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Inhibitory effects of levetiracetam on the high-voltage-activated L-type Ca2+ channels in hippocampal CA3 neurons of spontaneously epileptic rat (SER)
Brain Research Bulletin 90 (2013) 142–148
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Inhibitory effects of levetiracetam on the high-voltage-activated L-type Ca2+ channels in hippocampal CA3 neurons of spontaneously epileptic rat (SER) Hai-Dun Yan a , Kumatoshi Ishihara b,e,∗ , Takahiro Seki a , Ryosuke Hanaya c,f , Kaoru Kurisu c , Kazunori Arita c,f , Tadao Serikawa d , Masashi Sasa a,g a
Department of Pharmacology, Hiroshima University School of Medicine, Hiroshima 734-8551, Japan Department of Pharmacotherapy, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan c Department of Neurosurgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan d Institute of Laboratory Animals, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan e Laboratory of Neuropharmacology, Faculty of Pharmaceutical Sciences, Hiroshima International University, Kure 737-0112, Japan f Department of Neurosurgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan g Nagisa Clinic, Hirakata 573-1183, Japan b
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Article history: Received 10 September 2012 Received in revised form 16 October 2012 Accepted 17 October 2012 Available online 27 October 2012 Keywords: Levetiracetam (LEV) Ca2+ current Spontaneously epileptic rat (SER) Hippocampal CA3 neuron Patch clamp
a b s t r a c t Levetiracetam (LEV) is a widely used antiepileptic agent for partial refractory epilepsy in humans. LEV has unique antiepileptic effects in that it does not inhibit electroshock- or pentylenetetrazol-induced convulsion, but does inhibit seizures in kindling animal and spontaneously epileptic rat (SER: zi/zi, tm/tm) that shows both tonic convulsion and absence-like seizures. LEV also has unique characteristics in terms of its antiepileptic mechanism; it has no activity on Na+ and K+ channels or on glutamate and GABAA receptors. Recently, we found that LEV inhibits the depolarization shift and accompanying repetitive firing induced by mossy fiber stimulation in CA3 neurons of SER hippocampal slices. Therefore, this study was performed to determine whether LEV could inhibit the voltage-activated L-type Ca2+ current of hippocampal CA3 neurons obtained from SER and the non-epileptic Wistar rat. As previously reported, SER CA3 neurons were classified into type 1 and type 2 neurons. The application of LEV (100 M) elevated the threshold for activation of the Ca2+ current, which was lowered in SER type 1 neurons and reduced the current size. Type 2 neurons of SER have a similar current–voltage relationship to Wistar rat neurons and the decay component of Ca2+ current during depolarization pulse in type 2 neurons was found to be smaller than that in Wistar rat neurons. LEV (100 M) also reduced Ca2+ current in SER type 2 neurons. The effects of LEV were examined on such type 2 SER hippocampal CA3 neurons, compared with those on Wistar rat CA3 neurons. Application of LEV (10 M) produced a significant decrease of amplitude of the Ca2+ current in SER neurons, although at this concentration of LEV there was no statistically significant decrease in the amplitude of Ca2+ current in Wistar rat neurons. Furthermore, LEV (100 nM–1 mM) reduced the Ca2+ current in a concentration-dependent manner in both SER and Wistar rat neurons, but the inhibition was much more potent in the former neurons than in the latter. Under the condition that the Ca2+ current had already been inhibited by LEV (10 M), the addition of nifedipine (10 M) did not cause further inhibition. Conversely, LEV had no effects on the current that had already been decreased by nifedipine (10 M) given before LEV treatment (10 M), indicating that LEV could act on the L-type Ca2+ channel. LEV elevated the threshold potential level for activation of the Ca2+ current and reduced the L-type Ca2+ current in type 1 neurons of SER, and the inhibitory action in type 2 neurons was much more potent than that in Wistar rat neurons, suggesting that these effects contribute, at least partly, to the antiepileptic action of LEV. © 2012 Elsevier Inc. All rights reserved.
1. Introduction
∗ Corresponding author at: Laboratory of Neuropharmacology, Faculty of Pharmaceutical Sciences, Hiroshima International University, 5-1-1 Hirokoshingai, Kure 737-0112, Japan. Tel.: +81 823 73 8980; fax: +81 823 73 8981. E-mail address: [email protected] (K. Ishihara). 0361-9230/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2012.10.006
Levetiracetam (LEV: (S)-␣-ethyl-2-oxo-pyrrolidine acetamide) is an antiepileptic drug widely used for human partial seizures with a unique profile (Pellock et al., 2012). LEV does not inhibit acute convulsion with pentyleneterazol or maximal electroshock (Löscher & Hönack, 1993; Klitgaad et al., 1998), but does inhibit seizures in electroshock- or pentylenetetrazol-kindled animals (Gower et al.,
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1992; Klitgaad et al., 1998; Löscher et al., 1998; Ohno et al., 2010). In addition, LEV also has efficacy against audiogenic seizures in genetically epilepsy-prone rat and absence-like seizures in the genetic absence epilepsy rat Strasbourg (GAERS) (Gower et al., 1995), as well as Groggy rat (Tokuda et al., 2010). Several studies have reported an ability of LEV to antagonize epileptic responses in rat hippocampal slices in vivo and in vitro (Birnstiel et al., 1997; Margineanu and Klitgaard, 2000). LEV has been reported to bind specifically with SV2A located in synaptic vesicles within presynaptic terminals and presumably to modify neurotransmitter release (Noyer et al., 1995; Lynch et al., 2004; Yang et al., 2007). In addition, patch-clamp studies have demonstrated that LEV inhibits the highvoltage-activated (HVA) Ca2+ (N-type) current (Niespodziany et al., 2001), but does not affect the low-voltage-activated Ca2+ (T-type) current (Zona et al., 2001) in hippocampal slices of non-epileptic rat. Recently, we found that LEV inhibits depolarization shift accompanying repetitive firing induced by mossy fiber stimulation in hippocampal CA3 neurons of spontaneously epileptic rat (SER; zi/zi, tm/tm) (Hanaya et al., 2011). SER shows both tonic convulsion and absence-like seizures after 8 weeks of age (Serikawa and Yamada, 1986; Sasa et al., 1988). The SER are a useful model animal for evaluation of the acute and chronic effects of novel antiepileptic drugs (Sasa et al., 1988; Serikawa et al., 1990), since the profiles of antiepileptic effects in this animal are parallel to those in human absence seizures and tonic convulsions. In SER, a single administration of LEV inhibited both tonic convulsions and absence-like seizures (Cai et al., 2005). With repeated administration of LEV (once daily for 5 days), inhibitory effects on both seizures were increased (Yan et al., 2005). In addition, when LEV was administered once daily between 5 and 8 weeks old before seizure appearance, the development of tonic convulsion and absence-like seizures in SER was inhibited, compared with that in the salinetreated SER group (Yan et al., 2005). These findings suggest that LEV may have antiepileptogenic and/or disease-modifying activity. This possibility is supported by our recent, histochemical studies that prophylactic treatment with LEV in SER at 4–8 weeks of ages before appearance of the seizures produced an decrease in brainderived neurotrophic factor (BDNF) expression and mossy fiber sprouting in hippocampus of the seizure-manifesting ages (over 8 weeks), thereby inhibiting neuronal degeneration and/or regeneration (Sugata et al., 2011). Very recently, phase 2 study has been performed by Klein et al. (2012) to evaluate the safety and feasibility of LEV treatment for prevention of posttraumatic