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Epileptiform burst discharges in hippocampal CA3 neurons of young but not mature Noda epileptic rats (NER)
Brain Research 950 (2002) 317–320 www.elsevier.com / locate / bres
Short communication
Epileptiform burst discharges in hippocampal CA3 neurons of young but not mature Noda epileptic rats (NER) Ryosuke Hanaya a , *, Masashi Sasa b , Yoshihiro Kiura a , Kumatoshi Ishihara c , Tadao Serikawa d , Kaoru Kurisu a a
Department of Neurosurgery, Hiroshima University School of Medicine, Minami-ku, Kasumi, Hiroshima 734 -8551, Japan b Department of Pharmacology, Hiroshima University School of Medicine, Hiroshima 734 -8551, Japan c Department of Pharmacotherapy, Institute of Pharmaceutical Science, Faculty of Medicine, Hiroshima University, Hiroshima 734 -8551, Japan d Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto 606 -8501, Japan Accepted 20 June 2002
Abstract Noda epileptic rat (NER), originally found in a colony of Crj; Wistar rats, shows spontaneous tonic–clonic convulsion characterized by the appearance of high voltage polyspikes in cortical and hippocampal EEG once every 2–3 days after 2–4 months of age. Electrophysiological studies using hippocampal slice preparations of NER were performed to determine whether hippocampal neurons have abnormal excitability. When a single stimulus (1–25 V) was delivered to the mossy fibers of NER at 4–6 weeks old before they showed any seizures, a long-lasting depolarization shift (DS) accompanied by repetitive firings and after-hyperpolarization following the abnormal firing was observed in seven of 14 hippocampal CA3 neurons. A lower stimulation intensity evoked DS and abnormal firing in three of nine CA3 neurons of NER at 10–15 weeks old which had already showed seizures at 10–15 weeks of age. However, the abnormal firing was not observed in any 10 neurons of the animals at more than 20 weeks old nor in Wistar rats. The input impedances of CA3 neurons in NER with abnormal firing were lower than those without abnormal firing and those in Wistar rats. The abnormal excitability obtained in NER at an age when it did not display any seizures suggests that the hippocampus may play a role in epileptogenicity in NER. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: basic mechanisms Keywords: Noda epileptic rat (NER); Epilepsy; Hippocampus; CA3 neuron; Abnormal excitement; Development
Noda epileptic rats (NER) are mutants found in an inbred colony of Crj; Wistar rats. After 2–4 months of age, NER spontaneously shows tonic–clonic convulsion characterized by the appearance of high voltage polyspikes in cortical and hippocampal EEG once every 2–3 days [13]. Sporadic spikes appear and gradually increase predominantly in the hippocampus from 11 weeks of age in parallel with the behavioral development of seizures. Acoustic priming from 3 weeks of age once every week consistently induces tonic–clonic convulsion in NER with sound stimuli after 11 weeks of age [7]. The seizures are *Corresponding author. Tel.: 181-82-257-5227; fax 181-82-2575229. E-mail address: [email protected] (R. Hanaya).
inhibited by conventional antiepileptics such as diazepam, valproate, phenobarbital, and carbamazepine, but rarely by phenytoin, zonisamide, and ethosuximide [16]. The spontaneously epileptic rat (SER) is a double mutant (zi / zi, tm / tm) obtained originally by mating a heterozygote tremor rat (tm / 1), a mutant found in the inbred colony of Kyoto–Wistar rats [20], with a homozygote zitter rat found in the Sprague–Dawley colony [14]. After 8 weeks of age, SER spontaneously shows both tonic convulsions with low voltage fast waves, and absence-like seizures characterized by sudden behavioral changes such as immobility and staring with the simultaneous appearance of paroxysms of 5–7-Hz spike–wave complexes in cortical and hippocampal EEG. In addition, mild stimuli such as a hand clap and blowing on the face easily induce
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03195-5
