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Regional expression of Fos-like immunoreactivity following seizures in Noda epileptic rat (NER)
Epilepsy Research (2009) 87, 70—76
journal homepage: www.elsevier.com/locate/epilepsyres
Regional expression of Fos-like immunoreactivity following seizures in Noda epileptic rat (NER) Yukihiro Ohno a,∗, Saki Shimizu a, Yuya Harada a, Maho Morishita a, Shizuka Ishihara a, Kenta Kumafuji b, Masashi Sasa c, Tadao Serikawa b a
Laboratory of Pharmacology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan c Nagisa Clinic, Osaka, Japan b
Received 3 June 2009; received in revised form 22 July 2009; accepted 29 July 2009 Available online 26 August 2009
KEYWORDS NER; Fos expression; Epilepsy; Tonic—clonic convulsion; Animal model
Summary Noda epileptic rat (NER) is a genetic rat model of epilepsy that exhibit spontaneous generalized tonic—clonic (GTC) seizures with paroxysmal discharges. We analyzed the regional expression of Fos-like immunoreactivity (Fos-IR) following GTC seizures in NER to clarify the brain regions involved in the seizure generation. GTC seizures in NER elicited a marked increase in Fos expression in the piriform cortex, perirhinal—entorhinal cortex, insular cortex and other cortices including the motor cortex. In the limbic regions, Fos-IR was highest in the amygdalar nuclei (e.g., basomedial amygdaloid nucleus), followed by the cingulate cortex and hippocampus (i.e., dentate gyrus and CA3). As compared to the above forebrain regions, NER either with or without GTC seizures exhibited only marginal Fos expression in the basal ganglia (e.g., accumbens, striatum and globus pallidus), diencephalon (e.g., thalamus and hypothalamus) and lower brain stem structures (e.g., pons-medulla oblongata). These results suggest that GTC seizures in NER are of forebrain origin and are evoked primarily by activation of the limbic and/or cortical seizure circuits. © 2009 Elsevier B.V. All rights reserved.
Introduction A variety of animal models that exhibit seizures resembling certain types of human epilepsy (e.g., absence and grand mal seizures) have been developed and used for epilepsy research. These models are useful not only for
∗
Corresponding author. Tel.: +81 72 690 1052 fax: +81 72 690 1053. E-mail address: [email protected] (Y. Ohno).
screening the efficacy of antiepileptic drugs, but also for understanding the pathophysiology in epileptic diseases. Specifically, animals with inherited and spontaneous epileptic seizures are of value for clarifying the genetic basis and/or causative genes underlying generation of seizures and epileptogenesis. At present, several animal models with human grand mal-like seizures are available such as EL mice (Suzuki and Nakamoto, 1977), spontaneous epileptic rats (SER) (Serikawa and Yamada, 1986), genetically epilepsyprone rats (GEPR) (Dailey et al., 1989), and more recently Ihara Epileptic Rat (IER) (Amano et al., 1996), Noda epilep-
0920-1211/$ — see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2009.07.012
Seizure-induced Fos expression in NER tic rat (NER) (Noda et al., 1998) and Wakayama Epileptic Rat (WER) (Tsubota et al., 2003). NER is an epileptic rat strain that has been developed by inbreeding rats with generalized tonic—clonic (GTC) seizures in a stock of Crj:Wistar (Noda et al., 1998). NER exhibit spontaneous GTC convulsions associated with paroxysmal EEG discharges without apparent pathological alterations in the brain (Noda et al., 1998; Iida et al., 1998). In addition, inheritance analysis revealed that the incidence of GTC seizures in NER is controlled by more than one gene (Maihara et al., 2000). These findings suggest that NER may serve as an animal model for idiopathic epilepsy in humans. Previous studies also demonstrated that NER shows altered levels of neurotransmitters (e.g., glutamate, GABA and monoamines) and neuropeptides (e.g., neuropeptide Y) or enhanced excitability of hippocampal CA3 neurons (Sejima et al., 1999; Jinde et al., 1999, 2002; Hanaya et al., 2002; Kiura et al., 2003; Chanut et al., 2006). Nonetheless, the pathophysiological basis underlying the GTC seizure generation in NER remains to be clarified. The expression of Fos-like immunoreactivity (Fos-IR) has been widely used as a biological marker of neural activation following various stimuli, including convulsive seizures. The intensity and expression patterns of Fos-IR vary depending on the differences in animal models of epilepsy, conditions of electrical stimulation and types of convulsive agents (e.g., pentylenetetrazole (PTZ), GABAA receptor antagonists and K+ channel blockers) (Shehab et al., 1992; Kovács et al., 2003; Eells et al., 2004; Morimoto et al., 2004; Bastlund et al., 2005). Thus, the analysis of Fos expression can discern regions of the brain activated by seizure episodes. In the present study, therefore, we analyzed the regional Fos expression following GTC convulsions in NER to clarify the brain regions involved in their seizure generation.