epilepsy. They have reported that LEV-treated adult patients had a 53% reduction in risk of posttraumatic epilepsy, compared with untreated ones, although the results were not statistically significant, probably due to several reasons such as study designs, open-label and nonrandomized study, differences of severity of injury and small participant number, etc. Our previous studies have also demonstrated that the hippocampal CA3 pyramidal neurons in SER show a long-lasting depolarization shift accompanied by repetitive firing of a single stimulus given to the mossy fibers (Ishihara et al., 1993), and this abnormal excitability is attributed to abnormalities of voltagedependent L-type Ca2+ channel of hippocampal CA3 neurons, since an increase in Ca2+ influxes and enhanced L-type Ca2+ channel activities have been observed (Momiyama et al., 1995; Amano et al., 2001a,b). Furthermore, this Ca2+ channel of the SER hippocampal neurons was found to have lower threshold potential and greater peak current with a slower inactivation than those of non-epileptic Wistar rat (Yan et al., 2007). These neurons with lower threshold, named type 1 neurons, constituted about 40% of the total in the SER hippocampal CA3 region. The remaining 60% of neurons, named type 2 neurons, in SER had a similar current–voltage relationship to Wistar rat neurons and had slower inactivation than in
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non-epileptic Wistar rat. Since our recent intracellular study showed the inhibitory effects of a therapeutic level of LEV on L-type Ca2+ channel (Hanaya et al., 2011), patch-clamp studies were performed to determine whether the antiepileptic action of LEV is due to modulation of Ca2+ channels in SER hippocampal CA3 neurons using acutely dissociated neurons. 2. Materials and methods 2.1. Animals and cell preparation SER and Wistar rats were bred in a pathogen-free separated facility by the National BioResource Project – Rat, in the Institute of Laboratory Animals, Faculty of Medicine, Kyoto University. They were provided with pellet food and water ad libitum in shoe box-type cages in a room maintained at 23 ± 2 ◦ C and 55 ± 5% humidity with lights on from 8 a.m. to 8 p.m. Acutely dissociated hippocampal CA3 neurons were prepared from 15 SER of both genders (10–13 weeks old) exhibiting convulsive seizures, and 8 age-matched control Wistar rats. After decapitation under anesthesia with diethyl ether, the brain was rapidly removed and placed in oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 113, KCl 3, NaH2 PO4 1, NaHCO3 25, CaCl2 2, MgCl2 1, and glucose 11 (pH 7.4). Transverse brain slices (500 m in thickness) were cut along the sagittal plane using a microslicer (DTK 1500, Dosaka, Kyoto, Japan). Hippocampal slices were between L 1.5 and 3.5 mm, corresponding to L 1.4–3.4 mm in Paxinos and Watson rat brain map (Paxinos and Watson, 1998). Thereafter, the hippocampal CA3 region dissected from brain slices by cutting with a small knife was incubated for 8–10 min at 34 ◦ C in ACSF containing 0.05% trypsin (type XI, Sigma, St. Louis, MO, USA) under perfusion with 95% O2 and 5% CO2 . After washing five times, the minislices were stored in ACSF, and bubbled with 95% O2 and 5% CO2 until use. Hippocampal CA3 neurons, dissociated by gently triturating with fire-polished Pasteur pipettes, were plated on cover glasses (12 mm in diameter), and then transferred to a recording chamber (2-ml volume). Most neurons had a pyramid-like shape, although the exact cell type could not be clearly identified. 2.2. Solution and drugs All experiments were performed at room temperature. Patch pipettes with 3–6 M resistance were filled with a solution containing (in mM): CsCl 120, tetraethylammonium chloride (TEA-Cl) 20, MgCl2 2, CaCl2 1, ethylene glycol bis(aminoethyl-ether)-N,N,N ,N -tetraacetic acid (EGTA, Sigma) 11, HEPES 10, and K2 ATP 2, and pH was adjusted to 7.3 with Tris-[hydroxymethyl]-aminomethane (Sigma). The recording chamber was continuously perfused with an external solution at a rate of 2–4 ml/min. The external solution contained (in mM): NaCl 154, KCl 5, TEA-Cl 10, CaCl2 2, MgCl2 1, HEPES 5, glucose 10, and tetrodotoxin (TTX, Wako Pure Chemical, Osaka, Japan) 0.0003, and then pH was adjusted to 7.3 with NaOH. LEV (UCB S.A. Pharma Sector, Belgium) was applied with bath perfusion. 2.3. Whole-cell recording and analysis Tight seals (>1 G) were formed on cell bodies of chosen neurons and whole-cell recordings were obtained by rupturing the cell membrane with negative pressure. Voltage-clamp recordings were made using an EPC-7 amplifier (EPC-7, List-Medical Electronic, Darmstadt, Germany) with low-pass filtering at 3 kHz, displayed on an oscilloscope (VC-10, Nihon Kohden, Tokyo, Japan), and digitized at 10 kHz for storage. Recordings were considered acceptable if capacitance could be properly compensated, and if holding current was stable. After the whole-cell recordings were obtained, the series resistance was 10–20 M, which was monitored by measuring the size of the capacitate current generated by small (5 mV, 100 ms) voltage steps. For our recording, tight G seals were formed on the neurons, and rapidly decaying currents and slower capacitance transient in a whole-cell recording were canceled by using ‘C-fast’ and ‘C-slow’ control. Conductance in series with the capacitance (C-SLOW) was adjusted by ‘G-SERIES’ control. Cells were excluded from analysis if the series resistance changed by >15%. At least 10 min was allowed for equilibration of pipette solution with the intracellular milieu before commencing recordings. Voltage-dependent Ca2+ currents were induced using a depolarizing pulse with 100-ms duration applied to the hippocampal CA3 neurons from a holding potential of −90 mV to +50 mV with 10 mV increments. After the amplitude of Ca2+ currents was stabilized, external CaCl2 was replaced with equimolar BaCl2 as charge carrier. The currents were measured using the conventional whole-cell patch-clamp recording method. Electrophysiological signals obtained using a patch-clamp amplifier (EPC-7, List-Medical Electronic, Darmstadt, Germany) were displayed on an oscilloscope (VC-10, Nihon Kohden, Tokyo, Japan). The current signals were stored on a personal computer (FMV-Desk Power TI16, Fujitsu, Tokyo, Japan) and a video cassette recorder (SLV-779 HF, Sony, Tokyo, Japan) via DigiData interface (1200 series, Axon Instrument, Foster City, CA, USA). A digital data recorder (VR-10B, Instrutech Corporation, Long Island, NY, USA) was employed for subsequent analysis using pCLAMP 8.0 software (Axon Instrument). Depolarizing pulses to induce the current were generated using a personal computer and pCLAMP software. Leak current was
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Fig. 1. Effects of LEV on inward Ca2+ current in SER type 1 neurons. (A) Typical example of the current traces obtained by depolarizing from a holding potential of −90 mV to test potentials ranging from −50 mV to −10 mV for 100 ms duration in hippocampal CA3 neurons. Left and right traces are current traces before and during addition of LEV (100 M), respectively. (B) Current–voltage relationship of Ca2+ current in SER type 1 neurons in the presence or absence of LEV (100 M). Each point and bar represent mean and SEM (n = 6). *, **: p < 0.05, p < 0.01 vs. before drug treatment (paired t-test).
subtracted on-line with a P/N protocol by pCLAMP software. An approximate value of whole-cell capacitance was determined by adjusting the amplified capacitance. All data in the text and figures are expressed as mean values ± SEM, and the statistical significance of differences was determined using paired or unpaired Student’s t-test. One-way ANOVA was used for comparison between Wistar and SER groups in concentration–response curve experiments. Other details have been described elsewhere (Yan et al., 2007).