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convulsions [17,18]. This animal is useful for the evaluation of the acute and chronic effects of antiepileptics, since the antiepileptic profile of the conventional antiepileptic for tonic and absence-like seizures in SER is similar to those of human grand mal and petit mal epilepsy, respectively [17,19]. We have reported that in hippocampal CA3 pyramidal neurons of mature SER, which already show both tonic convulsion and absence-like seizures, a long-lasting depolarization shift accompanied by repetitive firing is induced by a single stimulus applied to the mossy fibers [8], and this abnormal excitation of the neurons is attributable to abnormalities of the Ca 21 channels [12]. Hyperexcitability of the neurons was also found in hippocampal CA1 neurons of the tottering mouse [10], and hippocampal CA3 neurons in kindled rats [9]. Genetically epileptic prone rats (GEPR), an animal model of spontaneous seizure and audiogenic seizure, also showed abnormal hippocampal excitability in vitro [3]. Therefore, the activity of hippocampal CA3 pyramidal neurons in NER was examined using conventional intracellular recording methods to determine whether these neurons show abnormal excitability. NERs were obtained from the Research Institute for Animal Science in Biochemistry and Toxicology (Kanagawa, Japan), and were inbred at the Institute for Laboratory Animals of our university. Wistar rats as controls were purchased from Charles River Japan (Yokohama, Japan). All animals were kept individually in shoebox-type cages in a room maintained at 2362 8C and 5565% humidity. They were provided with standard rat chow (F-2, Funabashi Form, Japan) and tap water ad libitum. Nineteen NER of both sexes (age: 4–54 weeks) and nine age-matched Wistar rats were used for this study. After decapitation, the brain was placed rapidly in cold oxygenated Ca 21 -free medium (in mM; 113 NaCl, 3 KCl, 1 NaH 2 PO 4 , 25 NaHCO 3 , 5 MgCl 2 , 11 glucose), and hippocampal slices were cut at a thickness of 400 mm with a microslicer (DTK-100, Dosaka EM, Japan). After 1–2 h incubation in artificial cerebrospinal fluid (ACSF (in mM); 113 NaCl, 3 KCl, 1 NaH 2 PO 4 , 25 NaHCO 3 , 2 CaCl 2 , 1 MgCl 2 , 11 glucose; pH 7.2) at 34 8C, one slice was transferred into the recording chamber. ACSF was continuously perfused over the slice at 1.5–2 ml / min at 30–32 8C. The ACSF was continuously bubbled with a mixture of 95% O 2 and 5% CO 2 . A single stimulus (0.1 ms duration) was applied to the mossy fibers every 5 s through a bipolar stimulating electrode placed in the granular cell layer of the dentate gyrus. The stimulus was increased to 1, 4, 10, 15, 20 and 25 V intensity to induce depolarization shift (DS). Intracellular recordings were made from the hippocampal CA3 pyramidal neurons using a glass microelectrode (electrical resistance; 40–100 MV) filled with 3 M KCl. Other details of the methods have been described elsewhere [5].
These responses were displayed on a digital oscilloscope (VC-10, Nihon Kohden, Japan) after amplification (MEZ8201, Nihon Kohden), and stored on a personal computer (Macintosh Color Classic or PowerMacintosh G4, Apple, USA) using the MacLab or PowerLab system (AD Instruments, Australia). The responses were continuously recorded on a thermal array recorder (RTA-1100, Nihon Kohden). The significance of the differences was determined by the Student’s t-test. The data were obtained from the neurons with a resting membrane potential of more than 245 mV. They were composed of 33 hippocampal CA3 neurons obtained from 19 NER. The intracellular activities of 18 CA3 neurons of nine normal Wistar rats were also recorded as controls. In NER at 4–6 weeks old when the animal did not yet show any spontaneous seizures, a long-lasting DS with repetitive firing followed by after-hyperpolarization was elicited by a single stimulation of the mossy fibers in seven (50%) of 14 hippocampal CA3 neurons tested (Fig. 1). In these seven neurons, a single action potential was induced by the stimulation at 5.1460.72 V, and DS with repetitive firing was induced by the stimulation at 13.1762.03 V. A significantly higher voltage was required to elicit DS and the abnormal firing. The mean duration of DS was 38.2667.91 ms in the seven neurons with DS. In the remaining seven neurons, only one action potential without
Fig. 1. Action potentials (a) and after-hyperpolarization (b) induced by mossy fiber stimulation in the hippocampal CA3 neurons of NER at 4 weeks old with no seizures, and in normal Wistar rats. Only a single action potential was induced by a single stimulation of mossy fibers in normal Wistar rats aged 4 weeks old.
R. Hanaya et al. / Brain Research 950 (2002) 317–320
Fig. 2. Action potentials in the hippocampal CA3 neurons of NER at 14 weeks (a) and 26 weeks old (b). (a) Depolarization shift accompanied with repetitive firing was induced by a single stimulation of mossy fibers in NER at 14 weeks old, which began to show spontaneous tonic–clonic convulsion. (b) Only a single action potential was induced by a single stimulation of mossy fibers in NER aged 26 weeks old.
