. Kobayashi M, Wen X, Buckmaster PS. 2003. Reduced inhibition and increased output of layer II neurons in the medial entorhinal cortex in a model of temporal lobe epilepsy. J Neurosci 23:8471–8479. Lehmann TN, Gabriel S, Kovacs R, Eilers A, Kivi A, Schulze K, Lanksch WR, Meencke HJ, Heinemann U. 2000. Alterations of neuronal connectivity in area CA1 of hippocampal slices from temporal lobe epilepsy patients and from pilocarpine-treated epileptic rats. Epilepsia 41(Suppl):S190–S194. Lehmann TN, Gabriel S, Eilers A, Njunting M, Kovacs R, Schulze K, Lanksch WR, Heinemann U. 2001. Fluorescent tracer in pilocarpinetreated rats shows widespread aberrant hippocampal neuronal connectivity. Eur J Neurosci 14:83–95. Lerner-Natoli M. 1987. Serotonin and kindling development. Int J Neurosci 36:139–151. Magloczky Z, Acsady L, Freund TF. 1994. Principal cells are the postsynaptic targets of supramammillary afferents in the hippocampus of the rat. Hippocampus 4:322–334. Magloczky Z, Freund TF. 1993. Selective neuronal death in the contralateral hippocampus following unilateral kainate injections into the CA3 subfield. Neuroscience 56:317–335. Magloczky Z, Freund TF. 1995. Delayed cell death in the contralateral hippocampus following kainate injection into the CA3 subfield. Neuroscience 66:847–860. Miller JW, Turner GM, Gray BC. 1994. Anticonvulsant effects of the experimental induction of hippocampal theta activity. Epilepsy Res 18:195–204. Nehlig A, Vergnes M, Boyet S, Marescaux C. 1998. Metabolic activity is increased in discrete brain regions before the occurrence of spike-andwave discharges in weanling rats with genetic absence epilepsy. Brain Res Dev Brain Res 108:69–75. Nitecka L, Tremblay E, Charton G, Bouillot JP, Berger ML, Ben-Ari Y. 1984. Maturation of kainic acid seizure-brain damage syndrome in the rat. II. Histopathological sequelae. Neuroscience 13:1073–1094. Paxinos G, Franklin KBJ. 2001. The mouse brain in stereotaxic coordinates. 2nd Edition. San Diego: Academic Press. Perez Y, Morin F, Beaulieu C, Lacaille JC. 1996. Axonal sprouting of CA1 pyramidal cells in hyperexcitable hippocampal slices of kainatetreated rats. Eur J Neurosci 8:736–748. Risold PY, Swanson LW. 1997. Connections of the rat lateral septal complex. Brain Res Brain Res Rev 24:115–195.


331

Saji M, Kobayashi S, Ohno K, Sekino Y. 2000. Interruption of supramammillohippocampal afferents prevents the genesis and spread of limbic seizures in the hippocampus via a disinhibition mechanism. Neuroscience 97:437–445. Savic I, Lindstrom P, Gulyas B, Halldin C, Andree B, Farde L. 2004. Limbic reductions of 5-HT1A receptor binding in human temporal lobe epilepsy. Neurology 62:1343–1351. Tang FR, Chia SC, Chen PM, Gao H, Lee WL, Yeo TS, Burgunder JM, Probst A, Sim MK, Ling EA. 2004a. Metabotropic glutamate receptor2/3 in the hippocampus of patients with mesial temporal lobe epilepsy, of rats and mice after pilocarpine-induced status epilepticus. Epilepsy Res 59:167–180. Tang FR, Chia SC, Zhang S, Chen PM, Gao H, Liu CP, Khanna S, Lee WL. 2005. Glutamate receptor 1 immunopositive neurons in the gliotic CA1 area of the mouse hippocampus after pilocarpine-induced status epilepticus. Eur J Neurosci 21:2361–2374. Tang FR, Lee WL. 2001. Expression of the group II and III metabotropic glutamate receptors in the hippocampus of patients with mesial temporal lobe epilepsy. J Neurocytol 30:135–141. Tang FR, Lee WL, Gao H, Chen Y, Loh YT, Chia SC. 2004b. Expression of different isoforms of protein kinase C in the rat hippocampus after pilocarpine-induced status epilepticus with special reference to CA1 area and the dentate gyrus. Hippocampus 14:87–98. Tang FR, Lee WL, Yang J, Sim MK, Ling EA. 2001a. Metabotropic glutamate receptor 8 in the rat hippocampus after pilocarpine-induced status epilepticus. Neurosci Lett 300:137–140. Tang FR, Lee WL, Yang J, Sim MK, Ling EA. 2001b. Expression of metabotropic glutamate receptor 1 in the hippocampus of rat pilocarpine model of status epilepticus. Epilepsy Res 46:179–189. Tang FR, Lee WL, Yeo TT. 2001c. Expression of the group I metabotropic glutamate receptor in the hippocampus of patients with mesial temporal lobe epilepsy. J Neurocytol 30:403–411. Tremblay E, Nitecka L, Berger ML, Ben-Ari Y. 1984. Maturation of kainic acid seizure-brain damage syndrome in the rat. I. Clinical, electrographic and metabolic observations. Neuroscience 13:1051–1072. Turski WA, Cavalheiro EA, Turski L, Kleinrok Z. 1983. Intrahippocampal bethanechol in rats: behavioral, electroencephalographic and neuropathological correlates. Behav Brain Res 7:361–370. Vertes RP. 1992. PHA-L analysis of projections from the supramammillary nucleus in the rat. J Comp Neurol 326:595–622. Whishaw IQ, Robinson TE, Schallert T, De Ryck M, Ramirez VD. 1978. Electrical activity of the hippocampus and neocortex in rats depleted of brain dopamine and norepinephrine: relations to behavior and effects of atropine. Exp Neurol 62:748–767. Zhang S, Khanna S, Tang FR. 2004. Rewiring of the dentate gyrus of dorsal hippocampus in the mouse model of temporal lobe epilepsy. Proceedings of International Biomedical Science Conference, Kunming, China.