Methods Animals Fifteen NER of either sex were used. NER were obtained from the National BioResource Project for the Rat (NBRPR#0368) in Japan. The animals were kept and bred at the Institute of Laboratory of Animals, Graduate School of Medicine, Kyoto University in airconditioned rooms (24 ± 2 ◦ C and 50 ± 10% relative humidity) under a 14-h light/10-h dark cycle (light on: 7:00 a.m.). The housing conditions of NER and the animal care methods complied with the Guidelines for Animal Experiments of Kyoto University. The experimental protocols of this study were also approved by the Experimental Animal Research Committee at Osaka University of Pharmaceutical Sciences. Observation of seizures and brain tissue preparations NER were normally housed in a group of 3 or 4 per cage, and the incidence of spontaneous seizures was monitored for 60 min once a week (Monday) from 14 weeks of age. Eleven out of 15 NER exhibited spontaneous GTC seizures twice or more during the observation period, and they were subjected to the following experiments. NER that showed only one (3 out of 15) or no seizure (1 out of 15) were excluded. External stimuli such as handling animals and placing them in a novel environment can facilitate the induction of spontaneous seizures. On the day of the experiment, we therefore placed NER individually in an observation box (area: 30 cm × 30 cm, height: 45 cm) for 30 min to induce seizures and the incidence of seizures
71 was monitored. NER, which exhibited GTC seizures during the observation period, were deeply anesthetized with pentobarbital (80 mg/kg, i.p.) 2 h after the seizure incidence. Animals were transcardially perfused with ice-cold phosphate-buffered saline (PBS) and then with 4% formaldehyde solution. The brain was removed from the skull and placed in fresh fixative for at least 24 h. After postfixation, coronal sections (30 m thickness) were cut from each brain using a Microslicer (DSK-3000, Kyoto, Japan). NER that did not develop any seizures were sacrificed immediately after the 30min-observation period. The brain samples of these animals were prepared in the same manner as described above and were used as the interictal control (GTC−). Fos immunohistochemistry The staining of Fos-immunoreactivity (IR) was performed by the method published previously (Ohno et al., 2008, 2009). Briefly, slices were washed with PBS containing 0.3% Triton X-100, and incubated for 2 h in the presence of 2% normal rabbit serum, and then again in the presence of 2% normal rabbit serum and goat c-Fos antiserum (diluted 1:4000, Santa Cruz Biotechnology Inc., Santa Cruz, CA) for an additional 18—36 h. After washing with PBS, the sections were incubated with a biotinylated rabbit anti-goat IgG secondary antibody (diluted 1:1000, Vector Laboratories, Burlingame, CA) for 2 h. The sections were then incubated with PBS containing 0.3% hydrogen peroxide for 30 min to inactivate the endogenous peroxidase. Thereafter, the sections were washed with PBS and incubated for 2 h with avidin-biotinylated horseradish peroxidase complex (Vectastain ABC Kit). Fos-IR was visualized by the diaminobenzidine-nickel staining method. Fos expression was quantified by counting the number of FosIR positive nuclei in various regions of the brain as shown in Fig. 1 (also see Paxinos and Watson, 2007), which includes the following regions: (1) the cerebral cortices; the medial prefrontal cortex (mPFC), motor cortex (MC), sensory cortex (SC), agranular insular cortex (AIC), piriform cortex (Pir), perirhinal—entorhinal (PRh—Ent) cortex, auditory cortex (AuC), (2) the limbic areas; CA1, CA3 and dentate gyrus (DG) of the hippocampus, basomedial amygdaloid nucleus (BMA), lateral amygdaloid nucleus ventromedialis (LaVM), posteromedial cortical amygdaloid nucleus (PMCo), cingulated cortex (Cg) and lateral septum (LS), (3) the basal ganglia; core (AcC) and shell (AcS) regions of the nucleus accumbens, dorsolateral (dlST) and dorsomedial (dmST) and globus pallidus (GP), (4) the diencephalon; paratenial (PT), anteromedial (AM), centromedial (CM) and ventromedial (VM) thalamus, lateral habenula (LHb), anterior (AH), posterior (PH) and dorsomedial (DMH) hypothalamus, (5) the pons/medulla oblongata; central gray (CG), locus coeruleus (LC), pontine reticular nucleus caudalis (PnC) and gigantocellular reticular nucleus (GiR). The number of Fos-IR positive nuclei was counted within a 350 m × 350 m grid laid over each of the above brain regions (Fig. 1) by observers who were blinded regarding the seizure incidence.
Statistical analysis Data are expressed as the mean ± S.E.M. Statistical significance of differences in the number of Fos-IR positive nuclei between NER with GTC seizures (GTC+) and the interictal control (GTC−) was determined by the Student’s t-test. A p-value of less than 0.05 was considered statistically significant.
Results GTC seizures in NER Since external stimuli such as handling animals and placing them in a novel environment are cues facilitating induc-
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Seizure-induced Fos expression in the cerebral cortex and limbic areas The baseline level (GTC−) of Fos expression in NER was generally very low in all brain regions. GTC seizures in NER elicited wide-spread expression of Fos-IR in the cerebral cortex, especially with high densities in the MC, SC, AIC, Pir, AuC and PRh—Ent (Figs. 2 and 3). Fos-IR positive cells distributed to the entire brain regions from the frontal to the occipito-temporal cortices. In the fronto-parietal regions, GTC seizure-induced Fos expression was highest in the Pir, which was followed by the AIC, MC and SC, while the number of Fos-IR positive cells in the mPFC was relatively low (Fig. 3). In occipito-temporal cortices, the Pir and PRh—Ent showed a very high Fos expression, which was followed by AuC (Figs. 2 and 3). The number of Fos-IR positive cells in the Pir and PRh—Ent were higher than those of MC or SC. In the limbic regions, GTC seizures in NER specifically induced Fos expression in the amygdala, Cg and hippocampus (Figs. 2 and 4). The highest Fos expression for GTC seizures was found in the amygdalar nuclei, BMA and PMCo. The Fos induction in the hippocampus occurred only in the DG and CA3, but not in the CA1 and CA2 regions (Figs. 2 and 4). In addition, Fos-IR positive cells in the DG were mainly localized in the dentate hilus and only sparsely in the molecular layer, but were absent from the granular cell layer (Fig. 2).
