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Suppression by topiramate of epileptiform burst discharges in hippocampal CA3 neurons of spontaneously epileptic rat in vitro
Brain Research 789 Ž1998. 274–282
Research report
Suppression by topiramate of epileptiform burst discharges in hippocampal CA3 neurons of spontaneously epileptic rat in vitro Ryosuke Hanaya a , Masashi Sasa b, ) , Hisamitsu Ujihara c , Kumatoshi Ishihara b , Tadao Serikawa d , Koji Iida a , Tomohide Akimitsu a , Kazunori Arita a , Kaoru Kurisu a a b
Department of Neurosurgery, Hiroshima UniÕersity School of Medicine, Hiroshima, Japan Department of Pharmacology, Hiroshima UniÕersity School of Medicine, Hiroshima, Japan c Department of Neuropsychiatry, Kochi Medical School, Kochi, Japan d Institute of Laboratory Animals, Faculty of Medicine, Kyoto UniÕersity, Kyoto, Japan Accepted 23 December 1997
Abstract Topiramate, a novel antiepileptic drug, inhibits the seizures of spontaneously epileptic rat ŽSER., a double mutant Ž zirzi, tmrtm. which exhibits both tonic convulsion and absence-like seizures from the age of 8-weeks. Hippocampal CA3 pyramidal neurons in SER show a long-lasting depolarization shift with accompanying repetitive firing when a single electrostimulation is delivered to the mossy fibers in vitro. The effects of topiramate on the excitability of CA3 pyramidal neurons in SER were examined to elucidate the mechanism underlying the antiepileptic action. Intracellular recordings were performed in 23 hippocampal slice preparations of 16 SER aged 8–17 weeks. Topiramate Ž10–100 m M. dose-dependently inhibited the depolarizing shifts with repetitive firing induced by mossy fiber stimulation without affecting the first spike and resting membrane potentials in hippocampal CA3 neurons of SER. Higher dose of topiramate Ž100 m M. sometimes inhibited the first spike, and decreased excitatory postsynaptic potentials in the SER CA3 neurons. However, topiramate up to 100 m M did not affect the single action potential elicited by the stimulation in the hippocampal CA3 neurons of age-matched Wistar rat devoid of the seizure. Application of topiramate Ž100 m M. did not significantly affect the firing induced by depolarizing pulse applied in the CA3 neurons of the SER. In addition, topiramate Ž100 m M. had no effects on the Ca2q spike induced by intracellularly applied depolarizing pulse in the presence of tetrodotoxin and tetraethylammonium. In contrast, a dose-dependent inhibition of depolarization and repetitive firing induced by bath application of glutamate in CA3 pyramidal neurons was obtained with topiramate Ž10–100 m M.. Furthermore, topiramate Ž100 m M. decreased the number of miniature postsynaptic potential of CA3 pyramidal neurons of SER. In patch clamp whole cell recording using acutely dissociated hippocampal CA3 neurons from SER aged 8-weeks and age-matched normal Wistar rats, there were no remarkable effects on voltage dependent Ca2q current with topiramate up to 300 m M in either animal; the current was completely blocked by Cd 2q at a concentration of 1 mM. These findings suggest that topiramate inhibits release of glutamate from the nerve terminals andror abnormal firing of the CA3 pyramidal neurons of SER by mainly blocking glutamate receptors in the neurons. q 1998 Elsevier Science B.V. Keywords: Topiramate; Antiepileptic drug; Glutamatergic transmission; Hippocampal CA3 neuron; Abnormal excitation; Spontaneously epileptic rat ŽSER.
1. Introduction Topiramate, a novel antiepileptic drug, is a derivative of which differs from known antiepileptics structurally. This drug inhibited convulsions induced by electroconvulsive shock in mice and rats w8x as well as amygdala-kindled seizures and posttraumatic epilepsy in D-fructose,
) Corresponding author. Department of Pharmacology, Hiroshima University School of Medicine, Minami-ku, Kasumi, Hiroshima 734, Japan. Fax: q81-82-257-5144; E-mail: [email protected]
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 1 1 6 - 4
rats w4,20x. In preliminary studies on human epilepsy, topiramate was effective in the treatment of partial seizures w1,21x. Recently, a placebo-controlled dose-ranging trial showed that the drug was also effective for refractory partial seizure w5,13x. In cultured neurons, topiramate has been reported to block voltage-dependent sodium channels w3x, enhance GABA-mediated chloride flux and GABAevoked current w2,22x, and block kainate-induced current in cultured neurons w17x. Spontaneously epileptic rat ŽSER; zirzi, tmrtm. is a double mutant obtained by mating heterozygote tremor rat
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Ž tmrq ., a mutant found in the inbred colony of Kyoto– Wistar rats w23x, with a homozygote zitter rat Ž zirzi . found in a Sprague–Dawley colony w14x. After 8 weeks of age, SER spontaneously shows tonic convulsions and absencelike seizures which are characterized by sudden immobility and staring. Cortical and hippocampal EEG showed that the tonic convulsions and absence-like seizures are accompanied by low voltage fast waves and by 5–7 Hz spikewave-like complexes, respectively w15,16x. The antiepileptic profile of antiepileptic drugs in SER parallels that of human absence seizures and tonic convulsions w15x. We have reported that hippocampal CA3 pyramidal neurons in SER show a long-lasting Ž100–200 ms. depolarizing shift accompanied by repetitive firing with a single stimulation of the mossy fibers w6x, and this abnormal excitability attributes to abnormalities of the Ca2q channels w10x. We have reported that topiramate inhibits both tonic convulsion and absence-like seizures in SER w11x, and reduces the abnormally high extracellular levels of glutamate and aspartate in this animal w7x. Thus, conventional intracellular and patch clamp studies were performed to elucidate the mechanism underlying the antiepileptic effect of topiramate using the hippocampal slices of SER and acutely dissociated hippocampal CA3 neurons of these animals, respectively.