3. Results 3.1. Effects of levetiracetam on Ca2+ current in type 1 neurons of SER As reported previously (Yan et al., 2007), hippocampal CA3 neurons of SER were classified as type 1 and type 2 neurons. In type 1 neurons, initiation and maximal Ca2+ current voltages, which were −50 and −30 mV, respectively, were lower than those (−40 and −10 mV, respectively) in Wistar rats (Fig. 1). In type 2 neurons, the current–voltage relationship (I–V curve) of Ca2+ current was similar to that in Wistar rat neurons. However, type 2 neurons had prolonged inactivation during application of an intracellular depolarization pulse, compared with Wistar rat neurons. The respective mean capacitances of type 1 neurons and type 2 neurons in SER and Wistar rat neurons were 7.08 ± 0.21 pF (n = 6), 7.18 ± 0.09 pF (n = 28), and 7.29 ± 0.10 pF (n = 13), respectively. There were no significant differences among these values, indicating that the sizes of these group neurons did not differ. The effects of LEV were examined in 6 type 1 neurons of SER. Fig. 1A shows current traces obtained in hippocampal CA3 neurons when the voltage was clamped at −90 mV, and then depolarizing pulses (duration; 100 ms) as a test potential ranging from −50 mV to −10 mV were given. Ca2+ currents were initiated at about −50 mV and the peak currents were observed as low as −30 mV (Fig. 1). When LEV at a concentration of 100 M was applied in the bath, 3–6 min later, the threshold of Ca2+ currents was elevated from −50 mV to −40 mV, and the maximum current was observed at −20 or −10 mV (Fig. 1). The currents completely
recovered to the original level by washout (data not shown). The I–V curve for the averaged peak Ca2+ current against the test potentials was obtained at a holding voltage of −90 mV in the presence and absence of LEV (100 M) (Fig. 1B). In the presence of LEV, the I–V curves shifted to the right concomitantly with a decrease in peak amplitude. After application of LEV, the current amplitude at the −40 mV level was significantly (p < 0.05) reduced to 27.1 ± 9.0% (n = 6) of the maximum current from 42.6 ± 4.4%. The peak amplitude of the current was also significantly (p < 0.01, paired t-test) decreased to −391.6 ± 66.9 pA at −10 mV, compared with that before application of LEV (−531.9 ± 88.8 pA, at −30 mV) (Fig. 1B). 3.2. Effects of LEV on Ca2+ currents in SER type 2 and Wistar rat neurons In type 2 neurons, the voltage was clamped at −90 mV, and then a depolarizing pulse to −10 mV elicited the maximal inward Ca2+ current (Fig. 2). LEV at a concentration of 100 M produced a reduction of the Ca2+ current 3–6 min after its application, and complete recovery of the current by washout was observed. The I–V curves were obtained by depolarizing the neurons for 100 ms from a holding potential of −90 mV to 50 mV with 10 mV increments. In SER type 2 neurons, LEV did not affect either the threshold potential for activating Ca2+ current or the voltage for appearance of the peak current (Figs. 2 and 3A). However, the current amplitudes were decreased between −20 and 20 mV test potential levels; the peak amplitude at −10 mV was significantly (p < 0.01) decreased from −845.7 ± 111.3 pA (n = 7) to −523.1 ± 99.2 pA after application of LEV at 100 M (Fig. 2). Furthermore, the current amplitudes were significantly decreased between −10 and 20 mV test potential levels; the peak amplitude at 0 mV was significantly (p < 0.01) decreased from −536.2 ± 94.1 pA (n = 5) to −355.5 ± 82.7 pA after application of LEV at 10 M (Fig. 3A). In Wistar rat neurons, LEV (10 M) did not affect either the threshold potential for activating Ca2+ current or the voltage for appearance of the peak current (Fig. 3B). The current amplitudes were slightly decreased between
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Fig. 2. Effects of LEV on inward Ca2+ current in SER type 2 neurons. (A) Typical example of the current traces obtained by depolarizing from a holding potential of −90 mV to test potentials ranging from −50 mV to 0 mV for 100 ms duration in hippocampal CA3 neurons. Left and right traces are current traces before and during addition of LEV (100 M), respectively. (B) Current–voltage relationship of Ca2+ current in SER type 2 neurons in the presence or absence of LEV (100 M). Each point and bar represent mean and SEM (n = 7). *, **: p < 0.05, p < 0.01 vs. before drug treatment (paired t-test).
−10 and 20 mV test potential levels; the peak amplitude at −10 mV was decreased from −624.0 ± 202.1 pA (n = 4) to −492.1 ± 137.0 pA after application of LEV at 10 M (Fig. 3B). However the difference of the values was not statistically significant. 3.3. Concentration–effect relationship of LEV on Ca2+ currents in SER type 2 and Wistar rat neurons Slight inhibitory effects of LEV were also observed in nonepileptic Wistar rat neurons, although these were much weaker than those for SER type 2 neurons. To compare the difference of inhibitory action of LEV between SER type 2 and Wistar rat neurons, the concentration–response relationships for the inhibitory action
of LEV were examined on the Ca2+ current evoked by a depolarizing pulse of −10 mV from a holding potential of −90 mV. Relationships between the concentration of LEV (100 nM–1 mM) and inhibition of peak Ca2+ current amplitude were observed in both SER type 2 and Wistar rat neurons in concentration-dependent manners (Fig. 4). The concentration–response curve in SER type 2 neurons shifted to the left, and the inhibitory effects of LEV at concentrations from 100 nM to 1 mM were significantly more potent than those in Wistar rat neurons (Fig. 4) (F(1,50) = 20.96, p < 0.01): the potency of LEV on type 2 neurons was almost one hundred times greater than that on Wistar rat neurons. In addition, the maximum inhibition (Rmax ) by as much as 1 mM LEV was 55.4 ± 8.5% (n = 5) in SER type 2 neurons; however it was only 29.2 ± 6.0% (n = 7) in
Fig. 3. Effects of LEV on inward Ca2+ current in SER type 2 neurons and Wistar rat neurons. (A) Current–voltage relationship of Ca2+ current in SER type 2 neurons in the presence or absence of LEV (10 M). (B) Current–voltage relationship of Ca2+ current in Wistar rat neurons in the presence or absence of LEV (10 M). Each point and bar represent mean and SEM (n = 5 or 4). *, **: p < 0.05, p < 0.01 vs. before drug treatment (paired t-test).
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Fig. 4. Concentration–response relationships for the inhibitory action of LEV on the Ca2+ current in Wistar (square) rat and SER type 2 neurons (circle). The current induced from a holding potential of −90 mV to −10 mV for 100 ms duration. Each point represents components (%) of the currents that were inhibited after addition of LEV at different concentrations. Points were fitted by the Hill equation. ANOVA result: F(1,50) = 20.964, p < 0.01. *p < 0.05 (unpaired t-test).