Figure 1 Schematic illustrations of the brain sections selected for quantitative analysis of Fos-IR positive cells. An antero-posterior coordinate (distance from the Bregma) is shown on the left top of each brain section. Filled boxes in each section indicate the sample areas analyzed; mPFC: the medial prefrontal cortex, MC: motor cortex, SC: sensory cortex, AIC: agranular insuler cortex, Pir: piriform cortex, AcC and AcS: core and shell regions of nucleus accumbens, respectively, Cg: cingulated cortex, LS: lateral septum, dlST and dmST: dorsolateral and dorsomedial striatum, respective, GP: globus pallidus, PT, AM, CM and VM: paratenial, anteromedial, centromedial and ventromedial thalamus, respectively, LHb: lateral habenula, AH, PH and DMH: anterior, posterior and dorsomedial hypothalamus, respectively, PRh—Ent: perirhinal—entorhinal cortex, AuC: auditory cortex, DG: dentate gyrus of the hippocampus, BMA: basomedial amygdaloid nucleus, LaVM: lateral amygdaloid nucleus ventromedialis, PMCo: posteromedial cortical amygdaloid nucleus, CG: central gray, LC: locus coeruleus, PnC: pontine reticular nucleus caudalis and GiR: gigantocellular reticular nucleus.
tion of spontaneous seizures, we placed NER individually in an observation box for 30 min to induce seizures. Under these conditions, 6 out of 11 NER examined exhibited one or more GTC seizures during the 30 min-observation. The mean number and duration of GTC seizures were 1.30 ± 0.21 times/30 min and 50.3 ± 3.23 s, respectively. GTC seizures in NER usually accompanied wild running and/or bouncing, and were normally followed by a postictal flaccid stage after seizure cessation.
Seizure-induced Fos expression in the basal ganglia and brain stem GTC seizures in NER hardly affected Fos expression in the basal ganglia. The numbers of Fos-IR positive cells in the basal ganglia were very low, and no significant differences in Fos expression were found between GTC+ and GTC− either in the nucleus accumbens (i.e., AcC and AcS), striatum (i.e., dlST and dmST) or GP (Fig. 5). Similarly, GTC seizures failed to affect Fos expression either in the diencephalon or lower brain stem structures (Fig. 5). The numbers of Fos-IR positive cells in most of the thalamic (i.e., PT, AM, CM or VM) or hypothalamic (i.e., AH, PH and DMH) nuclei were low both under postictal (GTC+) and interictal (GTC−) status in NER. In addition, Fos expression in the pons-medulla oblongata (i.e., CG, LC, PnC and GiR) was also very low in both the GTC+ and GTC− groups (Fig. 5).
Discussion Fos expression is widely used as a biological marker of neural activation to map the brain regions involved in seizure generation. We analyzed for the first time the regional expression of Fos-IR following GTC seizures in NER, a genetic rat model with spontaneous GTC seizures. The present results demonstrated that GTC seizures in NER elicited Fos expression specifically in the cerebral cortex such as MC, Pir, PRh—Ent and AIC, and certain limbic areas such as the amygdala, CA3 and DG of the hippocampus. In contrast, NER either with or without GTC convulsions exhibited only marginal Fos expression in the basal ganglia, diencephalon and lower brain stem structures. These results suggest that
Seizure-induced Fos expression in NER
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Figure 2 Representative photographs illustrating the Fos-IR positive cells in the cerebral cortex (A) and limbic regions (B) of NER. (A) Fos expression in the motor cortex (MC: Bregma 1.32), piriform cortex (Pir: Bregma −3.48), perirhinal—entorhinal cortex (PRh—Ent) of NER. (B) Fos expression in the CA1, dentate gyrus (DG) of the hippocampus, and vasomedial amygdaloid nucleus (BMA) of NER. The brain was removed 2 h after GTC seizure incidence (GTC+). GTC−: interictal control. Scale bar: 100 m.
GTC seizures in NER is of forebrain origin and are evoked primarily by activation of forebrain regions such as corticaland/or limbic-seizure circuits. In NER, the baseline level (GTC−) of Fos expression was very low, implying that the neuronal activity during the interictal status in NER is normal. GTC seizures in NER induced a marked and wide-spread increase in Fos expression in various regions of the cerebral cortex including MC and SC, which is consistent with previous study (Noda et al., 1998) that NER exhibited generalized paroxysmal discharges in cortical EEG during GTC seizures. In addition, intensive expression of Fos-IR was found in certain corticolimbic areas such as the Pir, AIC, PRh—Ent, amygdalar nuclei and hippocampus, all of which have been implicated in the generation of convulsive seizures and/or epileptogenesis (Morimoto et al., 2004). The distribution pattern of seizure-induced Fos-IR positive cells in NER partly resembled those of MES or PTZ-induced GTC seizures, which also enhanced Fos expression in the forebrain regions including the cerebral cortex, amygdala and hippocampus (Shehab et
al., 1992; Eells et al., 2004; Bastlund et al., 2005). However, Fos expression in NER was different from that in MES and PTZ seizures in some regards, such as NER evoked the most prominent Fos expression in the amygdala, whereas MES or PTZ seizures produced the highest Fos expression in the hippocampal DG (Shehab et al., 1992; Eells et al., 2004; Bastlund et al., 2005). Furthermore, Fos expression in NER seems to occur more specifically in the cortico-limbic areas as compared to that in MES and PTZ seizures. Previous studies showed that MES seizures induced Fos expression not only in the forebrain regions, but also in certain hypothalamus nuclei (e.g., DMH and VMH) and the lower brain stem (e.g., CG and GiR) (Shehab et al., 1992; Eells et al., 2004). PTZ seizures are also known to elevate Fos expression in the thalamic nuclei and lower brain stem structures (Shehab et al., 1992; Eells et al., 2004). Thus, the present results suggest that GTC seizures in NER are mainly of forebrain origin. This characteristic of NER is also different from other types of models such as the tonic seizures in GEPR evoked by audiogenic stimulation, where the activation of the brain stem
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Figure 4 Regional expression of Fos-IR induced by GTC seizures in the limbic regions of NER. The brain was removed 2 h after the occurrence of GTC (GTC+). DG: dentate gyrus, LaVM: lateral ventromedial amygdala, BMA: basomedial amygdaloid, PMCo: posteromedial cortical amygdala, Cg: cingulate cortex, LS: lateral septum. Each column represents the mean ± SEM. *P < 0.05, **P < 0.01, significantly different from the interictal control (GTC−).