DS was elicited by mossy fiber stimulation at 8.0061.27 V. However, in NER aged 10–15 weeks old, at which age the animals consistently showed convulsive seizures, the number of CA3 neurons with such DS accompanying repetitive firing elicited by mossy fiber stimulation was
319
decreased to 33.3% (three of nine neurons tested) (Fig. 2). In these three neurons, the mean voltage to induce DS was 13.3362.04 V. The voltage for inducing DS was not significantly different between NER aged 4–6 and 10–15 weeks. However, DS was significantly longer in the three neurons of NER aged 10–15 weeks than in the neurons of young NER (4–6 weeks) (Table 1). In the remaining six neurons tested, no DS was induced; no DS was induced by stimulus up to 25 V. After-hyperpolarization following DS was observed in all of seven and three neurons with DS: the mean maximum amplitude was 8.0160.36 (n57) and 6.5060.79 (n53) mV, and the mean duration was 3.6460.36 (n57) and 2.9360.11 (n53) s, in 4–6- and 10–15-week-old animals, respectively. In contrast, in NER after 15 weeks of age when they showed spontaneous seizures, the DS with repetitive firing was not induced by mossy fiber stimulation up to 25 V in any of 10 hippocampal CA3 neurons examined, and only a single action potential was elicited (Fig. 2). In Wistar rats aged 4–6 and more than 15 weeks old, only one action potential was elicited by mossy fiber stimulation up to 25 V in each of eight CA3 neurons examined (Fig. 1). The input impedance of CA3 neurons with abnormal firing in NER of all ages was significantly lower than that of those without abnormal firings. However, the resting membrane potential did not show any significant differences among NER of any age (Table 1). DS accompanying repetitive firing was found in hippocampal CA3 neurons of NER before and after the appearance of convulsive seizures. The DS in cortical and hippocampal neurons is involved in epileptic seizures of kindling animals, GAERS, tottering mouse and SER [3,4,8–10]. Such abnormal excitabilities of the CA3 neurons in NER showing epileptic seizures were similar to those found in SER in terms of the long-lasting duration of DS, including those induced by a single stimulation of the mossy fibers, although the number of neurons showing
Table 1 Physiological profiles of hippocampal CA3 neurons in NER and normal Wistar rats
NER (4–6 w) A (n57) B (n57) NER (10–15 w) A (n53) B (n56) NER (^15 w) A (n50) B (n510) Wistar rat (4–6 w, n58) (^20 w, n58)
Resting membrane potential (mV)
Input impedance (MV)
Number of spikes
Duration of depolarizing shift (ms)
258.462.8 264.762.6
25.263.1* 35.063.4
4.360.6 –
38.367.9 –
258.565.8 258.063.8
28.861.1** 39.960.4
5.060.7 –
60.465.8 –
– 260.968.3
– 41.862.6
– –
– –
255.962.3 257.164.3
38.963.1 40.462.8
– –
– –
Mean6S.E. A; Neurons showing abnormal firings induced by mossy fiber stimulation. B; Neurons showing single spikes induced by mossy fiber stimulation. * p,0.05; compared with B and Wistar rats. ** p,0.05; compared with B.
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R. Hanaya et al. / Brain Research 950 (2002) 317–320
such abnormalities was fewer in NER (30%) than SER (65%) [8]. We have previously demonstrated that the DS in hippocampal CA3 neurons of SER is due to enhanced Ca 21 influx, since the DS was inhibited by Ca 21 channel antagonists such as nicardipine and nifedipine and actual increases in the intracellular Ca 21 levels of CA3 neurons have been obtained by high K 1 and a single stimulation of the mossy fibers [1,2]. The DS obtained in the hippocampal CA3 neurons of NER was also suggested to be due to enhanced Ca 21 influx through Ca 21 channels as in SER, since the DS with repetitive firing was inhibited by nicardipine in some CA3 neurons of NER [6]. These findings suggest that focal hyperexcitability of neurons exists in the hippocampus at early postnatal ages. Animal models with abnormal excitability of the hippocampal CA3 neurons are known to show limbic seizures [11]. NER exhibits spontaneous tonic–clonic convulsion, standing with neck and forelimb clonus, wild jumping / running, opisthotonic postures and evolvement to tonic seizure proceeding to clonic convulsion followed by postictal flaccidity. This seizure pattern indicates the involvement of the forebrain and / or brainstem [7]. In addition, acoustic priming has been reported to activate the brainstem including the superior colliculus [15] and actually induce convulsive seizures in NER [7]. Sporadic spikes in the hippocampal CA3 area of NER followed by convulsive seizures were observed after 11 weeks of age [7]. Together with these findings, NER is thought to have epileptogenicity in the brainstem in addition to the hippocampus. It seems likely that the epileptogenic circuit including the brainstem–limbic system–cerebral cortex is completed with age and, thereafter, the abnormal excitability of the hippocampal CA3 neurons may be recovered by a mechanism as yet undetermined.