Wistar rat neurons. Furthermore, this inhibition at 1 mM was the ceiling in the latter neurons, although it did not appear to be the ceiling in the former neurons. These results indicate that SER type 2 neurons have higher sensitivity to LEV than Wistar rat neurons. 3.4. Effects of nifedipine on LEV-produced inhibition of Ca2+ current in SER type 2 neurons In order to elucidate which type of Ca2+ channel in SER type 2 neurons was responsible for LEV-induced inhibition, the effects of nifedipine, an antagonist of L-type Ca2+ channel, were examined before and after the application of LEV. A depolarizing pulse from a holding potential of −90 mV to −10 mV was applied to the neurons for 100 ms, and then the Ca2+ currents were continuously recorded for 6–7 min after application of the drugs. Fig. 5 shows the typical type 2 neuron responses to nifedipine and LEV. During application of nifedipine at 10 M, which is a concentration sufficient to inhibit the depolarization shift accompanying repetitive firing and Ca2+ influx in SER hippocampal neurons (Hanaya et al., 1998; Amano et al., 2001a), the mean current amplitude was significantly (p < 0.001) decreased from −486.9 ± 88.8 pA to −263.3 ± 44.5 pA (n = 7) (Fig. 6A). Under inhibition of the current, LEV at 10 M, which induces 50% inhibition of depolarization shift with repetitive firing in SER hippocampal neurons (Hanaya et al., 2011), was given to the bath, but there was no further inhibition of the current amplitude (−253.1 ± 42.7 pA, n = 7) (Figs. 5A and 6A). Conversely, 10 M LEV given first significantly (p < 0.001) reduced the mean current to −321.6 ± 69.4 pA (n = 8) from −574.4 ± 57.1 pA (n = 8) of the control level, and then 10 M nifedipine added subsequently in the presence of LEV did not induce further inhibition of the current (−311.8 ± 40.0 pA, n = 8) (Figs. 5B and 6B). These results indicate that LEV acts on the L-type Ca2+ channel in SER type 2 neurons. 4. Discussion As reported previously (Yan et al., 2007), type 1 and 2 neurons were found in the SER hippocampal CA3 neurons obtained herein using patch-clamp technique: type 1 neurons showed lowered threshold and greater Ca2+ current and type 2 neurons had similar threshold potential and Ca2+ current size to those in seizure-free
Wistar rat hippocampal CA3 neurons. These type 1 and 2 neurons are considered to correspond to group 1/2 and 3 neurons found in our previous intracellular recording method, respectively, since the neurons showing the long-lasting (over 100/below 100 ms) depolarization shift and no such depolarization shift were observed in the SER hippocampal CA3 neurons following the spike induced by mossy fiber stimulation (Ishihara et al., 1993; Momiyama et al., 1995; Hanaya et al., 1998). When LEV at 100 M was applied to the SER hippocampal CA3 type 1 neurons in the bath, the lowered threshold potential of Ca2+ channels was found to be raised to a level similar to that of seizure-free Wistar rats and the maximal Ca2+ current amplitude was decreased. These results suggest that LEV acts on Ca2+ channel to delay the opening and decrease enhanced Ca2+ influxes in SER type 1 neurons. The Ca2+ channel involved in the depolarization shift of SER CA3 neurons is thought to be the Ltype, since the channel was activated by high voltage and inhibited by L-type Ca2+ antagonist, nifedipine, in the present dissociated CA3 neurons and hippocampal slice preparations (Ishihara et al., 1993; Momiyama et al., 1995) and actually there was a blockade by nifedipine of Ca2+ influxes induced by mossy fiber stimulation as well as high K+ solution in SER hippocampal slice (Amano et al., 2001a,b). LEV at therapeutic concentration (10–100 M) was found to inhibit L-type Ca2+ channel currents of SER hippocampal CA3 neurons. These findings are in line with those reported by others that LEV inhibits high-voltage-activated Ca2+ current (N-type) in hippocampal pyramidal neurons (Niespodziany et al., 2001; Lukyanetz et al., 2002), but affects neither low-voltage-activated Ca2+ current in CA1 hippocampal neurons nor Na+ current in neocortical cultured neurons (Zona et al., 2001). The high-voltage-activated Ca2+ currents (L- and N-types) have also been reported to be inhibited by anti-epileptic drugs in isolated neurons including rat cortex, amygdala, and striatum (Stefani et al., 1997). Since our data showed that LEV reduced Ca2+ current in the hippocampal CA3 neurons of SER, LEV is considered to shorten Ca2+ channel opening time and/or decrease the number of available Ca2+ channels, thereby reducing Ca2+ influx. As a result, depolarization shift accompanying repetitive firing is inhibited, thereby suppressing hypersynchronization of neuron groups and inducing the antiepileptic effect of LEV. The inhibitory effects were much greater on Ca2+ currents of SER type 2 neurons than those of non-epileptic Wistar rat neurons, suggesting that LEV could be more sensitive to L-type Ca2+ channels with functional abnormality, like SER CA3 neurons, which would contribute to depolarization shift formation. The effects of LEV were concentration-dependent in SER neurons with 35–45% inhibition of the peak Ca2+ current at therapeutic doses (10–100 M). Therefore, an inhibition by LEV of depolarization shift results in depression of repetitive firing, thereby suppressing hypersynchronization of the neuron group, and then inducing antiepileptic action (Hanaya et al., 2011). In Wistar rat neurons, however, LEV had ceiling effects of 18–25% by doses as high as 1 mM in Wistar rat neurons. These results seem to suggest that LEV has negligible effects on L-type Ca2+ channels with normal function, like Wistar rat neurons. In addition, the present study also demonstrated that LEV did not produce further inhibition of the current amplitude, which had already been inhibited by nifedipine by approximately 30%, and conversely nifedipine had no effects on the current amplitude reduced by LEV already, suggesting that LEV might act on the same L-type Ca2+ channel as nifedipine. In conclusion, LEV has a high sensitivity for altering abnormalities of Ca2+ channel function in hippocampal CA3 neurons of SER. LEV raised threshold levels for activation of Ca2+ current, and depressed Ca2+ currents in a concentration-dependent manner, by inhibiting Ca2+ current involved in L-type Ca2+ channels. In addition to the modulatory effects of LEV on the synaptic transmission, these inhibitory effects of LEV on Ca2+ channel are suggested to
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Fig. 5. Typical example of effects of nifedipine on LEV-produced inhibition of Ca2+ current in SER type 2 neurons. LEV (10 M) was perfused after (A) or before (B) the addition of nifedipine (10 M). (a) Current traces obtained by depolarizing pulses (100 ms duration) to −10 mV from a holding potential of −90 mV. (b) Plots of changes in Ca2+ current amplitude against time. Blocks: period during application of respective drug.
Fig. 6. Effects of nifedipine on inhibition of Ca2+ current by LEV in SER type 2 neurons. Current induced by depolarizing pulses from a holding potential of −90 mV to −10 mV for 100 ms. Each column with vertical bars represents the mean amplitude and SEM. LEV (10 M) was perfused after (A) or before (B) the addition of nifedipine (10 M). ***p < 0.001, compared with the control (absence of LEV and nifedipine).
contribute, at least partly, to the inhibition of epileptic seizures in SER and probably humans. Disclosure of conflicts of interest None of the authors has any conflict of interest to disclose. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, and by UCB S.A., Belgium. Levetiracetam was generously supplied by UCB S.A., Belgium. This work was carried out with the equipment of the Research Facilities for
Laboratory Animal Science, Hiroshima University School of Medicine, Japan. We are grateful to the National BioResource Project – Rat (http://www.anim.med.kyoto-u.ac.jp/NBR/) for providing rat strains (SER).