the stimulation of Pir or PRh—Ent per se develops kindling, upon stimulation, even more rapidly than does the amygdala or hippocampus (Morimoto et al., 2004), suggesting that these structures may be causative sites of the GTC seizures in NER. However, this possibility is not likely since the kindling stimulation of Pir and PRh—Ent did not accompany hippocampal Fos expression (neuronal activation) even after the seizures were generalized (Sato et al., 1998; Morimoto Figure 3 Regional expression of Fos-IR induced by GTC seizures in the cerebral cortex of NER. The brain was removed 2 h after GTC seizure incidence (GTC+). MC: motor cortex, SC: sensory cortex at different Bregma (B) levels (from B 2.76 to B −3.48), mPFC: the medial prefrontal cortex, AIC: agranular insuler cortex, Pir: piriform cortex at two different levels (B 2.76 and B −1.20), AuC: auditory cortex, PRh—Ent: perirhinal—entorhinal cortex. Each column represents the mean ± SEM. *P < 0.05, **P < 0.01, significantly different from the interictal control (GTC−).
reticular formation is involved in their convulsions (Shehab et al., 1992; Eells et al., 2004; Morimoto et al., 2004), or seizures induced by known convulsive agents (e.g., nicotine and K+ channel blockers) (Kovács et al., 2003; Bastlund et al., 2005). It is therefore conceivable that GTC seizures in NER are evoked primarily by activation of the forebrain cortico-limbic circuits, but barely by the brain stem regions. In the forebrain regions of NER, Fos expression was most prominent in the Pir, PRh—Ent and amygdalar nuclei. The Pir and PRh—Ent have direct connections to the motor cortex, and these cortical circuits are known to be closely associated with seizure generation and kindling epileptogenesis (Morimoto et al., 2004). In fact, both Pir and PRh—Ent can be activated by focal (even single) stimulation of the amygdala. Repeated stimulation of the hippocampus also activated these structures after hippocampal kindling was generalized (Dragunow et al., 1988; Clark et al., 1991; Ferland et al., 1998; Sato et al., 1998). Thus, the Pir and PRh—Ent seem to participate in the generalization of limbic seizures to the cerebral cortices. On the other hand, it is also known that
Figure 5 Regional expression of Fos-IR induced by GTC seizures in the basal ganglia, diencephalon and pons/medulla oblongata of NER. The brain was removed 2 h after the occurrence of GTC (GTC+). AcC and AcS: core and shell regions of the nucleus accumbens, respectively, dlST and dmST: dorsolateral and dorsomedial striatum, respective, GP: globus pallidus, PT, AM, CM and VM: paratenial, anteromedial, centromedial and ventromedial thalamus, respectively, LHb: latetal habenula, AH, PH and DMH: anterior, posterior and dorsomedial hypothalamus, respectively, CG: central gray, LC: locus coeruleus, PnC: pontine reticular nucleus caudalis and GiR: gigantocellular reticular nucleus. GTC−: interictal control. Each column represents the mean ± SEM.
Seizure-induced Fos expression in NER et al., 2004). Alternatively, the limbic regions (i.e., amygdala and hippocampus) may be involved in the generation of GTC seizures in NER. In fact, GTC-induced Fos expression pattern in NER was similar to that induced by generalized amygdala kindling (Dragunow et al., 1988). Furthermore, our previous studies also demonstrated that NER CA3 neurons exhibit abnormal epileptiform discharges possibly due to dysfunction of T-type Ca2+ channels (Hanaya et al., 2002; Kiura et al., 2003). Further studies are required to clarify the functional alterations in the amygdalar and/or hippocampal seizure circuits in NER. In the present study, NER with no GTC seizures (GTC−) showed very low levels of Fos-IR in most of brain areas. Since Fos expression enhanced by seizures is known to continue for 4—8 h after the seizures onset (Jensen et al., 1993; Zimmer et al., 1997), above findings suggest that these NER did not experience seizures at least for several hours before the experiment. Nonetheless, this study cannot completely role out a possible influence of previously occurred seizures (e.g., experienced numbers and/or frequency) before the experiments on Fos expression. Further studies such as changes in Fos expression during different stage (before and after the onset) of seizure development will be required to delineate precise mechanism and brain regions underlying the seizure generation and/or epileptogenesis in NER.
Acknowledgements This work was partly supported by a Grant in Aid for Scientific Research 20240042 from the Ministry of Education, Science, Sports and Culture of Japan and by the Japan Epilepsy Research Foundation. The authors are thankful to the National BioResource Project for the Rat in Japan (http://www.anim.med.kyoto-u.ac.jp/NBR/) for providing NER.