References [1] H. Amano, T. Amano, H. Matsubayashi, K. Ishihara, T. Serikawa, M. Sasa, Enhanced calcium influx in hippocampal CA3 neurons of spontaneously epileptic rats (SER), Epilepsia 42 (2001) 345–350. [2] T. Amano, H. Amano, K. Matsubayashi, Ishihara, T. Serikawa and M. Sasa, Enhanced Ca(21) influx with mossy fiber stimulation in hippocampal CA3 neurons of spontaneously epileptic rats, Brain Res. 910 (2001) 199–203. [3] V. Armand, P. Hoffmann, M. Vergnes, U. Heinemann, Epileptiform activity induced by 4-aminopyridine in entorhinal cortex hippocampal slices of rats with a genetically determined absence epilepsy (GAERS), Brain Res. 841 (1999) 62–69. [4] A.C. Bragdon, D.M. Taylor, J.O. McNamara, W.A. Wilson, Abnormal hyperexcitability of hippocampal slices from kindled rats is transient, Brain Res. 453 (1988) 257–264.
[5] R. Hanaya, M. Sasa, H. Ujihara, K. Ishihara, T. Serikawa, K. Iida, T. Akimitsu, K. Arita, K. Kurisu, Suppression by topiramate of epileptic burst discharges in hippocampal CA3 neurons of spontaneously epileptic rat in vitro, Brain Res. 789 (1998) 274–282. [6] R. Hanaya, M. Sasa, K. Ishihara, K. Iida, T. Akimitsu, T. Serikawa, A. Noda, K. Kurisu, Abnormality of Ca 21 channels in CA3 pyramidal neurons of Noda epileptic rats (NERs), Epilepsia 39 (1998) 55. [7] K. Iida, M. Sasa, T. Serikawa, A. Noda, K. Ishihara, T. Akimitsu, R. Hanaya, K. Arita, K. Kurisu, Induction of convulsive seizures by acoustic priming in a new genetically defined model of epilepsy (Noda epileptic rat: NER), Epilepsy Res. 30 (1998) 115–126. [8] K. Ishihara, M. Sasa, T. Momiyama, H. Ujihara, J. Nakamura, T. Serikawa, J. Yamada, S. Takaori, Abnormal excitability of hippocampal CA3 pyramidal neurons of spontaneously epileptic rats (SER), double mutant, Exp. Neurol. 119 (1993) 287–290. [9] G.L. King, R. Dingledine, J.L. Giacchino, J.O. McNamara, Abnormal neuronal excitability in hippocampal slices from kindled rats, J. Neurophysiol. 54 (1985) 1295–1304. [10] G. Kostopoulos, C. Psarropoulou, H.L. Haas, Membrane properties, response to amines and to tetanic stimulation of hippocampal neurons in the genetically epileptic mutant mouse tottering, Exp. Brain Res. 72 (1988) 45–50. [11] D.C. McIntyre, M.E. Kelly, The parahippocampal cortices and kindling, Ann. NY Acad. Sci. 911 (2000) 343–354. [12] T. Momiyama, K. Ishihara, T. Serikawa, K. Moritake, M. Sasa, Effect of nicardipine on abnormal excitability of CA3 pyramidal cells in hippocampal slices of spontaneously epileptic rats, Eur. J. Pharmacol. 280 (1995) 119–123. [13] A. Noda, R. Hashizume, T. Maihara, Y. Tomizawa, Y. Ito, M. Inoue, K. Kobayashi, Y. Asano, M. Sasa, T. Serikawa, NER rat strain: a new type of genetic model in epilepsy research, Epilepsia 39 (1998) 99–107. [14] S. Rhem, P. Mehraein, A.P. Anzil and, F. Deerberg, A new rat mutant with defective overhairs and spongy degeneration of the central nervous system; clinical pathological studies, Lab. Anim. Sci. 32 (1982) 70–73. [15] T. Sakamoto, H. Niki, Acoustic priming lowers the threshold for electrically induced seizures in mice inferior colliculus, but not in the deep layers of superior colliculus, Brain Res. 898 (2001) 358–363. [16] M. Sasa, R. Hanaya, K. Iida, T. Akimitsu, K. Kurisu, A. Noda, T. Serikawa, A novel animal epilepsy model (NER), Jpn. J. Psychopharmacol. 17 (1997) 35–38. [17] M. Sasa, Y. Ohno, H. Ujihara, Y. Fujita, M. Yoshimura, S. Takaori, T. Serikawa, J. Yamada, Effect of antiepileptic drugs on absence-like and tonic seizures in the spontaneously epileptic rat, a double mutant rat, Epilepsia 29 (1998) 505–513. [18] T. Serikawa, J. Yamada, Epileptic seizures in rats homozygous for two mutations, zitter and tremor, J. Hered. 77 (1986) 441–444. [19] T. Serikawa, K. Kogishi, J. Yamada, Y. Ohno, H. Ujihara, Y. Fujita, M. Sasa, S. Takaori, Long-term effects of continual intake of phenobarbital on the spontaneously epileptic rat, Epilepsia 31 (1990) 9–14. [20] J. Yamada, T. Serikawa, J. Ishiko, T. Inui, H. Takada, Y. Kawai, A. Okaniwa, Rats with congenital tremor and curled whisker and hair, Exp. Anim. 34 (1985) 183–188.