2. Materials and methods 2.1. Intracellular recording in hippocampal slices Sixteen mature SER of both sexes Ž8–17 weeks. exhibiting convulsive seizures and eight age-matched Wistar rats were used. After decapitation, the brain was rapidly placed in cold oxygenated Ca2q free-medium Žin mM: 113 NaCl, 3 KCl, 1 NaH 2 PO4 , 25 NaHCO 3 , 5 MgCl 2 , 11 glucose., and hippocampal slices were cut to 400 m m with a microslicer ŽDTK-1000, Dosaka, Japan.. After 1–2 h incubation in artificial cerebrospinal fluid ŽACSF: in mM; 113 NaCl, 3 KCl, 1 NaH 2 PO4 , 25 NaHCO 3 , 2 CaCl 2 , 1 MgCl 2 , 11 glucose; pH 7.2. at 348C, one slice was transferred into the recording chamber. ACSF was continuously perfused over the slice at 1.5–2 mlrmin at 30–328C. The ACSF was continuously bubbled with a mixture of 95% O 2 and 5% CO 2 . A bipolar stimulating electrode was placed in the granule cell layer of the dentate gyrus and a stimulus Ž0.1 ms duration and 6–10 V. was given to the mossy fiber every 5 s. Intracellular recordings were made from the hippocampal CA3 pyramidal neurons using a glass microelectrode Želectrical resistance; 40–100 M V . filled with 3 M KCl. Under the same conditions, a depolarizing pulse Ž1 nA intensity, 120 ms duration. in the cell was applied through the recording electrode to induce repetitive firing. To evoke Ca2q spikes, 1 m M sodium tetrodotoxin ŽTTX. and 10 mM tetraethylammoniumchloride ŽTEA. were added to ACSF, and 10 mM NaCl was
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deleted from ACSF for osmotic pressure adjustment of the solution. Under such a blockade of Naq and Kq channels, a Ca2q spike was elicited by applying a depolarizing pulse Ž1 nA, 120 ms. in the cell through the recording electrode filled with 3 M KCl. Glutamate ŽKanto, Japan. was applied to the bath to induce the depolarization of hippocampal CA3 pyramidal neurons in SER and normal Wistar rat. Topiramate Ž1–100 m M. dissolved in the ACSF was applied to the bath. 2.2. Patch-clamp whole cell recording in acutely dissociated hippocampal CA3 neurons Hippocampal slices of SER were made according to the method mentioned above. Thereafter, the hippocampus region was dissected from brain slices by cutting gently with a small knife. These slices were digested enzymatically by incubating them for 1.5 h at 358C in L-15 Medium ŽSigma, USA. containing 0.1% trypsin ŽSigma type XI. under perfusion with 100% O 2 . The minislices were sequentially washed 3 times with L-15 Medium containing 20% horse serum and washed three more times with only L-15 Medium. This minislice was reserved in L-15 Medium bubbling with 100% O 2 . Hippocampal neurons were dissociated into single cells by pipetting in L-15 Medium containing 0.01% DNAase. The dissociated neurons were transferred into a recording chamber. After 10 min, the chamber was continuously perfused with a solution Žin mM; 5 BaCl 2 , 130 TEA-Cl, 10 HEPES, and 0.0003 TTX; pH 7.4. at 1.5–2 mlrmin at 25–308C. The solution was bubbled with the 100% O 2 . The recording electrode was filled with internal solution Žin mM; 28 Trisma base, 70 Trisma PO4 , 11 EGTA, 40 TEA-Cl, 2.5 MgCl 2 , 5 ATP-K, 0.3 GTP-Na; pH 7.2.. For whole cell recordings, the electrode was sealed against the cell and the underlying membranes were broken by suction pulses, allowing the recording andror clamping of the intracellular membrane potential. Topiramate Ž300 m M., nicardipine Ž10 and 100 nM; Research Biochemicals, USA., and cadmium Ž1 mM. were directly ejected on the neurons through the U-tube placed near the recorded neuron w19x. The patch microelectrode had a resistance of 2–4 M V, and the series resistance was 5–15 M V under whole cell recording. Whole cell recording was done using the EPC-7 patch clamp system ŽList Electronics, Germany.. To obtain a topiramate-induced current–voltage Ž I–V . relation curve, I–V curves were constructed by application of a voltage command that went from y60 to q52 mV for 160 ms. 2.3. Recording and analysis These responses were displayed on the digital oscilloscope ŽVC-10, Nihon Kohden, Japan. after amplification ŽMEZ-8201, Nihon Kohden., and stored on a personal computer ŽMacintosh color classic, Apple, USA. using MacLab system ŽAD Instruments, Australia.. The re-
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sponses were continuously recorded on a thermal array recorder ŽRTA-1100, Nihon Kohden. andror on an FM data recorder ŽA-65, Sony, Japan.. Amplitude histograms of miniature postsynaptic potential ŽmPSP. were made by measuring mPSP with an amplitude of more than 1 mV. Statistical significance of the differences of topiramate was determined by a one-way analysis of variance ŽANOVA. followed by Bonferroni–Dunn’s test. Prior to those tests, statistical differences of variances were tested by Bartlett’s test. And the differences between pre- and posttreatment of topiramate was determined by paired Student’s t-test. 3. Results 3.1. Intracellular recording The data were obtained from the neurons with resting membrane potential of more than y45 mV. They were
composed of 23 and 10 hippocampal CA3 neurons obtained from 16 mature SER and 8 normal Wistar rats, respectively. 3.2. Effects of topiramate on depolarization shift with repetitiÕe firing The effects of topiramate were examined in 11 CA3 neurons with depolarization shift and repetitive firing elicited by mossy fiber stimulation in SER, and five CA3 neurons of normal Wistar rats. The mean duration of depolarization shift and number of firings induced by a single stimulation of mossy fiber in SER was 62.12 " 5.20 ms and 7.00 " 0.71 Ž n s 11. ŽFig. 1A.. In contrast, no such depolarization shift was observed in any of the five neurons of Wistar rat and only one action potential was obtained with mossy fiber stimulation in the five neurons ŽFig. 2.. Topiramate at a dose of 1 m M applied in the bath did not affect mossy fiber stimulation-induced abnormal firing. However, topiramate at 10–100 m M dose-depen-
Fig. 1. Effects of topiramate Ž10 and 100 m M. on intracellularly recorded action potentials ŽA. and after-hyperpolarization ŽB. elicited by mossy fiber stimulation in hippocampal CA3 neurons of SER. Arrows indicate the stimuli applied to the mossy fibers.