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Regional expression of Fos-like immunoreactivity following seizures in Noda epileptic rat (NER) Yukihiro Ohno a,∗, Saki Shimizu a, Yuya Harada a, Maho Morishita a, Shizuka Ishihara a, Kenta Kumafuji b, Masashi Sasa c, Tadao Serikawa b a
Laboratory of Pharmacology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan c Nagisa Clinic, Osaka, Japan b
Received 3 June 2009; received in revised form 22 July 2009; accepted 29 July 2009 Available online 26 August 2009
KEYWORDS NER; Fos expression; Epilepsy; Tonic—clonic convulsion; Animal model
Summary Noda epileptic rat (NER) is a genetic rat model of epilepsy that exhibit spontaneous generalized tonic—clonic (GTC) seizures with paroxysmal discharges. We analyzed the regional expression of Fos-like immunoreactivity (Fos-IR) following GTC seizures in NER to clarify the brain regions involved in the seizure generation. GTC seizures in NER elicited a marked increase in Fos expression in the piriform cortex, perirhinal—entorhinal cortex, insular cortex and other cortices including the motor cortex. In the limbic regions, Fos-IR was highest in the amygdalar nuclei (e.g., basomedial amygdaloid nucleus), followed by the cingulate cortex and hippocampus (i.e., dentate gyrus and CA3). As compared to the above forebrain regions, NER either with or without GTC seizures exhibited only marginal Fos expression in the basal ganglia (e.g., accumbens, striatum and globus pallidus), diencephalon (e.g., thalamus and hypothalamus) and lower brain stem structures (e.g., pons-medulla oblongata). These results suggest that GTC seizures in NER are of forebrain origin and are evoked primarily by activation of the limbic and/or cortical seizure circuits. © 2009 Elsevier B.V. All rights reserved.
Introduction A variety of animal models that exhibit seizures resembling certain types of human epilepsy (e.g., absence and grand mal seizures) have been developed and used for epilepsy research. These models are useful not only for
∗
Corresponding author. Tel.: +81 72 690 1052 fax: +81 72 690 1053. E-mail address: [email protected] (Y. Ohno).
screening the efficacy of antiepileptic drugs, but also for understanding the pathophysiology in epileptic diseases. Specifically, animals with inherited and spontaneous epileptic seizures are of value for clarifying the genetic basis and/or causative genes underlying generation of seizures and epileptogenesis. At present, several animal models with human grand mal-like seizures are available such as EL mice (Suzuki and Nakamoto, 1977), spontaneous epileptic rats (SER) (Serikawa and Yamada, 1986), genetically epilepsyprone rats (GEPR) (Dailey et al., 1989), and more recently Ihara Epileptic Rat (IER) (Amano et al., 1996), Noda epilep-
0920-1211/$ — see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2009.07.012
Seizure-induced Fos expression in NER tic rat (NER) (Noda et al., 1998) and Wakayama Epileptic Rat (WER) (Tsubota et al., 2003). NER is an epileptic rat strain that has been developed by inbreeding rats with generalized tonic—clonic (GTC) seizures in a stock of Crj:Wistar (Noda et al., 1998). NER exhibit spontaneous GTC convulsions associated with paroxysmal EEG discharges without apparent pathological alterations in the brain (Noda et al., 1998; Iida et al., 1998). In addition, inheritance analysis revealed that the incidence of GTC seizures in NER is controlled by more than one gene (Maihara et al., 2000). These findings suggest that NER may serve as an animal model for idiopathic epilepsy in humans. Previous studies also demonstrated that NER shows altered levels of neurotransmitters (e.g., glutamate, GABA and monoamines) and neuropeptides (e.g., neuropeptide Y) or enhanced excitability of hippocampal CA3 neurons (Sejima et al., 1999; Jinde et al., 1999, 2002; Hanaya et al., 2002; Kiura et al., 2003; Chanut et al., 2006). Nonetheless, the pathophysiological basis underlying the GTC seizure generation in NER remains to be clarified. The expression of Fos-like immunoreactivity (Fos-IR) has been widely used as a biological marker of neural activation following various stimuli, including convulsive seizures. The intensity and expression patterns of Fos-IR vary depending on the differences in animal models of epilepsy, conditions of electrical stimulation and types of convulsive agents (e.g., pentylenetetrazole (PTZ), GABAA receptor antagonists and K+ channel blockers) (Shehab et al., 1992; Kovács et al., 2003; Eells et al., 2004; Morimoto et al., 2004; Bastlund et al., 2005). Thus, the analysis of Fos expression can discern regions of the brain activated by seizure episodes. In the present study, therefore, we analyzed the regional Fos expression following GTC convulsions in NER to clarify the brain regions involved in their seizure generation.