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Inhibitory effects of levetiracetam on the high-voltage-activated L-type Ca2+ channels in hippocampal CA3 neurons of spontaneously epileptic rat (SER) Hai-Dun Yan a , Kumatoshi Ishihara b,e,∗ , Takahiro Seki a , Ryosuke Hanaya c,f , Kaoru Kurisu c , Kazunori Arita c,f , Tadao Serikawa d , Masashi Sasa a,g a
Department of Pharmacology, Hiroshima University School of Medicine, Hiroshima 734-8551, Japan Department of Pharmacotherapy, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan c Department of Neurosurgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan d Institute of Laboratory Animals, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan e Laboratory of Neuropharmacology, Faculty of Pharmaceutical Sciences, Hiroshima International University, Kure 737-0112, Japan f Department of Neurosurgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan g Nagisa Clinic, Hirakata 573-1183, Japan b
a r t i c l e
i n f o
Article history: Received 10 September 2012 Received in revised form 16 October 2012 Accepted 17 October 2012 Available online 27 October 2012 Keywords: Levetiracetam (LEV) Ca2+ current Spontaneously epileptic rat (SER) Hippocampal CA3 neuron Patch clamp
a b s t r a c t Levetiracetam (LEV) is a widely used antiepileptic agent for partial refractory epilepsy in humans. LEV has unique antiepileptic effects in that it does not inhibit electroshock- or pentylenetetrazol-induced convulsion, but does inhibit seizures in kindling animal and spontaneously epileptic rat (SER: zi/zi, tm/tm) that shows both tonic convulsion and absence-like seizures. LEV also has unique characteristics in terms of its antiepileptic mechanism; it has no activity on Na+ and K+ channels or on glutamate and GABAA receptors. Recently, we found that LEV inhibits the depolarization shift and accompanying repetitive firing induced by mossy fiber stimulation in CA3 neurons of SER hippocampal slices. Therefore, this study was performed to determine whether LEV could inhibit the voltage-activated L-type Ca2+ current of hippocampal CA3 neurons obtained from SER and the non-epileptic Wistar rat. As previously reported, SER CA3 neurons were classified into type 1 and type 2 neurons. The application of LEV (100 M) elevated the threshold for activation of the Ca2+ current, which was lowered in SER type 1 neurons and reduced the current size. Type 2 neurons of SER have a similar current–voltage relationship to Wistar rat neurons and the decay component of Ca2+ current during depolarization pulse in type 2 neurons was found to be smaller than that in Wistar rat neurons. LEV (100 M) also reduced Ca2+ current in SER type 2 neurons. The effects of LEV were examined on such type 2 SER hippocampal CA3 neurons, compared with those on Wistar rat CA3 neurons. Application of LEV (10 M) produced a significant decrease of amplitude of the Ca2+ current in SER neurons, although at this concentration of LEV there was no statistically significant decrease in the amplitude of Ca2+ current in Wistar rat neurons. Furthermore, LEV (100 nM–1 mM) reduced the Ca2+ current in a concentration-dependent manner in both SER and Wistar rat neurons, but the inhibition was much more potent in the former neurons than in the latter. Under the condition that the Ca2+ current had already been inhibited by LEV (10 M), the addition of nifedipine (10 M) did not cause further inhibition. Conversely, LEV had no effects on the current that had already been decreased by nifedipine (10 M) given before LEV treatment (10 M), indicating that LEV could act on the L-type Ca2+ channel. LEV elevated the threshold potential level for activation of the Ca2+ current and reduced the L-type Ca2+ current in type 1 neurons of SER, and the inhibitory action in type 2 neurons was much more potent than that in Wistar rat neurons, suggesting that these effects contribute, at least partly, to the antiepileptic action of LEV. © 2012 Elsevier Inc. All rights reserved.
1. Introduction
∗ Corresponding author at: Laboratory of Neuropharmacology, Faculty of Pharmaceutical Sciences, Hiroshima International University, 5-1-1 Hirokoshingai, Kure 737-0112, Japan. Tel.: +81 823 73 8980; fax: +81 823 73 8981. E-mail address: [email protected] (K. Ishihara). 0361-9230/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2012.10.006
Levetiracetam (LEV: (S)-␣-ethyl-2-oxo-pyrrolidine acetamide) is an antiepileptic drug widely used for human partial seizures with a unique profile (Pellock et al., 2012). LEV does not inhibit acute convulsion with pentyleneterazol or maximal electroshock (Löscher & Hönack, 1993; Klitgaad et al., 1998), but does inhibit seizures in electroshock- or pentylenetetrazol-kindled animals (Gower et al.,
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1992; Klitgaad et al., 1998; Löscher et al., 1998; Ohno et al., 2010). In addition, LEV also has efficacy against audiogenic seizures in genetically epilepsy-prone rat and absence-like seizures in the genetic absence epilepsy rat Strasbourg (GAERS) (Gower et al., 1995), as well as Groggy rat (Tokuda et al., 2010). Several studies have reported an ability of LEV to antagonize epileptic responses in rat hippocampal slices in vivo and in vitro (Birnstiel et al., 1997; Margineanu and Klitgaard, 2000). LEV has been reported to bind specifically with SV2A located in synaptic vesicles within presynaptic terminals and presumably to modify neurotransmitter release (Noyer et al., 1995; Lynch et al., 2004; Yang et al., 2007). In addition, patch-clamp studies have demonstrated that LEV inhibits the highvoltage-activated (HVA) Ca2+ (N-type) current (Niespodziany et al., 2001), but does not affect the low-voltage-activated Ca2+ (T-type) current (Zona et al., 2001) in hippocampal slices of non-epileptic rat. Recently, we found that LEV inhibits depolarization shift accompanying repetitive firing induced by mossy fiber stimulation in hippocampal CA3 neurons of spontaneously epileptic rat (SER; zi/zi, tm/tm) (Hanaya et al., 2011). SER shows both tonic convulsion and absence-like seizures after 8 weeks of age (Serikawa and Yamada, 1986; Sasa et al., 1988). The SER are a useful model animal for evaluation of the acute and chronic effects of novel antiepileptic drugs (Sasa et al., 1988; Serikawa et al., 1990), since the profiles of antiepileptic effects in this animal are parallel to those in human absence seizures and tonic convulsions. In SER, a single administration of LEV inhibited both tonic convulsions and absence-like seizures (Cai et al., 2005). With repeated administration of LEV (once daily for 5 days), inhibitory effects on both seizures were increased (Yan et al., 2005). In addition, when LEV was administered once daily between 5 and 8 weeks old before seizure appearance, the development of tonic convulsion and absence-like seizures in SER was inhibited, compared with that in the salinetreated SER group (Yan et al., 2005). These findings suggest that LEV may have antiepileptogenic and/or disease-modifying activity. This possibility is supported by our recent, histochemical studies that prophylactic treatment with LEV in SER at 4–8 weeks of ages before appearance of the seizures produced an decrease in brainderived neurotrophic factor (BDNF) expression and mossy fiber sprouting in hippocampus of the seizure-manifesting ages (over 8 weeks), thereby inhibiting neuronal degeneration and/or regeneration (Sugata et al., 2011). Very recently, phase 2 study has been performed by Klein et al. (2012) to evaluate the safety and feasibility of LEV treatment for prevention of posttraumatic epilepsy. They have reported that LEV-treated adult patients had a 53% reduction in risk of posttraumatic epilepsy, compared with untreated