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Table 1 Effect of topiramate on repetitive firing induced by mossy fiber stimulation No. of neurons
SER Normal Wistar rat
9 5
No. of spikes Before
Topiramate 10 m M
100 m M
7.00 " 0.87 a 1
2.00 " 1.41b Ž n s 5.
0.57 " 0.46 b Ž n s 7. 1
The values are mean " S.E.M. a P - 0.01, as compared with the value of normal Wistar rat. b P - 0.01, as compared with the value before drug application.
Fig. 2. Failure of effects of topiramate Ž100 m M. on intracellularly recorded action potentials elicited by mossy fiber stimulation in hippocampal CA3 neurons of a normal Wistar rat. Arrows indicate the stimuli applied to the mossy fibers.
Fig. 3. Failure of effects of topiramate Ž100 m M. on sodium spikes induced by intracellularly applied depolarizing pulse Ž1 nA, 120 ms. in hippocampal CA3 neuron of SER.
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Fig. 4. Failure of effects of topiramate Ž100 m M. on Ca2q spikes induced by intracellularly applied depolarizing pulse Ž1 nA, 120 ms. in hippocampal CA3 neuron of SER in the presence of tetrodotoxin Ž1 m M. and tetraethylammonium Ž10 mM..
Fig. 5. Effects of topiramate Ž100 m M. on glutamate-induced depolarization in hippocampal CA3 neurons of SER. Glutamate-induced depolarization and spikes Ž1.. Effects of topiramate Ž100 m M. on glutamate-induced depolarization Ž1 mM. Ž2.. Effects of glutamate Ž1 mM. 7 min after washing the preparation Ž3.. The upper part of action potentials was cut off ŽA.. Action potentials recorded with faster sweep in left chart ŽB..
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Fig. 6. The effect of topiramate Ž100 m M. on miniature postsynaptic potentials in hippocampal CA3 neurons of SER. Filled and vacant columns show pre- and posttreatment of topiramate Ž100 m M., respectively. Each column and bar represents the mean and S.E.M., respectively Ž ns6.. ) P - 0.05, as compared with the pretreatment of topiramate.
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dently inhibited the depolarizing shift and repetitive firings in all nine neurons tested within 2 min after applying it to ACSF ŽFig. 1A, B.. Out of nine neurons, three were tested for both 10 and 100 m M topiramate and two were examined for either 10 or 100 m M ŽTable 1.. The first action potential induced by mossy fiber stimulation was also blocked by 100 m M topiramate in five of seven neurons ŽFig. 1A., while blockade of the first spike was not obtained with 10 m M topiramate in any neurons tested. After-hyperpolarization following the depolarizing shift and repetitive firings also was inhibited by 10–100 m M topiramate ŽFig. 1B.. A complete recovery of the responses was obtained by washing with ACSF for 3–5 min. In contrast to SER CA3 neurons, the action potential elicited by a mossy fiber stimulation in the CA3 neuron in Wistar rats remained unaffected with topiramate at doses up to 100 m M in all five neurons tested ŽFig. 2, Table 1..
Fig. 7. The effect of topiramate and nicardipine on acutely dissociated hippocampal CA3 neurons of SER. ŽA. Effect of topiramate and nicardipine on the inward current. ŽB. Current–voltage curve of peak currents in the presence and absence of topiramate Ž300 m M.. ŽC. Current–voltage curve of peak currents in the presence and absence of nicardipine Ž10 and 100 nM..
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Table 2 Effect of topiramate on glutamate-induced depolarization in hippocampal CA3 neurons of SER No. of neurons
Glutamate-induced depolarization ŽmV. Before
SER Normal Wistar rat
7 6
8.87 " 0.70 a 4.92 " 0.50
Topiramate 10 m M
100 m M
5.33 " 1.13 b Ž n s 3. 3.97 " 1.06 b Ž n s 3.
0.57 " 0.62 c,d 0.40 " 0.45 c,d
The value are mean " S.E.M. a P - 0.01, as compared with the value in normal Wistar rat. b P - 0.05, as compared with the value before drug application. c P - 0.01, as compared with the value before drug application. d P - 0.01, as compared with the value of 10 m M topiramate application.
3.3. Effects on depolarizing pulse-induced repetitiÕe firing Intracellular application of depolarizing current with a duration of 120 ms induced firing in SER hippocampal CA3 neurons. The repetitive firing was not affected by topiramate at the dose of 100 m M in six of eight neurons tested ŽFig. 3., and not affected by 10 m M topiramate at all. In the other two neurons, the firing was partially inhibited by topiramate. The mean number of spikes was
7.00 " 1.07 and 6.25 " 0.66 before and 5 min after application of 100 m M topiramate, respectively. However, there was no significant difference between these values. 3.4. Effects on Ca 2 q spikes Ca2q spikes were induced by intracellular application of depolarizing pulse lasting 120 ms under blockade of Naq and Kq channels in the presence of TTX and TEA,
Fig. 8. The effect of cadmium on acutely dissociated hippocampal CA3 neurons of SER. ŽA. Effect of cadmium and nicardipine on the inward current. ŽB. Current–voltage curve of peak currents in the presence and absence of cadmium Ž1 mM. and nicardipine Ž10 nM..
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respectively. The effects of topiramate on Ca2q spikes were examined in three hippocampal neurons of SER. Topiramate at a dose up to 100 m M did not affect the Ca2q spikes in any hippocampal CA3 neurons tested ŽFig. 4..
nicardipine Ž10–100 nM. and Cd 2q Ž1 mM. ŽFig. 7C and Fig. 8..