Short communication
Epileptiform burst discharges in hippocampal CA3 neurons of young but not mature Noda epileptic rats (NER) Ryosuke Hanaya a , *, Masashi Sasa b , Yoshihiro Kiura a , Kumatoshi Ishihara c , Tadao Serikawa d , Kaoru Kurisu a a
Department of Neurosurgery, Hiroshima University School of Medicine, Minami-ku, Kasumi, Hiroshima 734 -8551, Japan b Department of Pharmacology, Hiroshima University School of Medicine, Hiroshima 734 -8551, Japan c Department of Pharmacotherapy, Institute of Pharmaceutical Science, Faculty of Medicine, Hiroshima University, Hiroshima 734 -8551, Japan d Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto 606 -8501, Japan Accepted 20 June 2002
Abstract Noda epileptic rat (NER), originally found in a colony of Crj; Wistar rats, shows spontaneous tonic–clonic convulsion characterized by the appearance of high voltage polyspikes in cortical and hippocampal EEG once every 2–3 days after 2–4 months of age. Electrophysiological studies using hippocampal slice preparations of NER were performed to determine whether hippocampal neurons have abnormal excitability. When a single stimulus (1–25 V) was delivered to the mossy fibers of NER at 4–6 weeks old before they showed any seizures, a long-lasting depolarization shift (DS) accompanied by repetitive firings and after-hyperpolarization following the abnormal firing was observed in seven of 14 hippocampal CA3 neurons. A lower stimulation intensity evoked DS and abnormal firing in three of nine CA3 neurons of NER at 10–15 weeks old which had already showed seizures at 10–15 weeks of age. However, the abnormal firing was not observed in any 10 neurons of the animals at more than 20 weeks old nor in Wistar rats. The input impedances of CA3 neurons in NER with abnormal firing were lower than those without abnormal firing and those in Wistar rats. The abnormal excitability obtained in NER at an age when it did not display any seizures suggests that the hippocampus may play a role in epileptogenicity in NER. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: basic mechanisms Keywords: Noda epileptic rat (NER); Epilepsy; Hippocampus; CA3 neuron; Abnormal excitement; Development
Noda epileptic rats (NER) are mutants found in an inbred colony of Crj; Wistar rats. After 2–4 months of age, NER spontaneously shows tonic–clonic convulsion characterized by the appearance of high voltage polyspikes in cortical and hippocampal EEG once every 2–3 days [13]. Sporadic spikes appear and gradually increase predominantly in the hippocampus from 11 weeks of age in parallel with the behavioral development of seizures. Acoustic priming from 3 weeks of age once every week consistently induces tonic–clonic convulsion in NER with sound stimuli after 11 weeks of age [7]. The seizures are *Corresponding author. Tel.: 181-82-257-5227; fax 181-82-2575229. E-mail address: [email protected] (R. Hanaya).
inhibited by conventional antiepileptics such as diazepam, valproate, phenobarbital, and carbamazepine, but rarely by phenytoin, zonisamide, and ethosuximide [16]. The spontaneously epileptic rat (SER) is a double mutant (zi / zi, tm / tm) obtained originally by mating a heterozygote tremor rat (tm / 1), a mutant found in the inbred colony of Kyoto–Wistar rats [20], with a homozygote zitter rat found in the Sprague–Dawley colony [14]. After 8 weeks of age, SER spontaneously shows both tonic convulsions with low voltage fast waves, and absence-like seizures characterized by sudden behavioral changes such as immobility and staring with the simultaneous appearance of paroxysms of 5–7-Hz spike–wave complexes in cortical and hippocampal EEG. In addition, mild stimuli such as a hand clap and blowing on the face easily induce
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03195-5
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convulsions [17,18]. This animal is useful for the evaluation of the acute and chronic effects of antiepileptics, since the antiepileptic profile of the conventional antiepileptic for tonic and absence-like seizures in SER is similar to those of human grand mal and petit mal epilepsy, respectively [17,19]. We have reported that in hippocampal CA3 pyramidal neurons of mature SER, which already show both tonic convulsion and absence-like seizures, a long-lasting depolarization shift accompanied by repetitive firing is induced by a single stimulus applied to the mossy fibers [8], and this abnormal excitation of the neurons is