ones, although the results were not statistically significant, probably due to several reasons such as study designs, open-label and nonrandomized study, differences of severity of injury and small participant number, etc. Our previous studies have also demonstrated that the hippocampal CA3 pyramidal neurons in SER show a long-lasting depolarization shift accompanied by repetitive firing of a single stimulus given to the mossy fibers (Ishihara et al., 1993), and this abnormal excitability is attributed to abnormalities of voltagedependent L-type Ca2+ channel of hippocampal CA3 neurons, since an increase in Ca2+ influxes and enhanced L-type Ca2+ channel activities have been observed (Momiyama et al., 1995; Amano et al., 2001a,b). Furthermore, this Ca2+ channel of the SER hippocampal neurons was found to have lower threshold potential and greater peak current with a slower inactivation than those of non-epileptic Wistar rat (Yan et al., 2007). These neurons with lower threshold, named type 1 neurons, constituted about 40% of the total in the SER hippocampal CA3 region. The remaining 60% of neurons, named type 2 neurons, in SER had a similar current–voltage relationship to Wistar rat neurons and had slower inactivation than in
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non-epileptic Wistar rat. Since our recent intracellular study showed the inhibitory effects of a therapeutic level of LEV on L-type Ca2+ channel (Hanaya et al., 2011), patch-clamp studies were performed to determine whether the antiepileptic action of LEV is due to modulation of Ca2+ channels in SER hippocampal CA3 neurons using acutely dissociated neurons. 2. Materials and methods 2.1. Animals and cell preparation SER and Wistar rats were bred in a pathogen-free separated facility by the National BioResource Project – Rat, in the Institute of Laboratory Animals, Faculty of Medicine, Kyoto University. They were provided with pellet food and water ad libitum in shoe box-type cages in a room maintained at 23 ± 2 ◦ C and 55 ± 5% humidity with lights on from 8 a.m. to 8 p.m. Acutely dissociated hippocampal CA3 neurons were prepared from 15 SER of both genders (10–13 weeks old) exhibiting convulsive seizures, and 8 age-matched control Wistar rats. After decapitation under anesthesia with diethyl ether, the brain was rapidly removed and placed in oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 113, KCl 3, NaH2 PO4 1, NaHCO3 25, CaCl2 2, MgCl2 1, and glucose 11 (pH 7.4). Transverse brain slices (500 m in thickness) were cut along the sagittal plane using a microslicer (DTK 1500, Dosaka, Kyoto, Japan). Hippocampal slices were between L 1.5 and 3.5 mm, corresponding to L 1.4–3.4 mm in Paxinos and Watson rat brain map (Paxinos and Watson, 1998). Thereafter, the hippocampal CA3 region dissected from brain slices by cutting with a small knife was incubated for 8–10 min at 34 ◦ C in ACSF containing 0.05% trypsin (type XI, Sigma, St. Louis, MO, USA) under perfusion with 95% O2 and 5% CO2 . After washing five times, the minislices were stored in ACSF, and bubbled with 95% O2 and 5% CO2 until use. Hippocampal CA3 neurons, dissociated by gently triturating with fire-polished Pasteur pipettes, were plated on cover glasses (12 mm in diameter), and then transferred to a recording chamber (2-ml volume). Most neurons had a pyramid-like shape, although the exact cell type could not be clearly identified. 2.2. Solution and drugs All experiments were performed at room temperature. Patch pipettes with 3–6 M resistance were filled with a solution containing (in mM): CsCl 120, tetraethylammonium chloride (TEA-Cl) 20, MgCl2 2, CaCl2 1, ethylene glycol bis(aminoethyl-ether)-N,N,N ,N -tetraacetic acid (EGTA, Sigma) 11, HEPES 10, and K2 ATP 2, and pH was adjusted to 7.3 with Tris-[hydroxymethyl]-aminomethane (Sigma). The recording chamber was continuously perfused with an external solution at a rate of 2–4 ml/min. The external solution contained (in mM): NaCl 154, KCl 5, TEA-Cl 10, CaCl2 2, MgCl2 1, HEPES 5, glucose 10, and tetrodotoxin (TTX, Wako Pure Chemical, Osaka, Japan) 0.0003, and then pH was adjusted to 7.3 with NaOH. LEV (UCB S.A. Pharma Sector, Belgium) was applied with bath perfusion. 2.3. Whole-cell recording and analysis Tight seals (>1 G) were formed on cell bodies of chosen neurons and whole-cell recordings were obtained by rupturing the cell membrane with negative pressure. Voltage-clamp recordings were made using an EPC-7 amplifier (EPC-7, List-Medical Electronic, Darmstadt, Germany) with low-pass filtering at 3 kHz, displayed on an oscilloscope (VC-10, Nihon Kohden, Tokyo, Japan), and digitized at 10 kHz for storage. Recordings were considered acceptable if capacitance could be properly compensated, and if holding current was stable. After the whole-cell recordings were obtained, the series resistance was 10–20 M, which was monitored by measuring the size of the capacitate current generated by small (5 mV, 100 ms) voltage steps. For our recording, tight G seals were formed on the neurons, and rapidly decaying currents and slower capacitance transient in a whole-cell recording were canceled by using ‘C-fast’ and ‘C-slow’ control. Conductance in series with the capacitance (C-SLOW) was adjusted by ‘G-SERIES’ control. Cells were excluded from analysis if the series resistance changed by >15%. At least 10 min was allowed for equilibration of pipette solution with the intracellular milieu before commencing recordings. Voltage-dependent Ca2+ currents were induced using a depolarizing pulse with 100-ms duration applied to the hippocampal CA3 neurons from a holding potential of −90 mV to +50 mV with 10 mV increments. After the amplitude of Ca2+ currents was stabilized, external CaCl2 was replaced with equimolar BaCl2 as charge carrier. The currents were measured using the conventional whole-cell patch-clamp recording method. Electrophysiological signals obtained using a patch-clamp amplifier (EPC-7, List-Medical Electronic, Darmstadt, Germany) were displayed on an oscilloscope (VC-10, Nihon Kohden, Tokyo, Japan). The current signals were stored on a personal computer (FMV-Desk Power TI16, Fujitsu, Tokyo, Japan) and a video cassette recorder (SLV-779 HF, Sony, Tokyo, Japan) via DigiData interface (1200 series, Axon Instrument, Foster City, CA, USA). A digital data recorder (VR-10B, Instrutech Corporation, Long Island, NY, USA) was employed for subsequent analysis using pCLAMP 8.0 software (Axon Instrument). Depolarizing pulses to induce the current were generated using a personal computer and pCLAMP software. Leak current was
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Fig. 1. Effects of LEV on inward Ca2+ current in SER type 1 neurons. (A) Typical example of the current traces obtained by depolarizing from a holding potential of −90 mV to test potentials ranging from −50 mV to −10 mV for 100 ms duration in hippocampal CA3 neurons. Left and right traces are current traces before and during addition of LEV (100 M), respectively. (B) Current–voltage relationship of Ca2+ current in SER type 1 neurons in the presence or absence of LEV (100 M). Each point and bar represent mean and SEM (n = 6). *, **: p < 0.05, p < 0.01 vs. before drug treatment (paired t-test).
subtracted on-line with a P/N protocol by pCLAMP software. An approximate value of whole-cell capacitance was determined by adjusting the amplified capacitance. All data in the text and figures are expressed as mean values ± SEM, and the statistical significance of differences was determined using paired or unpaired Student’s t-test. One-way ANOVA was used for comparison between Wistar and SER groups in concentration–response curve experiments. Other details have been described elsewhere (Yan et al., 2007).