3.5. Effects on glutamate-induced depolarization and firing
Topiramate at a concentration of 10 m M inhibited the depolarizing shift accompanying repetitive firing induced by a single stimulus applied to the mossy fiber in hippocampal CA3 neurons of SER without affecting the first spikes. These findings indicate that topiramate inhibits glutamatergic transmission from mossy fiber to CA3 neurons, since the neurotransmitter involved in this pathway is glutamate w12x. Topiramate at a concentration that does not affect the first spike elicited by mossy fiber stimulation, inhibited the glutamate-induced depolarization accompanying firing. Therefore, it is suggested that the drug blocks the glutamate receptor andror related channels. This conclusion is supported by the biochemical finding that topiramate possesses affinity for glutamate receptors w9x, and the electrophysiological observation that kainate-induced current was inhibited by the drug in cultured hippocampal neurons w17x, although NMDA-induced current remained unaffected w18x. It is noteworthy that CA3 neurons in SER were much more sensitive to glutamate than those in normal Wistar rats herein. Higher dose Ž100 m M. of topiramate also inhibited the first spikes and EPSP elicited by mossy fiber stimulation in SER hippocampal CA3 neurons. This finding also suggests that higher dose of topiramate inhibits release of glutamate from the mossy fiber terminals andror blocked the postsynaptic glutamate receptors, since the drug had no effects on the depolarizing pulse-induced firing, that is due to Naq channel activation in the SER. Topiramate did not affect the Ca2q channels that show abnormalities in the SER CA3 neurons w6,10x, since the drug did not affect the Ca2q spikes induced by depolarizing pulse applied in the neuron in the presence of TTX and TEA. The whole cell recording also demonstrated that Ca2q inward currents in acutely dissociated hippocampal CA3 neurons were not affected by topiramate up to 300 m M. That topiramate may inhibit the release of glutamate are in line with the findings of an in vivo microdialysis that extracellular glutamate levels in the hippocampus of SER were 2- to 3-fold higher than those of normal Wistar rats, and topiramate lowered the level to the normal control value w7x. Furthermore that topiramate inhibited mPSPs, also indicates the ability of topiramate to inhibit the release of glutamate from the nerve terminal. In contrast to the CA3 neurons in SER, the spike elicited by mossy fiber stimulation in the neurons of normal Wistar rat was not affected by topiramate at concentrations up to 100 m M, although the glutamate-induced depolarization, which was much smaller than that in SER, was inhibited by high concentration Ž100 m M. of the drug. Therefore, it is suggested that glutamatergic transmission in normal animals is not affected by topiramate, although the drug
Glutamate was applied to the CA3 neurons which exhibited long-lasting depolarization shift with repetitive firing by a single stimulation of mossy fiber. Under these conditions, glutamate at a concentration of 1–10 mM produced depolarization with repetitive firing in a dose– dependent manner, and induced long-lasting depolarization shift with repetitive firings similar to those elicited by a single stimulation of mossy fiber ŽFig. 5.. The effects of topiramate were examined on the depolarization and repetitive firing induced by 1 mM glutamate in eight hippocampal neurons of SER, since the recovery was rapidly obtained with washing. The glutamate-induced depolarization of SER hippocampal neurons was inhibited by bath application of 10–100 m M topiramate ŽFig. 5A.. Bath application of glutamate at a concentration of 1 mM also induced depolarization accompanying repetitive firing in the six hippocampal CA3 neurons of the normal Wistar rats tested ŽTable 2.. However, the depolarization induced by glutamate Ž1 mM. in normal Wistar rat was significantly smaller than that in SER. The glutamate Ž1 mM.-induced depolarization and repetitive firing in the normal Wistar rat were also inhibited by topiramate at 100 m M ŽTable 2.. Topiramate-induced inhibition was abolished 5–10 min after washing of the preparation with ACSF ŽFig. 5.. 3.6. Effects on miniature postsynaptic potential Effects of topiramate on mPSP were examined in 6 hippocampal CA3 neurons. Topiramate Ž100 m M. applied to the bath significantly Ž P - 0.05. decreased the number of mPSP of SER CA3 neurons 2–4 min after application to the ACSF ŽFig. 6.. 3.7. Effects on Ca 2 q current of dissociated hippocampal CA3 pyramidal neurons Whole cell recording was performed in three neurons from SER and normal Wistar rat. The Ca2q channel inward current was obtained by decreasing the membrane potential at 8 mV-steps from the holding potential of y100 mV to q52 mV. The current was obtained at y60 mV, and the maximum current was detected when the membrane potential registered y20 mV. Topiramate Ž300 m M. did not affect the peak current and steady-state currents in any of the three CA3 neurons tested in SER ŽFig. 7A, B.. However the inward current in the hippocampal neurons of SER were dose-dependently inhibited by
4. Discussion
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has antagonistic activities against glutamate receptors in SER hippocampal neurons. 5. Conclusion Topiramate is suggested to act on the nerve terminals to inhibit the glutamate release andror on the postsynaptic glutamate receptors to antagonize the response in the hippocampal CA3 neurons of SER. Acknowledgements This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan. We are grateful to Kyowa Hakko Kogyo, Tokyo, Japan for a gift of topiramate. References w1x C. Aranguiz, J. McJilton, M. Vega, R.E. Ramsay, Safety and effectiveness of three oral doses of topiramate in the treatment of patients with refractory partial epilepsy, Epilepsia 32 Ž1991. 11, Žsuppl. 3.. w2x S.D. Brown, H.H. Wolf, E.A. Swinyard, R.E. Twyman, H.S. White, The novel anticonvulsant topiramate enhances GABA-mediated chloride flux, Epilepsia 34 Ž1993. 122–123, Žsuppl. 2.. w3x D.A. Coulter, S. Sonbati, R.J. Delorenzo, Selective effects of topiramate on sustained repetitive firing and spontaneous bursting in cultured hippocampal neurons, Epilepsia 34 Ž1993. 123, Žsuppl. 2.. w4x H. Edmonds, D. Jiang, P. Zhang, J.L. Vaught, Topiramate in a rat model of posttraumatic epilepsy, Epilepsia 32 Ž1991. 15, Žsuppl. 3.. w5x E. Faught, B.J. Wilder, R.E. Ramsay, R.A. Reife, L.D. Kramer, G.W. Pledger, R.M. Karim, the Topiramate YD Study group, Topiramate placebo-controlled dose-ranging trial in refractory partial epilepsy using 200-, 400-, and 600-mg daily dosages, Neurology 46 Ž1996. 1684–1690. w6x 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 ŽSER., a double mutant, Exp. Neurol. 119 Ž1993. 287–290. w7x T. Kanda, M. Kurokawa, S. Tamura, J. Nakamura, A. Ishii, Y. Kuwana, T. Serikawa, J. Yamada, K. Ishihara, M. Sasa, Topiramate reduces abnormally high extracellular levels of glutamate and aspartate in the hippocampus of spontaneously epileptic rats ŽSER., Life Sci. 59 Ž1996. 1607–1616.