attributable to abnormalities of the Ca 21 channels [12]. Hyperexcitability of the neurons was also found in hippocampal CA1 neurons of the tottering mouse [10], and hippocampal CA3 neurons in kindled rats [9]. Genetically epileptic prone rats (GEPR), an animal model of spontaneous seizure and audiogenic seizure, also showed abnormal hippocampal excitability in vitro [3]. Therefore, the activity of hippocampal CA3 pyramidal neurons in NER was examined using conventional intracellular recording methods to determine whether these neurons show abnormal excitability. NERs were obtained from the Research Institute for Animal Science in Biochemistry and Toxicology (Kanagawa, Japan), and were inbred at the Institute for Laboratory Animals of our university. Wistar rats as controls were purchased from Charles River Japan (Yokohama, Japan). All animals were kept individually in shoebox-type cages in a room maintained at 2362 8C and 5565% humidity. They were provided with standard rat chow (F-2, Funabashi Form, Japan) and tap water ad libitum. Nineteen NER of both sexes (age: 4–54 weeks) and nine age-matched Wistar rats were used for this study. After decapitation, the brain was placed rapidly in cold oxygenated Ca 21 -free medium (in mM; 113 NaCl, 3 KCl, 1 NaH 2 PO 4 , 25 NaHCO 3 , 5 MgCl 2 , 11 glucose), and hippocampal slices were cut at a thickness of 400 mm with a microslicer (DTK-100, Dosaka EM, Japan). After 1–2 h incubation in artificial cerebrospinal fluid (ACSF (in mM); 113 NaCl, 3 KCl, 1 NaH 2 PO 4 , 25 NaHCO 3 , 2 CaCl 2 , 1 MgCl 2 , 11 glucose; pH 7.2) at 34 8C, one slice was transferred into the recording chamber. ACSF was continuously perfused over the slice at 1.5–2 ml / min at 30–32 8C. The ACSF was continuously bubbled with a mixture of 95% O 2 and 5% CO 2 . A single stimulus (0.1 ms duration) was applied to the mossy fibers every 5 s through a bipolar stimulating electrode placed in the granular cell layer of the dentate gyrus. The stimulus was increased to 1, 4, 10, 15, 20 and 25 V intensity to induce depolarization shift (DS). Intracellular recordings were made from the hippocampal CA3 pyramidal neurons using a glass microelectrode (electrical resistance; 40–100 MV) filled with 3 M KCl. Other details of the methods have been described elsewhere [5].
These responses were displayed on a digital oscilloscope (VC-10, Nihon Kohden, Japan) after amplification (MEZ8201, Nihon Kohden), and stored on a personal computer (Macintosh Color Classic or PowerMacintosh G4, Apple, USA) using the MacLab or PowerLab system (AD Instruments, Australia). The responses were continuously recorded on a thermal array recorder (RTA-1100, Nihon Kohden). The significance of the differences was determined by the Student’s t-test. The data were obtained from the neurons with a resting membrane potential of more than 245 mV. They were composed of 33 hippocampal CA3 neurons obtained from 19 NER. The intracellular activities of 18 CA3 neurons of nine normal Wistar rats were also recorded as controls. In NER at 4–6 weeks old when the animal did not yet show any spontaneous seizures, a long-lasting DS with repetitive firing followed by after-hyperpolarization was elicited by a single stimulation of the mossy fibers in seven (50%) of 14 hippocampal CA3 neurons tested (Fig. 1). In these seven neurons, a single action potential was induced by the stimulation at 5.1460.72 V, and DS with repetitive firing was induced by the stimulation at 13.1762.03 V. A significantly higher voltage was required to elicit DS and the abnormal firing. The mean duration of DS was 38.2667.91 ms in the seven neurons with DS. In the remaining seven neurons, only one action potential without
Fig. 1. Action potentials (a) and after-hyperpolarization (b) induced by mossy fiber stimulation in the hippocampal CA3 neurons of NER at 4 weeks old with no seizures, and in normal Wistar rats. Only a single action potential was induced by a single stimulation of mossy fibers in normal Wistar rats aged 4 weeks old.
R. Hanaya et al. / Brain Research 950 (2002) 317–320
Fig. 2. Action potentials in the hippocampal CA3 neurons of NER at 14 weeks (a) and 26 weeks old (b). (a) Depolarization shift accompanied with repetitive firing was induced by a single stimulation of mossy fibers in NER at 14 weeks old, which began to show spontaneous tonic–clonic convulsion. (b) Only a single action potential was induced by a single stimulation of mossy fibers in NER aged 26 weeks old.