3. Results 3.1. Effects of levetiracetam on Ca2+ current in type 1 neurons of SER As reported previously (Yan et al., 2007), hippocampal CA3 neurons of SER were classified as type 1 and type 2 neurons. In type 1 neurons, initiation and maximal Ca2+ current voltages, which were −50 and −30 mV, respectively, were lower than those (−40 and −10 mV, respectively) in Wistar rats (Fig. 1). In type 2 neurons, the current–voltage relationship (I–V curve) of Ca2+ current was similar to that in Wistar rat neurons. However, type 2 neurons had prolonged inactivation during application of an intracellular depolarization pulse, compared with Wistar rat neurons. The respective mean capacitances of type 1 neurons and type 2 neurons in SER and Wistar rat neurons were 7.08 ± 0.21 pF (n = 6), 7.18 ± 0.09 pF (n = 28), and 7.29 ± 0.10 pF (n = 13), respectively. There were no significant differences among these values, indicating that the sizes of these group neurons did not differ. The effects of LEV were examined in 6 type 1 neurons of SER. Fig. 1A shows current traces obtained in hippocampal CA3 neurons when the voltage was clamped at −90 mV, and then depolarizing pulses (duration; 100 ms) as a test potential ranging from −50 mV to −10 mV were given. Ca2+ currents were initiated at about −50 mV and the peak currents were observed as low as −30 mV (Fig. 1). When LEV at a concentration of 100 M was applied in the bath, 3–6 min later, the threshold of Ca2+ currents was elevated from −50 mV to −40 mV, and the maximum current was observed at −20 or −10 mV (Fig. 1). The currents completely
recovered to the original level by washout (data not shown). The I–V curve for the averaged peak Ca2+ current against the test potentials was obtained at a holding voltage of −90 mV in the presence and absence of LEV (100 M) (Fig. 1B). In the presence of LEV, the I–V curves shifted to the right concomitantly with a decrease in peak amplitude. After application of LEV, the current amplitude at the −40 mV level was significantly (p < 0.05) reduced to 27.1 ± 9.0% (n = 6) of the maximum current from 42.6 ± 4.4%. The peak amplitude of the current was also significantly (p < 0.01, paired t-test) decreased to −391.6 ± 66.9 pA at −10 mV, compared with that before application of LEV (−531.9 ± 88.8 pA, at −30 mV) (Fig. 1B). 3.2. Effects of LEV on Ca2+ currents in SER type 2 and Wistar rat neurons In type 2 neurons, the voltage was clamped at −90 mV, and then a depolarizing pulse to −10 mV elicited the maximal inward Ca2+ current (Fig. 2). LEV at a concentration of 100 M produced a reduction of the Ca2+ current 3–6 min after its application, and complete recovery of the current by washout was observed. The I–V curves were obtained by depolarizing the neurons for 100 ms from a holding potential of −90 mV to 50 mV with 10 mV increments. In SER type 2 neurons, LEV did not affect either the threshold potential for activating Ca2+ current or the voltage for appearance of the peak current (Figs. 2 and 3A). However, the current amplitudes were decreased between −20 and 20 mV test potential levels; the peak amplitude at −10 mV was significantly (p < 0.01) decreased from −845.7 ± 111.3 pA (n = 7) to −523.1 ± 99.2 pA after application of LEV at 100 M (Fig. 2). Furthermore, the current amplitudes were significantly decreased between −10 and 20 mV test potential levels; the peak amplitude at 0 mV was significantly (p < 0.01) decreased from −536.2 ± 94.1 pA (n = 5) to −355.5 ± 82.7 pA after application of LEV at 10 M (Fig. 3A). In Wistar rat neurons, LEV (10 M) did not affect either the threshold potential for activating Ca2+ current or the voltage for appearance of the peak current (Fig. 3B). The current amplitudes were slightly decreased between
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Fig. 2. Effects of LEV on inward Ca2+ current in SER type 2 neurons. (A) Typical example of the current traces obtained by depolarizing from a holding potential of −90 mV to test potentials ranging from −50 mV to 0 mV for 100 ms duration in hippocampal CA3 neurons. Left and right traces are current traces before and during addition of LEV (100 M), respectively. (B) Current–voltage relationship of Ca2+ current in SER type 2 neurons in the presence or absence of LEV (100 M). Each point and bar represent mean and SEM (n = 7). *, **: p < 0.05, p < 0.01 vs. before drug treatment (paired t-test).
−10 and 20 mV test potential levels; the peak amplitude at −10 mV was decreased from −624.0 ± 202.1 pA (n = 4) to −492.1 ± 137.0 pA after application of LEV at 10 M (Fig. 3B). However the difference of the values was not statistically significant. 3.3. Concentration–effect relationship of LEV on Ca2+ currents in SER type 2 and Wistar rat neurons Slight inhibitory effects of LEV were also observed in nonepileptic Wistar rat neurons, although these were much weaker than those for SER type 2 neurons. To compare the difference of inhibitory action of LEV between SER type 2 and Wistar rat neurons, the concentration–response relationships for the inhibitory action
of LEV were examined on the Ca2+ current evoked by a depolarizing pulse of −10 mV from a holding potential of −90 mV. Relationships between the concentration of LEV (100 nM–1 mM) and inhibition of peak Ca2+ current amplitude were observed in both SER type 2 and Wistar rat neurons in concentration-dependent manners (Fig. 4). The concentration–response curve in SER type 2 neurons shifted to the left, and the inhibitory effects of LEV at concentrations from 100 nM to 1 mM were significantly more potent than those in Wistar rat neurons (Fig. 4) (F(1,50) = 20.96, p < 0.01): the potency of LEV on type 2 neurons was almost one hundred times greater than that on Wistar rat neurons. In addition, the maximum inhibition (Rmax ) by as much as 1 mM LEV was 55.4 ± 8.5% (n = 5) in SER type 2 neurons; however it was only 29.2 ± 6.0% (n = 7) in
Fig. 3. Effects of LEV on inward Ca2+ current in SER type 2 neurons and Wistar rat neurons. (A) Current–voltage relationship of Ca2+ current in SER type 2 neurons in the presence or absence of LEV (10 M). (B) Current–voltage relationship of Ca2+ current in Wistar rat neurons in the presence or absence of LEV (10 M). Each point and bar represent mean and SEM (n = 5 or 4). *, **: p < 0.05, p < 0.01 vs. before drug treatment (paired t-test).
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Fig. 4. Concentration–response relationships for the inhibitory action of LEV on the Ca2+ current in Wistar (square) rat and SER type 2 neurons (circle). The current induced from a holding potential of −90 mV to −10 mV for 100 ms duration. Each point represents components (%) of the currents that were inhibited after addition of LEV at different concentrations. Points were fitted by the Hill equation. ANOVA result: F(1,50) = 20.964, p < 0.01. *p < 0.05 (unpaired t-test).