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Research report
Suppression by topiramate of epileptiform burst discharges in hippocampal CA3 neurons of spontaneously epileptic rat in vitro Ryosuke Hanaya a , Masashi Sasa b, ) , Hisamitsu Ujihara c , Kumatoshi Ishihara b , Tadao Serikawa d , Koji Iida a , Tomohide Akimitsu a , Kazunori Arita a , Kaoru Kurisu a a b
Department of Neurosurgery, Hiroshima UniÕersity School of Medicine, Hiroshima, Japan Department of Pharmacology, Hiroshima UniÕersity School of Medicine, Hiroshima, Japan c Department of Neuropsychiatry, Kochi Medical School, Kochi, Japan d Institute of Laboratory Animals, Faculty of Medicine, Kyoto UniÕersity, Kyoto, Japan Accepted 23 December 1997
Abstract Topiramate, a novel antiepileptic drug, inhibits the seizures of spontaneously epileptic rat ŽSER., a double mutant Ž zirzi, tmrtm. which exhibits both tonic convulsion and absence-like seizures from the age of 8-weeks. Hippocampal CA3 pyramidal neurons in SER show a long-lasting depolarization shift with accompanying repetitive firing when a single electrostimulation is delivered to the mossy fibers in vitro. The effects of topiramate on the excitability of CA3 pyramidal neurons in SER were examined to elucidate the mechanism underlying the antiepileptic action. Intracellular recordings were performed in 23 hippocampal slice preparations of 16 SER aged 8–17 weeks. Topiramate Ž10–100 m M. dose-dependently inhibited the depolarizing shifts with repetitive firing induced by mossy fiber stimulation without affecting the first spike and resting membrane potentials in hippocampal CA3 neurons of SER. Higher dose of topiramate Ž100 m M. sometimes inhibited the first spike, and decreased excitatory postsynaptic potentials in the SER CA3 neurons. However, topiramate up to 100 m M did not affect the single action potential elicited by the stimulation in the hippocampal CA3 neurons of age-matched Wistar rat devoid of the seizure. Application of topiramate Ž100 m M. did not significantly affect the firing induced by depolarizing pulse applied in the CA3 neurons of the SER. In addition, topiramate Ž100 m M. had no effects on the Ca2q spike induced by intracellularly applied depolarizing pulse in the presence of tetrodotoxin and tetraethylammonium. In contrast, a dose-dependent inhibition of depolarization and repetitive firing induced by bath application of glutamate in CA3 pyramidal neurons was obtained with topiramate Ž10–100 m M.. Furthermore, topiramate Ž100 m M. decreased the number of miniature postsynaptic potential of CA3 pyramidal neurons of SER. In patch clamp whole cell recording using acutely dissociated hippocampal CA3 neurons from SER aged 8-weeks and age-matched normal Wistar rats, there were no remarkable effects on voltage dependent Ca2q current with topiramate up to 300 m M in either animal; the current was completely blocked by Cd 2q at a concentration of 1 mM. These findings suggest that topiramate inhibits release of glutamate from the nerve terminals andror abnormal firing of the CA3 pyramidal neurons of SER by mainly blocking glutamate receptors in the neurons. q 1998 Elsevier Science B.V. Keywords: Topiramate; Antiepileptic drug; Glutamatergic transmission; Hippocampal CA3 neuron; Abnormal excitation; Spontaneously epileptic rat ŽSER.
1. Introduction Topiramate, a novel antiepileptic drug, is a derivative of which differs from known antiepileptics structurally. This drug inhibited convulsions induced by electroconvulsive shock in mice and rats w8x as well as amygdala-kindled seizures and posttraumatic epilepsy in D-fructose,
) Corresponding author. Department of Pharmacology, Hiroshima University School of Medicine, Minami-ku, Kasumi, Hiroshima 734, Japan. Fax: q81-82-257-5144; E-mail: [email protected]
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 1 1 6 - 4
rats w4,20x. In preliminary studies on human epilepsy, topiramate was effective in the treatment of partial seizures w1,21x. Recently, a placebo-controlled dose-ranging trial showed that the drug was also effective for refractory partial seizure w5,13x. In cultured neurons, topiramate has been reported to block voltage-dependent sodium channels w3x, enhance GABA-mediated chloride flux and GABAevoked current w2,22x, and block kainate-induced current in cultured neurons w17x. Spontaneously epileptic rat ŽSER; zirzi, tmrtm. is a double mutant obtained by mating heterozygote tremor rat
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Ž tmrq ., a mutant found in the inbred colony of Kyoto– Wistar rats w23x, with a homozygote zitter rat Ž zirzi . found in a Sprague–Dawley colony w14x. After 8 weeks of age, SER spontaneously shows tonic convulsions and absencelike seizures which are characterized by sudden immobility and staring. Cortical and hippocampal EEG showed that the tonic convulsions and absence-like seizures are accompanied by low voltage fast waves and by 5–7 Hz spikewave-like complexes, respectively w15,16x. The antiepileptic profile of antiepileptic drugs in SER parallels that of human absence seizures and tonic convulsions w15x. We have reported that hippocampal CA3 pyramidal neurons in SER show a long-lasting Ž100–200 ms. depolarizing shift accompanied by repetitive firing with a single stimulation of the mossy fibers w6x, and this abnormal excitability attributes to abnormalities of the Ca2q channels w10x. We have reported that topiramate inhibits both tonic convulsion and absence-like seizures in SER w11x, and reduces the abnormally high extracellular levels of glutamate and aspartate in this animal w7x. Thus, conventional intracellular and patch clamp studies were performed to elucidate the mechanism underlying the antiepileptic effect of topiramate using the hippocampal slices of SER and acutely dissociated hippocampal CA3 neurons of these animals, respectively.