Methods Animals Fifteen NER of either sex were used. NER were obtained from the National BioResource Project for the Rat (NBRPR#0368) in Japan. The animals were kept and bred at the Institute of Laboratory of Animals, Graduate School of Medicine, Kyoto University in airconditioned rooms (24 ± 2 ◦ C and 50 ± 10% relative humidity) under a 14-h light/10-h dark cycle (light on: 7:00 a.m.). The housing conditions of NER and the animal care methods complied with the Guidelines for Animal Experiments of Kyoto University. The experimental protocols of this study were also approved by the Experimental Animal Research Committee at Osaka University of Pharmaceutical Sciences. Observation of seizures and brain tissue preparations NER were normally housed in a group of 3 or 4 per cage, and the incidence of spontaneous seizures was monitored for 60 min once a week (Monday) from 14 weeks of age. Eleven out of 15 NER exhibited spontaneous GTC seizures twice or more during the observation period, and they were subjected to the following experiments. NER that showed only one (3 out of 15) or no seizure (1 out of 15) were excluded. External stimuli such as handling animals and placing them in a novel environment can facilitate the induction of spontaneous seizures. On the day of the experiment, we therefore placed NER individually in an observation box (area: 30 cm × 30 cm, height: 45 cm) for 30 min to induce seizures and the incidence of seizures
71 was monitored. NER, which exhibited GTC seizures during the observation period, were deeply anesthetized with pentobarbital (80 mg/kg, i.p.) 2 h after the seizure incidence. Animals were transcardially perfused with ice-cold phosphate-buffered saline (PBS) and then with 4% formaldehyde solution. The brain was removed from the skull and placed in fresh fixative for at least 24 h. After postfixation, coronal sections (30 m thickness) were cut from each brain using a Microslicer (DSK-3000, Kyoto, Japan). NER that did not develop any seizures were sacrificed immediately after the 30min-observation period. The brain samples of these animals were prepared in the same manner as described above and were used as the interictal control (GTC−). Fos immunohistochemistry The staining of Fos-immunoreactivity (IR) was performed by the method published previously (Ohno et al., 2008, 2009). Briefly, slices were washed with PBS containing 0.3% Triton X-100, and incubated for 2 h in the presence of 2% normal rabbit serum, and then again in the presence of 2% normal rabbit serum and goat c-Fos antiserum (diluted 1:4000, Santa Cruz Biotechnology Inc., Santa Cruz, CA) for an additional 18—36 h. After washing with PBS, the sections were incubated with a biotinylated rabbit anti-goat IgG secondary antibody (diluted 1:1000, Vector Laboratories, Burlingame, CA) for 2 h. The sections were then incubated with PBS containing 0.3% hydrogen peroxide for 30 min to inactivate the endogenous peroxidase. Thereafter, the sections were washed with PBS and incubated for 2 h with avidin-biotinylated horseradish peroxidase complex (Vectastain ABC Kit). Fos-IR was visualized by the diaminobenzidine-nickel staining method. Fos expression was quantified by counting the number of FosIR positive nuclei in various regions of the brain as shown in Fig. 1 (also see Paxinos and Watson, 2007), which includes the following regions: (1) the cerebral cortices; the medial prefrontal cortex (mPFC), motor cortex (MC), sensory cortex (SC), agranular insular cortex (AIC), piriform cortex (Pir), perirhinal—entorhinal (PRh—Ent) cortex, auditory cortex (AuC), (2) the limbic areas; CA1, CA3 and dentate gyrus (DG) of the hippocampus, basomedial amygdaloid nucleus (BMA), lateral amygdaloid nucleus ventromedialis (LaVM), posteromedial cortical amygdaloid nucleus (PMCo), cingulated cortex (Cg) and lateral septum (LS), (3) the basal ganglia; core (AcC) and shell (AcS) regions of the nucleus accumbens, dorsolateral (dlST) and dorsomedial (dmST) and globus pallidus (GP), (4) the diencephalon; paratenial (PT), anteromedial (AM), centromedial (CM) and ventromedial (VM) thalamus, lateral habenula (LHb), anterior (AH), posterior (PH) and dorsomedial (DMH) hypothalamus, (5) the pons/medulla oblongata; central gray (CG), locus coeruleus (LC), pontine reticular nucleus caudalis (PnC) and gigantocellular reticular nucleus (GiR). The number of Fos-IR positive nuclei was counted within a 350 m × 350 m grid laid over each of the above brain regions (Fig. 1) by observers who were blinded regarding the seizure incidence.
Statistical analysis Data are expressed as the mean ± S.E.M. Statistical significance of differences in the number of Fos-IR positive nuclei between NER with GTC seizures (GTC+) and the interictal control (GTC−) was determined by the Student’s t-test. A p-value of less than 0.05 was considered statistically significant.
Results GTC seizures in NER Since external stimuli such as handling animals and placing them in a novel environment are cues facilitating induc-
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Seizure-induced Fos expression in the cerebral cortex and limbic areas The baseline level (GTC−) of Fos expression in NER was generally very low in all brain regions. GTC seizures in NER elicited wide-spread expression of Fos-IR in the cerebral cortex, especially with high densities in the MC, SC, AIC, Pir, AuC and PRh—Ent (Figs. 2 and 3). Fos-IR positive cells distributed to the entire brain regions from the frontal to the occipito-temporal cortices. In the fronto-parietal regions, GTC seizure-induced Fos expression was highest in the Pir, which was followed by the AIC, MC and SC, while the number of Fos-IR positive cells in the mPFC was relatively low (Fig. 3). In occipito-temporal cortices, the Pir and PRh—Ent showed a very high Fos expression, which was followed by AuC (Figs. 2 and 3). The number of Fos-IR positive cells in the Pir and PRh—Ent were higher than those of MC or SC. In the limbic regions, GTC seizures in NER specifically induced Fos expression in the amygdala, Cg and hippocampus (Figs. 2 and 4). The highest Fos expression for GTC seizures was found in the amygdalar nuclei, BMA and PMCo. The Fos induction in the hippocampus occurred only in the DG and CA3, but not in the CA1 and CA2 regions (Figs. 2 and 4). In addition, Fos-IR positive cells in the DG were mainly localized in the dentate hilus and only sparsely in the molecular layer, but were absent from the granular cell layer (Fig. 2).