DS was elicited by mossy fiber stimulation at 8.0061.27 V. However, in NER aged 10–15 weeks old, at which age the animals consistently showed convulsive seizures, the number of CA3 neurons with such DS accompanying repetitive firing elicited by mossy fiber stimulation was
319
decreased to 33.3% (three of nine neurons tested) (Fig. 2). In these three neurons, the mean voltage to induce DS was 13.3362.04 V. The voltage for inducing DS was not significantly different between NER aged 4–6 and 10–15 weeks. However, DS was significantly longer in the three neurons of NER aged 10–15 weeks than in the neurons of young NER (4–6 weeks) (Table 1). In the remaining six neurons tested, no DS was induced; no DS was induced by stimulus up to 25 V. After-hyperpolarization following DS was observed in all of seven and three neurons with DS: the mean maximum amplitude was 8.0160.36 (n57) and 6.5060.79 (n53) mV, and the mean duration was 3.6460.36 (n57) and 2.9360.11 (n53) s, in 4–6- and 10–15-week-old animals, respectively. In contrast, in NER after 15 weeks of age when they showed spontaneous seizures, the DS with repetitive firing was not induced by mossy fiber stimulation up to 25 V in any of 10 hippocampal CA3 neurons examined, and only a single action potential was elicited (Fig. 2). In Wistar rats aged 4–6 and more than 15 weeks old, only one action potential was elicited by mossy fiber stimulation up to 25 V in each of eight CA3 neurons examined (Fig. 1). The input impedance of CA3 neurons with abnormal firing in NER of all ages was significantly lower than that of those without abnormal firings. However, the resting membrane potential did not show any significant differences among NER of any age (Table 1). DS accompanying repetitive firing was found in hippocampal CA3 neurons of NER before and after the appearance of convulsive seizures. The DS in cortical and hippocampal neurons is involved in epileptic seizures of kindling animals, GAERS, tottering mouse and SER [3,4,8–10]. Such abnormal excitabilities of the CA3 neurons in NER showing epileptic seizures were similar to those found in SER in terms of the long-lasting duration of DS, including those induced by a single stimulation of the mossy fibers, although the number of neurons showing
Table 1 Physiological profiles of hippocampal CA3 neurons in NER and normal Wistar rats
NER (4–6 w) A (n57) B (n57) NER (10–15 w) A (n53) B (n56) NER (^15 w) A (n50) B (n510) Wistar rat (4–6 w, n58) (^20 w, n58)
Resting membrane potential (mV)
Input impedance (MV)
Number of spikes
Duration of depolarizing shift (ms)
258.462.8 264.762.6
25.263.1* 35.063.4
4.360.6 –
38.367.9 –
258.565.8 258.063.8
28.861.1** 39.960.4
5.060.7 –
60.465.8 –
– 260.968.3
– 41.862.6
– –
– –
255.962.3 257.164.3
38.963.1 40.462.8
– –
– –
Mean6S.E. A; Neurons showing abnormal firings induced by mossy fiber stimulation. B; Neurons showing single spikes induced by mossy fiber stimulation. * p,0.05; compared with B and Wistar rats. ** p,0.05; compared with B.
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such abnormalities was fewer in NER (30%) than SER (65%) [8]. We have previously demonstrated that the DS in hippocampal CA3 neurons of SER is due to enhanced Ca 21 influx, since the DS was inhibited by Ca 21 channel antagonists such as nicardipine and nifedipine and actual increases in the intracellular Ca 21 levels of CA3 neurons have been obtained by high K 1 and a single stimulation of the mossy fibers [1,2]. The DS obtained in the hippocampal CA3 neurons of NER was also suggested to be due to enhanced Ca 21 influx through Ca 21 channels as in SER, since the DS with repetitive firing was inhibited by nicardipine in some CA3 neurons of NER [6]. These findings suggest that focal hyperexcitability of neurons exists in the hippocampus at early postnatal ages. Animal models with abnormal excitability of the hippocampal CA3 neurons are known to show limbic seizures [11]. NER exhibits spontaneous tonic–clonic convulsion, standing with neck and forelimb clonus, wild jumping / running, opisthotonic postures and evolvement to tonic seizure proceeding to clonic convulsion followed by postictal flaccidity. This seizure pattern indicates the involvement of the forebrain and / or brainstem [7]. In addition, acoustic priming has been reported to activate the brainstem including the superior colliculus [15] and actually induce convulsive seizures in NER [7]. Sporadic spikes in the hippocampal CA3 area of NER followed by convulsive seizures were observed after 11 weeks of age [7]. Together with these findings, NER is thought to have epileptogenicity in the brainstem in addition to the hippocampus. It seems likely that the epileptogenic circuit including the brainstem–limbic system–cerebral cortex is completed with age and, thereafter, the abnormal excitability of the hippocampal CA3 neurons may be recovered by a mechanism as yet undetermined.
References [1] H. Amano, T. Amano, H. Matsubayashi, K. Ishihara, T. Serikawa, M. Sasa, Enhanced calcium influx in hippocampal CA3 neurons of spontaneously epileptic rats (SER), Epilepsia 42 (2001) 345–350. [2] T. Amano, H. Amano, K. Matsubayashi, Ishihara, T. Serikawa and M. Sasa, Enhanced Ca(21) influx with mossy fiber stimulation in hippocampal CA3 neurons of spontaneously epileptic rats, Brain Res. 910 (2001) 199–203. [3] V. Armand, P. Hoffmann, M. Vergnes, U. Heinemann, Epileptiform activity induced by 4-aminopyridine in entorhinal cortex hippocampal slices of rats with a genetically determined absence epilepsy (GAERS), Brain Res. 841 (1999) 62–69. [4] A.C. Bragdon, D.M. Taylor, J.O. McNamara, W.A. Wilson, Abnormal hyperexcitability of hippocampal slices from kindled rats is transient, Brain Res. 453 (1988) 257–264.