2. Materials and methods 2.1. Intracellular recording in hippocampal slices Sixteen mature SER of both sexes Ž8–17 weeks. exhibiting convulsive seizures and eight age-matched Wistar rats were used. After decapitation, the brain was rapidly placed in cold oxygenated Ca2q free-medium Žin mM: 113 NaCl, 3 KCl, 1 NaH 2 PO4 , 25 NaHCO 3 , 5 MgCl 2 , 11 glucose., and hippocampal slices were cut to 400 m m with a microslicer ŽDTK-1000, Dosaka, Japan.. After 1–2 h incubation in artificial cerebrospinal fluid ŽACSF: in mM; 113 NaCl, 3 KCl, 1 NaH 2 PO4 , 25 NaHCO 3 , 2 CaCl 2 , 1 MgCl 2 , 11 glucose; pH 7.2. at 348C, one slice was transferred into the recording chamber. ACSF was continuously perfused over the slice at 1.5–2 mlrmin at 30–328C. The ACSF was continuously bubbled with a mixture of 95% O 2 and 5% CO 2 . A bipolar stimulating electrode was placed in the granule cell layer of the dentate gyrus and a stimulus Ž0.1 ms duration and 6–10 V. was given to the mossy fiber every 5 s. Intracellular recordings were made from the hippocampal CA3 pyramidal neurons using a glass microelectrode Želectrical resistance; 40–100 M V . filled with 3 M KCl. Under the same conditions, a depolarizing pulse Ž1 nA intensity, 120 ms duration. in the cell was applied through the recording electrode to induce repetitive firing. To evoke Ca2q spikes, 1 m M sodium tetrodotoxin ŽTTX. and 10 mM tetraethylammoniumchloride ŽTEA. were added to ACSF, and 10 mM NaCl was
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deleted from ACSF for osmotic pressure adjustment of the solution. Under such a blockade of Naq and Kq channels, a Ca2q spike was elicited by applying a depolarizing pulse Ž1 nA, 120 ms. in the cell through the recording electrode filled with 3 M KCl. Glutamate ŽKanto, Japan. was applied to the bath to induce the depolarization of hippocampal CA3 pyramidal neurons in SER and normal Wistar rat. Topiramate Ž1–100 m M. dissolved in the ACSF was applied to the bath. 2.2. Patch-clamp whole cell recording in acutely dissociated hippocampal CA3 neurons Hippocampal slices of SER were made according to the method mentioned above. Thereafter, the hippocampus region was dissected from brain slices by cutting gently with a small knife. These slices were digested enzymatically by incubating them for 1.5 h at 358C in L-15 Medium ŽSigma, USA. containing 0.1% trypsin ŽSigma type XI. under perfusion with 100% O 2 . The minislices were sequentially washed 3 times with L-15 Medium containing 20% horse serum and washed three more times with only L-15 Medium. This minislice was reserved in L-15 Medium bubbling with 100% O 2 . Hippocampal neurons were dissociated into single cells by pipetting in L-15 Medium containing 0.01% DNAase. The dissociated neurons were transferred into a recording chamber. After 10 min, the chamber was continuously perfused with a solution Žin mM; 5 BaCl 2 , 130 TEA-Cl, 10 HEPES, and 0.0003 TTX; pH 7.4. at 1.5–2 mlrmin at 25–308C. The solution was bubbled with the 100% O 2 . The recording electrode was filled with internal solution Žin mM; 28 Trisma base, 70 Trisma PO4 , 11 EGTA, 40 TEA-Cl, 2.5 MgCl 2 , 5 ATP-K, 0.3 GTP-Na; pH 7.2.. For whole cell recordings, the electrode was sealed against the cell and the underlying membranes were broken by suction pulses, allowing the recording andror clamping of the intracellular membrane potential. Topiramate Ž300 m M., nicardipine Ž10 and 100 nM; Research Biochemicals, USA., and cadmium Ž1 mM. were directly ejected on the neurons through the U-tube placed near the recorded neuron w19x. The patch microelectrode had a resistance of 2–4 M V, and the series resistance was 5–15 M V under whole cell recording. Whole cell recording was done using the EPC-7 patch clamp system ŽList Electronics, Germany.. To obtain a topiramate-induced current–voltage Ž I–V . relation curve, I–V curves were constructed by application of a voltage command that went from y60 to q52 mV for 160 ms. 2.3. Recording and analysis These responses were displayed on the digital oscilloscope ŽVC-10, Nihon Kohden, Japan. after amplification ŽMEZ-8201, Nihon Kohden., and stored on a personal computer ŽMacintosh color classic, Apple, USA. using MacLab system ŽAD Instruments, Australia.. The re-
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sponses were continuously recorded on a thermal array recorder ŽRTA-1100, Nihon Kohden. andror on an FM data recorder ŽA-65, Sony, Japan.. Amplitude histograms of miniature postsynaptic potential ŽmPSP. were made by measuring mPSP with an amplitude of more than 1 mV. Statistical significance of the differences of topiramate was determined by a one-way analysis of variance ŽANOVA. followed by Bonferroni–Dunn’s test. Prior to those tests, statistical differences of variances were tested by Bartlett’s test. And the differences between pre- and posttreatment of topiramate was determined by paired Student’s t-test. 3. Results 3.1. Intracellular recording The data were obtained from the neurons with resting membrane potential of more than y45 mV. They were
composed of 23 and 10 hippocampal CA3 neurons obtained from 16 mature SER and 8 normal Wistar rats, respectively. 3.2. Effects of topiramate on depolarization shift with repetitiÕe firing The effects of topiramate were examined in 11 CA3 neurons with depolarization shift and repetitive firing elicited by mossy fiber stimulation in SER, and five CA3 neurons of normal Wistar rats. The mean duration of depolarization shift and number of firings induced by a single stimulation of mossy fiber in SER was 62.12 " 5.20 ms and 7.00 " 0.71 Ž n s 11. ŽFig. 1A.. In contrast, no such depolarization shift was observed in any of the five neurons of Wistar rat and only one action potential was obtained with mossy fiber stimulation in the five neurons ŽFig. 2.. Topiramate at a dose of 1 m M applied in the bath did not affect mossy fiber stimulation-induced abnormal firing. However, topiramate at 10–100 m M dose-depen-
Fig. 1. Effects of topiramate Ž10 and 100 m M. on intracellularly recorded action potentials ŽA. and after-hyperpolarization ŽB. elicited by mossy fiber stimulation in hippocampal CA3 neurons of SER. Arrows indicate the stimuli applied to the mossy fibers.