Figure 1 Schematic illustrations of the brain sections selected for quantitative analysis of Fos-IR positive cells. An antero-posterior coordinate (distance from the Bregma) is shown on the left top of each brain section. Filled boxes in each section indicate the sample areas analyzed; mPFC: the medial prefrontal cortex, MC: motor cortex, SC: sensory cortex, AIC: agranular insuler cortex, Pir: piriform cortex, AcC and AcS: core and shell regions of nucleus accumbens, respectively, Cg: cingulated cortex, LS: lateral septum, dlST and dmST: dorsolateral and dorsomedial striatum, respective, GP: globus pallidus, PT, AM, CM and VM: paratenial, anteromedial, centromedial and ventromedial thalamus, respectively, LHb: lateral habenula, AH, PH and DMH: anterior, posterior and dorsomedial hypothalamus, respectively, PRh—Ent: perirhinal—entorhinal cortex, AuC: auditory cortex, DG: dentate gyrus of the hippocampus, BMA: basomedial amygdaloid nucleus, LaVM: lateral amygdaloid nucleus ventromedialis, PMCo: posteromedial cortical amygdaloid nucleus, CG: central gray, LC: locus coeruleus, PnC: pontine reticular nucleus caudalis and GiR: gigantocellular reticular nucleus.
tion of spontaneous seizures, we placed NER individually in an observation box for 30 min to induce seizures. Under these conditions, 6 out of 11 NER examined exhibited one or more GTC seizures during the 30 min-observation. The mean number and duration of GTC seizures were 1.30 ± 0.21 times/30 min and 50.3 ± 3.23 s, respectively. GTC seizures in NER usually accompanied wild running and/or bouncing, and were normally followed by a postictal flaccid stage after seizure cessation.
Seizure-induced Fos expression in the basal ganglia and brain stem GTC seizures in NER hardly affected Fos expression in the basal ganglia. The numbers of Fos-IR positive cells in the basal ganglia were very low, and no significant differences in Fos expression were found between GTC+ and GTC− either in the nucleus accumbens (i.e., AcC and AcS), striatum (i.e., dlST and dmST) or GP (Fig. 5). Similarly, GTC seizures failed to affect Fos expression either in the diencephalon or lower brain stem structures (Fig. 5). The numbers of Fos-IR positive cells in most of the thalamic (i.e., PT, AM, CM or VM) or hypothalamic (i.e., AH, PH and DMH) nuclei were low both under postictal (GTC+) and interictal (GTC−) status in NER. In addition, Fos expression in the pons-medulla oblongata (i.e., CG, LC, PnC and GiR) was also very low in both the GTC+ and GTC− groups (Fig. 5).
Discussion Fos expression is widely used as a biological marker of neural activation to map the brain regions involved in seizure generation. We analyzed for the first time the regional expression of Fos-IR following GTC seizures in NER, a genetic rat model with spontaneous GTC seizures. The present results demonstrated that GTC seizures in NER elicited Fos expression specifically in the cerebral cortex such as MC, Pir, PRh—Ent and AIC, and certain limbic areas such as the amygdala, CA3 and DG of the hippocampus. In contrast, NER either with or without GTC convulsions exhibited only marginal Fos expression in the basal ganglia, diencephalon and lower brain stem structures. These results suggest that
Seizure-induced Fos expression in NER
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Figure 2 Representative photographs illustrating the Fos-IR positive cells in the cerebral cortex (A) and limbic regions (B) of NER. (A) Fos expression in the motor cortex (MC: Bregma 1.32), piriform cortex (Pir: Bregma −3.48), perirhinal—entorhinal cortex (PRh—Ent) of NER. (B) Fos expression in the CA1, dentate gyrus (DG) of the hippocampus, and vasomedial amygdaloid nucleus (BMA) of NER. The brain was removed 2 h after GTC seizure incidence (GTC+). GTC−: interictal control. Scale bar: 100 m.
GTC seizures in NER is of forebrain origin and are evoked primarily by activation of forebrain regions such as corticaland/or limbic-seizure circuits. In NER, the baseline level (GTC−) of Fos expression was very low, implying that the neuronal activity during the interictal status in NER is normal. GTC seizures in NER induced a marked and wide-spread increase in Fos expression in various regions of the cerebral cortex including MC and SC, which is consistent with previous study (Noda et al., 1998) that NER exhibited generalized paroxysmal discharges in cortical EEG during GTC seizures. In addition, intensive expression of Fos-IR was found in certain corticolimbic areas such as the Pir, AIC, PRh—Ent, amygdalar nuclei and hippocampus, all of which have been implicated in the generation of convulsive seizures and/or epileptogenesis (Morimoto et al., 2004). The distribution pattern of seizure-induced Fos-IR positive cells in NER partly resembled those of MES or PTZ-induced GTC seizures, which also enhanced Fos expression in the forebrain regions including the cerebral cortex, amygdala and hippocampus (Shehab et
al., 1992; Eells et al., 2004; Bastlund et al., 2005). However, Fos expression in NER was different from that in MES and PTZ seizures in some regards, such as NER evoked the most prominent Fos expression in the amygdala, whereas MES or PTZ seizures produced the highest Fos expression in the hippocampal DG (Shehab et al., 1992; Eells et al., 2004; Bastlund et al., 2005). Furthermore, Fos expression in NER seems to occur more specifically in the cortico-limbic areas as compared to that in MES and PTZ seizures. Previous studies showed that MES seizures induced Fos expression not only in the forebrain regions, but also in certain hypothalamus nuclei (e.g., DMH and VMH) and the lower brain stem (e.g., CG and GiR) (Shehab et al., 1992; Eells et al., 2004). PTZ seizures are also known to elevate Fos expression in the thalamic nuclei and lower brain stem structures (Shehab et al., 1992; Eells et al., 2004). Thus, the present results suggest that GTC seizures in NER are mainly of forebrain origin. This characteristic of NER is also different from other types of models such as the tonic seizures in GEPR evoked by audiogenic stimulation, where the activation of the brain stem
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Figure 4 Regional expression of Fos-IR induced by GTC seizures in the limbic regions of NER. The brain was removed 2 h after the occurrence of GTC (GTC+). DG: dentate gyrus, LaVM: lateral ventromedial amygdala, BMA: basomedial amygdaloid, PMCo: posteromedial cortical amygdala, Cg: cingulate cortex, LS: lateral septum. Each column represents the mean ± SEM. *P < 0.05, **P < 0.01, significantly different from the interictal control (GTC−).