[5] R. Hanaya, M. Sasa, H. Ujihara, K. Ishihara, T. Serikawa, K. Iida, T. Akimitsu, K. Arita, K. Kurisu, Suppression by topiramate of epileptic burst discharges in hippocampal CA3 neurons of spontaneously epileptic rat in vitro, Brain Res. 789 (1998) 274–282. [6] R. Hanaya, M. Sasa, K. Ishihara, K. Iida, T. Akimitsu, T. Serikawa, A. Noda, K. Kurisu, Abnormality of Ca 21 channels in CA3 pyramidal neurons of Noda epileptic rats (NERs), Epilepsia 39 (1998) 55. [7] K. Iida, M. Sasa, T. Serikawa, A. Noda, K. Ishihara, T. Akimitsu, R. Hanaya, K. Arita, K. Kurisu, Induction of convulsive seizures by acoustic priming in a new genetically defined model of epilepsy (Noda epileptic rat: NER), Epilepsy Res. 30 (1998) 115–126. [8] K. Ishihara, M. Sasa, T. Momiyama, H. Ujihara, J. Nakamura, T. Serikawa, J. Yamada, S. Takaori, Abnormal excitability of hippocampal CA3 pyramidal neurons of spontaneously epileptic rats (SER), double mutant, Exp. Neurol. 119 (1993) 287–290. [9] G.L. King, R. Dingledine, J.L. Giacchino, J.O. McNamara, Abnormal neuronal excitability in hippocampal slices from kindled rats, J. Neurophysiol. 54 (1985) 1295–1304. [10] G. Kostopoulos, C. Psarropoulou, H.L. Haas, Membrane properties, response to amines and to tetanic stimulation of hippocampal neurons in the genetically epileptic mutant mouse tottering, Exp. Brain Res. 72 (1988) 45–50. [11] D.C. McIntyre, M.E. Kelly, The parahippocampal cortices and kindling, Ann. NY Acad. Sci. 911 (2000) 343–354. [12] T. Momiyama, K. Ishihara, T. Serikawa, K. Moritake, M. Sasa, Effect of nicardipine on abnormal excitability of CA3 pyramidal cells in hippocampal slices of spontaneously epileptic rats, Eur. J. Pharmacol. 280 (1995) 119–123. [13] A. Noda, R. Hashizume, T. Maihara, Y. Tomizawa, Y. Ito, M. Inoue, K. Kobayashi, Y. Asano, M. Sasa, T. Serikawa, NER rat strain: a new type of genetic model in epilepsy research, Epilepsia 39 (1998) 99–107. [14] S. Rhem, P. Mehraein, A.P. Anzil and, F. Deerberg, A new rat mutant with defective overhairs and spongy degeneration of the central nervous system; clinical pathological studies, Lab. Anim. Sci. 32 (1982) 70–73. [15] T. Sakamoto, H. Niki, Acoustic priming lowers the threshold for electrically induced seizures in mice inferior colliculus, but not in the deep layers of superior colliculus, Brain Res. 898 (2001) 358–363. [16] M. Sasa, R. Hanaya, K. Iida, T. Akimitsu, K. Kurisu, A. Noda, T. Serikawa, A novel animal epilepsy model (NER), Jpn. J. Psychopharmacol. 17 (1997) 35–38. [17] M. Sasa, Y. Ohno, H. Ujihara, Y. Fujita, M. Yoshimura, S. Takaori, T. Serikawa, J. Yamada, Effect of antiepileptic drugs on absence-like and tonic seizures in the spontaneously epileptic rat, a double mutant rat, Epilepsia 29 (1998) 505–513. [18] T. Serikawa, J. Yamada, Epileptic seizures in rats homozygous for two mutations, zitter and tremor, J. Hered. 77 (1986) 441–444. [19] T. Serikawa, K. Kogishi, J. Yamada, Y. Ohno, H. Ujihara, Y. Fujita, M. Sasa, S. Takaori, Long-term effects of continual intake of phenobarbital on the spontaneously epileptic rat, Epilepsia 31 (1990) 9–14. [20] J. Yamada, T. Serikawa, J. Ishiko, T. Inui, H. Takada, Y. Kawai, A. Okaniwa, Rats with congenital tremor and curled whisker and hair, Exp. Anim. 34 (1985) 183–188.