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Table 1 Effect of topiramate on repetitive firing induced by mossy fiber stimulation No. of neurons
SER Normal Wistar rat
9 5
No. of spikes Before
Topiramate 10 m M
100 m M
7.00 " 0.87 a 1
2.00 " 1.41b Ž n s 5.
0.57 " 0.46 b Ž n s 7. 1
The values are mean " S.E.M. a P - 0.01, as compared with the value of normal Wistar rat. b P - 0.01, as compared with the value before drug application.
Fig. 2. Failure of effects of topiramate Ž100 m M. on intracellularly recorded action potentials elicited by mossy fiber stimulation in hippocampal CA3 neurons of a normal Wistar rat. Arrows indicate the stimuli applied to the mossy fibers.
Fig. 3. Failure of effects of topiramate Ž100 m M. on sodium spikes induced by intracellularly applied depolarizing pulse Ž1 nA, 120 ms. in hippocampal CA3 neuron of SER.
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Fig. 4. Failure of effects of topiramate Ž100 m M. on Ca2q spikes induced by intracellularly applied depolarizing pulse Ž1 nA, 120 ms. in hippocampal CA3 neuron of SER in the presence of tetrodotoxin Ž1 m M. and tetraethylammonium Ž10 mM..
Fig. 5. Effects of topiramate Ž100 m M. on glutamate-induced depolarization in hippocampal CA3 neurons of SER. Glutamate-induced depolarization and spikes Ž1.. Effects of topiramate Ž100 m M. on glutamate-induced depolarization Ž1 mM. Ž2.. Effects of glutamate Ž1 mM. 7 min after washing the preparation Ž3.. The upper part of action potentials was cut off ŽA.. Action potentials recorded with faster sweep in left chart ŽB..
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Fig. 6. The effect of topiramate Ž100 m M. on miniature postsynaptic potentials in hippocampal CA3 neurons of SER. Filled and vacant columns show pre- and posttreatment of topiramate Ž100 m M., respectively. Each column and bar represents the mean and S.E.M., respectively Ž ns6.. ) P - 0.05, as compared with the pretreatment of topiramate.
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dently inhibited the depolarizing shift and repetitive firings in all nine neurons tested within 2 min after applying it to ACSF ŽFig. 1A, B.. Out of nine neurons, three were tested for both 10 and 100 m M topiramate and two were examined for either 10 or 100 m M ŽTable 1.. The first action potential induced by mossy fiber stimulation was also blocked by 100 m M topiramate in five of seven neurons ŽFig. 1A., while blockade of the first spike was not obtained with 10 m M topiramate in any neurons tested. After-hyperpolarization following the depolarizing shift and repetitive firings also was inhibited by 10–100 m M topiramate ŽFig. 1B.. A complete recovery of the responses was obtained by washing with ACSF for 3–5 min. In contrast to SER CA3 neurons, the action potential elicited by a mossy fiber stimulation in the CA3 neuron in Wistar rats remained unaffected with topiramate at doses up to 100 m M in all five neurons tested ŽFig. 2, Table 1..
Fig. 7. The effect of topiramate and nicardipine on acutely dissociated hippocampal CA3 neurons of SER. ŽA. Effect of topiramate and nicardipine on the inward current. ŽB. Current–voltage curve of peak currents in the presence and absence of topiramate Ž300 m M.. ŽC. Current–voltage curve of peak currents in the presence and absence of nicardipine Ž10 and 100 nM..
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Table 2 Effect of topiramate on glutamate-induced depolarization in hippocampal CA3 neurons of SER No. of neurons
Glutamate-induced depolarization ŽmV. Before
SER Normal Wistar rat
7 6
8.87 " 0.70 a 4.92 " 0.50
Topiramate 10 m M
100 m M
5.33 " 1.13 b Ž n s 3. 3.97 " 1.06 b Ž n s 3.
0.57 " 0.62 c,d 0.40 " 0.45 c,d
The value are mean " S.E.M. a P - 0.01, as compared with the value in normal Wistar rat. b P - 0.05, as compared with the value before drug application. c P - 0.01, as compared with the value before drug application. d P - 0.01, as compared with the value of 10 m M topiramate application.
3.3. Effects on depolarizing pulse-induced repetitiÕe firing Intracellular application of depolarizing current with a duration of 120 ms induced firing in SER hippocampal CA3 neurons. The repetitive firing was not affected by topiramate at the dose of 100 m M in six of eight neurons tested ŽFig. 3., and not affected by 10 m M topiramate at all. In the other two neurons, the firing was partially inhibited by topiramate. The mean number of spikes was
7.00 " 1.07 and 6.25 " 0.66 before and 5 min after application of 100 m M topiramate, respectively. However, there was no significant difference between these values. 3.4. Effects on Ca 2 q spikes Ca2q spikes were induced by intracellular application of depolarizing pulse lasting 120 ms under blockade of Naq and Kq channels in the presence of TTX and TEA,
Fig. 8. The effect of cadmium on acutely dissociated hippocampal CA3 neurons of SER. ŽA. Effect of cadmium and nicardipine on the inward current. ŽB. Current–voltage curve of peak currents in the presence and absence of cadmium Ž1 mM. and nicardipine Ž10 nM..
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respectively. The effects of topiramate on Ca2q spikes were examined in three hippocampal neurons of SER. Topiramate at a dose up to 100 m M did not affect the Ca2q spikes in any hippocampal CA3 neurons tested ŽFig. 4..
nicardipine Ž10–100 nM. and Cd 2q Ž1 mM. ŽFig. 7C and Fig. 8..