the stimulation of Pir or PRh—Ent per se develops kindling, upon stimulation, even more rapidly than does the amygdala or hippocampus (Morimoto et al., 2004), suggesting that these structures may be causative sites of the GTC seizures in NER. However, this possibility is not likely since the kindling stimulation of Pir and PRh—Ent did not accompany hippocampal Fos expression (neuronal activation) even after the seizures were generalized (Sato et al., 1998; Morimoto Figure 3 Regional expression of Fos-IR induced by GTC seizures in the cerebral cortex of NER. The brain was removed 2 h after GTC seizure incidence (GTC+). MC: motor cortex, SC: sensory cortex at different Bregma (B) levels (from B 2.76 to B −3.48), mPFC: the medial prefrontal cortex, AIC: agranular insuler cortex, Pir: piriform cortex at two different levels (B 2.76 and B −1.20), AuC: auditory cortex, PRh—Ent: perirhinal—entorhinal cortex. Each column represents the mean ± SEM. *P < 0.05, **P < 0.01, significantly different from the interictal control (GTC−).
reticular formation is involved in their convulsions (Shehab et al., 1992; Eells et al., 2004; Morimoto et al., 2004), or seizures induced by known convulsive agents (e.g., nicotine and K+ channel blockers) (Kovács et al., 2003; Bastlund et al., 2005). It is therefore conceivable that GTC seizures in NER are evoked primarily by activation of the forebrain cortico-limbic circuits, but barely by the brain stem regions. In the forebrain regions of NER, Fos expression was most prominent in the Pir, PRh—Ent and amygdalar nuclei. The Pir and PRh—Ent have direct connections to the motor cortex, and these cortical circuits are known to be closely associated with seizure generation and kindling epileptogenesis (Morimoto et al., 2004). In fact, both Pir and PRh—Ent can be activated by focal (even single) stimulation of the amygdala. Repeated stimulation of the hippocampus also activated these structures after hippocampal kindling was generalized (Dragunow et al., 1988; Clark et al., 1991; Ferland et al., 1998; Sato et al., 1998). Thus, the Pir and PRh—Ent seem to participate in the generalization of limbic seizures to the cerebral cortices. On the other hand, it is also known that
Figure 5 Regional expression of Fos-IR induced by GTC seizures in the basal ganglia, diencephalon and pons/medulla oblongata of NER. The brain was removed 2 h after the occurrence of GTC (GTC+). AcC and AcS: core and shell regions of the nucleus accumbens, respectively, dlST and dmST: dorsolateral and dorsomedial striatum, respective, GP: globus pallidus, PT, AM, CM and VM: paratenial, anteromedial, centromedial and ventromedial thalamus, respectively, LHb: latetal habenula, AH, PH and DMH: anterior, posterior and dorsomedial hypothalamus, respectively, CG: central gray, LC: locus coeruleus, PnC: pontine reticular nucleus caudalis and GiR: gigantocellular reticular nucleus. GTC−: interictal control. Each column represents the mean ± SEM.
Seizure-induced Fos expression in NER et al., 2004). Alternatively, the limbic regions (i.e., amygdala and hippocampus) may be involved in the generation of GTC seizures in NER. In fact, GTC-induced Fos expression pattern in NER was similar to that induced by generalized amygdala kindling (Dragunow et al., 1988). Furthermore, our previous studies also demonstrated that NER CA3 neurons exhibit abnormal epileptiform discharges possibly due to dysfunction of T-type Ca2+ channels (Hanaya et al., 2002; Kiura et al., 2003). Further studies are required to clarify the functional alterations in the amygdalar and/or hippocampal seizure circuits in NER. In the present study, NER with no GTC seizures (GTC−) showed very low levels of Fos-IR in most of brain areas. Since Fos expression enhanced by seizures is known to continue for 4—8 h after the seizures onset (Jensen et al., 1993; Zimmer et al., 1997), above findings suggest that these NER did not experience seizures at least for several hours before the experiment. Nonetheless, this study cannot completely role out a possible influence of previously occurred seizures (e.g., experienced numbers and/or frequency) before the experiments on Fos expression. Further studies such as changes in Fos expression during different stage (before and after the onset) of seizure development will be required to delineate precise mechanism and brain regions underlying the seizure generation and/or epileptogenesis in NER.
Acknowledgements This work was partly supported by a Grant in Aid for Scientific Research 20240042 from the Ministry of Education, Science, Sports and Culture of Japan and by the Japan Epilepsy Research Foundation. The authors are thankful to the National BioResource Project for the Rat in Japan (http://www.anim.med.kyoto-u.ac.jp/NBR/) for providing NER.
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