Wistar rat neurons. Furthermore, this inhibition at 1 mM was the ceiling in the latter neurons, although it did not appear to be the ceiling in the former neurons. These results indicate that SER type 2 neurons have higher sensitivity to LEV than Wistar rat neurons. 3.4. Effects of nifedipine on LEV-produced inhibition of Ca2+ current in SER type 2 neurons In order to elucidate which type of Ca2+ channel in SER type 2 neurons was responsible for LEV-induced inhibition, the effects of nifedipine, an antagonist of L-type Ca2+ channel, were examined before and after the application of LEV. A depolarizing pulse from a holding potential of −90 mV to −10 mV was applied to the neurons for 100 ms, and then the Ca2+ currents were continuously recorded for 6–7 min after application of the drugs. Fig. 5 shows the typical type 2 neuron responses to nifedipine and LEV. During application of nifedipine at 10 M, which is a concentration sufficient to inhibit the depolarization shift accompanying repetitive firing and Ca2+ influx in SER hippocampal neurons (Hanaya et al., 1998; Amano et al., 2001a), the mean current amplitude was significantly (p < 0.001) decreased from −486.9 ± 88.8 pA to −263.3 ± 44.5 pA (n = 7) (Fig. 6A). Under inhibition of the current, LEV at 10 M, which induces 50% inhibition of depolarization shift with repetitive firing in SER hippocampal neurons (Hanaya et al., 2011), was given to the bath, but there was no further inhibition of the current amplitude (−253.1 ± 42.7 pA, n = 7) (Figs. 5A and 6A). Conversely, 10 M LEV given first significantly (p < 0.001) reduced the mean current to −321.6 ± 69.4 pA (n = 8) from −574.4 ± 57.1 pA (n = 8) of the control level, and then 10 M nifedipine added subsequently in the presence of LEV did not induce further inhibition of the current (−311.8 ± 40.0 pA, n = 8) (Figs. 5B and 6B). These results indicate that LEV acts on the L-type Ca2+ channel in SER type 2 neurons. 4. Discussion As reported previously (Yan et al., 2007), type 1 and 2 neurons were found in the SER hippocampal CA3 neurons obtained herein using patch-clamp technique: type 1 neurons showed lowered threshold and greater Ca2+ current and type 2 neurons had similar threshold potential and Ca2+ current size to those in seizure-free
Wistar rat hippocampal CA3 neurons. These type 1 and 2 neurons are considered to correspond to group 1/2 and 3 neurons found in our previous intracellular recording method, respectively, since the neurons showing the long-lasting (over 100/below 100 ms) depolarization shift and no such depolarization shift were observed in the SER hippocampal CA3 neurons following the spike induced by mossy fiber stimulation (Ishihara et al., 1993; Momiyama et al., 1995; Hanaya et al., 1998). When LEV at 100 M was applied to the SER hippocampal CA3 type 1 neurons in the bath, the lowered threshold potential of Ca2+ channels was found to be raised to a level similar to that of seizure-free Wistar rats and the maximal Ca2+ current amplitude was decreased. These results suggest that LEV acts on Ca2+ channel to delay the opening and decrease enhanced Ca2+ influxes in SER type 1 neurons. The Ca2+ channel involved in the depolarization shift of SER CA3 neurons is thought to be the Ltype, since the channel was activated by high voltage and inhibited by L-type Ca2+ antagonist, nifedipine, in the present dissociated CA3 neurons and hippocampal slice preparations (Ishihara et al., 1993; Momiyama et al., 1995) and actually there was a blockade by nifedipine of Ca2+ influxes induced by mossy fiber stimulation as well as high K+ solution in SER hippocampal slice (Amano et al., 2001a,b). LEV at therapeutic concentration (10–100 M) was found to inhibit L-type Ca2+ channel currents of SER hippocampal CA3 neurons. These findings are in line with those reported by others that LEV inhibits high-voltage-activated Ca2+ current (N-type) in hippocampal pyramidal neurons (Niespodziany et al., 2001; Lukyanetz et al., 2002), but affects neither low-voltage-activated Ca2+ current in CA1 hippocampal neurons nor Na+ current in neocortical cultured neurons (Zona et al., 2001). The high-voltage-activated Ca2+ currents (L- and N-types) have also been reported to be inhibited by anti-epileptic drugs in isolated neurons including rat cortex, amygdala, and striatum (Stefani et al., 1997). Since our data showed that LEV reduced Ca2+ current in the hippocampal CA3 neurons of SER, LEV is considered to shorten Ca2+ channel opening time and/or decrease the number of available Ca2+ channels, thereby reducing Ca2+ influx. As a result, depolarization shift accompanying repetitive firing is inhibited, thereby suppressing hypersynchronization of neuron groups and inducing the antiepileptic effect of LEV. The inhibitory effects were much greater on Ca2+ currents of SER type 2 neurons than those of non-epileptic Wistar rat neurons, suggesting that LEV could be more sensitive to L-type Ca2+ channels with functional abnormality, like SER CA3 neurons, which would contribute to depolarization shift formation. The effects of LEV were concentration-dependent in SER neurons with 35–45% inhibition of the peak Ca2+ current at therapeutic doses (10–100 M). Therefore, an inhibition by LEV of depolarization shift results in depression of repetitive firing, thereby suppressing hypersynchronization of the neuron group, and then inducing antiepileptic action (Hanaya et al., 2011). In Wistar rat neurons, however, LEV had ceiling effects of 18–25% by doses as high as 1 mM in Wistar rat neurons. These results seem to suggest that LEV has negligible effects on L-type Ca2+ channels with normal function, like Wistar rat neurons. In addition, the present study also demonstrated that LEV did not produce further inhibition of the current amplitude, which had already been inhibited by nifedipine by approximately 30%, and conversely nifedipine had no effects on the current amplitude reduced by LEV already, suggesting that LEV might act on the same L-type Ca2+ channel as nifedipine. In conclusion, LEV has a high sensitivity for altering abnormalities of Ca2+ channel function in hippocampal CA3 neurons of SER. LEV raised threshold levels for activation of Ca2+ current, and depressed Ca2+ currents in a concentration-dependent manner, by inhibiting Ca2+ current involved in L-type Ca2+ channels. In addition to the modulatory effects of LEV on the synaptic transmission, these inhibitory effects of LEV on Ca2+ channel are suggested to
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Fig. 5. Typical example of effects of nifedipine on LEV-produced inhibition of Ca2+ current in SER type 2 neurons. LEV (10 M) was perfused after (A) or before (B) the addition of nifedipine (10 M). (a) Current traces obtained by depolarizing pulses (100 ms duration) to −10 mV from a holding potential of −90 mV. (b) Plots of changes in Ca2+ current amplitude against time. Blocks: period during application of respective drug.
Fig. 6. Effects of nifedipine on inhibition of Ca2+ current by LEV in SER type 2 neurons. Current induced by depolarizing pulses from a holding potential of −90 mV to −10 mV for 100 ms. Each column with vertical bars represents the mean amplitude and SEM. LEV (10 M) was perfused after (A) or before (B) the addition of nifedipine (10 M). ***p < 0.001, compared with the control (absence of LEV and nifedipine).
contribute, at least partly, to the inhibition of epileptic seizures in SER and probably humans. Disclosure of conflicts of interest None of the authors has any conflict of interest to disclose. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, and by UCB S.A., Belgium. Levetiracetam was generously supplied by UCB S.A., Belgium. This work was carried out with the equipment of the Research Facilities for
Laboratory Animal Science, Hiroshima University School of Medicine, Japan. We are grateful to the National BioResource Project – Rat (http://www.anim.med.kyoto-u.ac.jp/NBR/) for providing rat strains (SER).
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