3.5. Effects on glutamate-induced depolarization and firing
Topiramate at a concentration of 10 m M inhibited the depolarizing shift accompanying repetitive firing induced by a single stimulus applied to the mossy fiber in hippocampal CA3 neurons of SER without affecting the first spikes. These findings indicate that topiramate inhibits glutamatergic transmission from mossy fiber to CA3 neurons, since the neurotransmitter involved in this pathway is glutamate w12x. Topiramate at a concentration that does not affect the first spike elicited by mossy fiber stimulation, inhibited the glutamate-induced depolarization accompanying firing. Therefore, it is suggested that the drug blocks the glutamate receptor andror related channels. This conclusion is supported by the biochemical finding that topiramate possesses affinity for glutamate receptors w9x, and the electrophysiological observation that kainate-induced current was inhibited by the drug in cultured hippocampal neurons w17x, although NMDA-induced current remained unaffected w18x. It is noteworthy that CA3 neurons in SER were much more sensitive to glutamate than those in normal Wistar rats herein. Higher dose Ž100 m M. of topiramate also inhibited the first spikes and EPSP elicited by mossy fiber stimulation in SER hippocampal CA3 neurons. This finding also suggests that higher dose of topiramate inhibits release of glutamate from the mossy fiber terminals andror blocked the postsynaptic glutamate receptors, since the drug had no effects on the depolarizing pulse-induced firing, that is due to Naq channel activation in the SER. Topiramate did not affect the Ca2q channels that show abnormalities in the SER CA3 neurons w6,10x, since the drug did not affect the Ca2q spikes induced by depolarizing pulse applied in the neuron in the presence of TTX and TEA. The whole cell recording also demonstrated that Ca2q inward currents in acutely dissociated hippocampal CA3 neurons were not affected by topiramate up to 300 m M. That topiramate may inhibit the release of glutamate are in line with the findings of an in vivo microdialysis that extracellular glutamate levels in the hippocampus of SER were 2- to 3-fold higher than those of normal Wistar rats, and topiramate lowered the level to the normal control value w7x. Furthermore that topiramate inhibited mPSPs, also indicates the ability of topiramate to inhibit the release of glutamate from the nerve terminal. In contrast to the CA3 neurons in SER, the spike elicited by mossy fiber stimulation in the neurons of normal Wistar rat was not affected by topiramate at concentrations up to 100 m M, although the glutamate-induced depolarization, which was much smaller than that in SER, was inhibited by high concentration Ž100 m M. of the drug. Therefore, it is suggested that glutamatergic transmission in normal animals is not affected by topiramate, although the drug
Glutamate was applied to the CA3 neurons which exhibited long-lasting depolarization shift with repetitive firing by a single stimulation of mossy fiber. Under these conditions, glutamate at a concentration of 1–10 mM produced depolarization with repetitive firing in a dose– dependent manner, and induced long-lasting depolarization shift with repetitive firings similar to those elicited by a single stimulation of mossy fiber ŽFig. 5.. The effects of topiramate were examined on the depolarization and repetitive firing induced by 1 mM glutamate in eight hippocampal neurons of SER, since the recovery was rapidly obtained with washing. The glutamate-induced depolarization of SER hippocampal neurons was inhibited by bath application of 10–100 m M topiramate ŽFig. 5A.. Bath application of glutamate at a concentration of 1 mM also induced depolarization accompanying repetitive firing in the six hippocampal CA3 neurons of the normal Wistar rats tested ŽTable 2.. However, the depolarization induced by glutamate Ž1 mM. in normal Wistar rat was significantly smaller than that in SER. The glutamate Ž1 mM.-induced depolarization and repetitive firing in the normal Wistar rat were also inhibited by topiramate at 100 m M ŽTable 2.. Topiramate-induced inhibition was abolished 5–10 min after washing of the preparation with ACSF ŽFig. 5.. 3.6. Effects on miniature postsynaptic potential Effects of topiramate on mPSP were examined in 6 hippocampal CA3 neurons. Topiramate Ž100 m M. applied to the bath significantly Ž P - 0.05. decreased the number of mPSP of SER CA3 neurons 2–4 min after application to the ACSF ŽFig. 6.. 3.7. Effects on Ca 2 q current of dissociated hippocampal CA3 pyramidal neurons Whole cell recording was performed in three neurons from SER and normal Wistar rat. The Ca2q channel inward current was obtained by decreasing the membrane potential at 8 mV-steps from the holding potential of y100 mV to q52 mV. The current was obtained at y60 mV, and the maximum current was detected when the membrane potential registered y20 mV. Topiramate Ž300 m M. did not affect the peak current and steady-state currents in any of the three CA3 neurons tested in SER ŽFig. 7A, B.. However the inward current in the hippocampal neurons of SER were dose-dependently inhibited by
4. Discussion
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has antagonistic activities against glutamate receptors in SER hippocampal neurons. 5. Conclusion Topiramate is suggested to act on the nerve terminals to inhibit the glutamate release andror on the postsynaptic glutamate receptors to antagonize the response in the hippocampal CA3 neurons of SER. Acknowledgements This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan. We are grateful to Kyowa Hakko Kogyo, Tokyo, Japan for a gift of topiramate. References w1x C. Aranguiz, J. McJilton, M. Vega, R.E. Ramsay, Safety and effectiveness of three oral doses of topiramate in the treatment of patients with refractory partial epilepsy, Epilepsia 32 Ž1991. 11, Žsuppl. 3.. w2x S.D. Brown, H.H. Wolf, E.A. Swinyard, R.E. Twyman, H.S. White, The novel anticonvulsant topiramate enhances GABA-mediated chloride flux, Epilepsia 34 Ž1993. 122–123, Žsuppl. 2.. w3x D.A. Coulter, S. Sonbati, R.J. Delorenzo, Selective effects of topiramate on sustained repetitive firing and spontaneous bursting in cultured hippocampal neurons, Epilepsia 34 Ž1993. 123, Žsuppl. 2.. w4x H. Edmonds, D. Jiang, P. Zhang, J.L. Vaught, Topiramate in a rat model of posttraumatic epilepsy, Epilepsia 32 Ž1991. 15, Žsuppl. 3.. w5x E. Faught, B.J. Wilder, R.E. Ramsay, R.A. Reife, L.D. Kramer, G.W. Pledger, R.M. Karim, the Topiramate YD Study group, Topiramate placebo-controlled dose-ranging trial in refractory partial epilepsy using 200-, 400-, and 600-mg daily dosages, Neurology 46 Ž1996. 1684–1690. w6x 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 ŽSER., a double mutant, Exp. Neurol. 119 Ž1993. 287–290. w7x T. Kanda, M. Kurokawa, S. Tamura, J. Nakamura, A. Ishii, Y. Kuwana, T. Serikawa, J. Yamada, K. Ishihara, M. Sasa, Topiramate reduces abnormally high extracellular levels of glutamate and aspartate in the hippocampus of spontaneously epileptic rats ŽSER., Life Sci. 59 Ž1996. 1607–1616.
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