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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2009 Nov 3;158(7):1753–1762. doi: 10.1111/j.1476-5381.2009.00463.x

Levetiracetam inhibits glutamate transmission through presynaptic P/Q-type calcium channels on the granule cells of the dentate gyrus

Chun-Yao Lee 1, Chih-Chuan Chen 2, Horng-Huei Liou 1,2
PMCID: PMC2801216  PMID: 19888964

Abstract

Background and purpose:

Levetiracetam is an effective anti-epileptic drug in the treatment of partial and generalized seizure. The purpose of the present study was to investigate whether levetiracetam regulates AMPA and NMDA receptor-mediated excitatory synaptic transmission and to determine its site of action in the dentate gyrus (DG), the area of the hippocampus that regulates seizure activities.

Experimental approach:

Whole-cell patch-clamp method was used to record the AMPA and NMDA receptor-mediated excitatory postsynaptic currents (EPSCAMPA and EPSCNMDA) in the presence of specific antagonists, from the granule cells in the DG in brain slice preparations from young Wistar rats (60–120 g).

Key results:

Levetiracetam (100 µM) inhibited both evoked EPSCAMPA and EPSCNMDA to an equal extent (80%), altered the paired-pulse ratio (from 1.39 to 1.25), decreased the frequency of asynchronous EPSC and prolonged the inter-event interval of miniature EPSCAMPA (from 2.7 to 4.6 s) without changing the amplitude, suggesting a presynaptic action of levetiracetam. The inhibitory effect of levetiracetam on evoked EPSCAMPA was blocked by ω-agatoxin TK (100 nM), a selective P/Q-type voltage-dependent calcium channel blocker, but not by nimodipine (10 µM) or ω-conotoxin (400 nM).

Conclusions and implications:

These results suggest that levetiracetam modulated the presynaptic P/Q-type voltage-dependent calcium channel to reduce glutamate release and inhibited the amplitude of EPSC in DG. This effect is most likely to contribute to the anti-epileptic action of levetiracetam in patients.

Keywords: levetiracetam, glutamate, EPSC, dentate gyrus, voltage-dependent calcium channel

Introduction

Levetiracetam ((S)-α-ethyl-2-exo-pyrrolidine acetamide) is a novel anti-epileptic drug in the treatment of partial and generalized seizure (Cereghino et al., 2000; Weber and Beran, 2004) and is effective in wide-spectrum neuropsychiatric disorders (Muralidharan and Bhagwagar, 2006; Mula et al., 2007). Several pharmacological properties of levetiracetam on ion channels have been reported, including blockade of high-voltage-activated voltage-dependent Ca2+ channels (HVA-VDCCs) to reduce neuronal depolarization in dissociated CA1 pyramidal neurons (Niespodziany et al., 2001; Lukyanetz et al., 2002; Pisani et al., 2004), modulation of K+ channels to stabilize the membrane potential (Madeja et al., 2003; Lee et al., 2008a) and enhancement of GABA- and glycine-gated currents to strengthen inhibitory tones (Rigo et al., 2002), but little or no effects on voltage-dependent Na+ and T-type Ca2+ channels (Zona et al., 2001); channel and receptor nomenclature follows Alexander et al. (2008). Besides acting on ion channels, levetiracetam is reported to inhibit epileptiform discharges in human neocortical slices (Gorji et al., 2002) and reduce neurotransmission in rat hippocampal slices (Yang et al., 2007). In addition, levetiracetam is known to specifically bind to SV2A (Lynch et al., 2004), a protein abundantly located on synaptic vesicles and interacting with synaptotagmin 1 to enhance neurotransmitter release (Janz et al., 1999). As levetiracetam is a novel anti-epileptic drug for partial and generalized seizures, outside the classical categorization, a greater understanding of the mechanisms by which levetiracetam suppresses seizures is relevant to the development of new therapeutic agents.

In the chronic animal model of temporal lobe epilepsy, dramatic pathological alterations in glutamate transmission in dentate gyrus (DG) are observed (Nadler, 2003). Levetiracetam significantly opposes pilocarpine-induced increase of neuronal excitability and counteracts seizure-induced alteration in excitatory amino acid concentration (Löscher et al., 1998; Klitgaard et al., 2003). These characteristics of levetiracetam indicate that reduction of neuronal excitability in epileptic states by levetiracetam is possibly due to down-regulation of glutamate transmission.

Glutamate is the most important excitatory neurotransmitter in the mammalian CNS. After release from presynaptic nerve terminals, glutamate binds to both pre- and postsynaptic metabotropic and ionotropic glutamate receptors. Activation of glutamate receptors maintains normal brain functions, but excessive levels of glutamate may cause neuronal damage and seizure activities. An elevated glutamate concentration in the hippocampus has been reported, before seizure onset and during seizures, in patients with epilepsy (During and Spencer, 1993). Reducing glutamate release (Cunningham et al., 2004) or blocking glutamate receptors (Kleckner et al., 1999) can decrease neuronal excitability and is recognized as an important mechanism of anti-epileptic drugs.

Although previous studies have revealed an action of levetiracetam on ion channels (Niespodziany et al., 2001; Lukyanetz et al., 2002; Madeja et al., 2003; Pisani et al., 2004; Lee et al., 2008a) and synaptic transmission (Gorji et al., 2002; Rigo et al., 2002; Yang et al., 2007), how levetiracetam influences glutamatergic transmission to reduce the excitability is still unclear. The aim of our present study was to investigate the effect of levetiracetam on the glutamatergic transmission system. We utilized whole-cell patch-clamp recording to investigate the action of levetiracetam on the excitatory postsynaptic currents (EPSC) in the granule cells in the DG, which controls the propagation of seizure activities (McNaughton and Barnes, 1997). We assessed the involvement of different VDCCs in the effects of levetiracetam on glutamatergic transmission. Our results suggested that levetiracetam decreased the amplitude of evoked EPSC (eEPSC) through interacting with presynaptic P/Q-type VDCC to reduce glutamate release, which is likely to contribute to the anti-epileptic action of levetiracetam observed in patients.

Methods

Brain slice preparation

All animal care and experimental procedures were in accordance with the Guide for the Care and Use of Laboratory Animals by the US National Institutes of Health and approved by the Animal Ethics Committee of National Taiwan University. All efforts were made to minimize animal suffering and to reduce the number of animals used. Male Wistar rats (4–6 weeks, 60–120 g) were housed in polycarbonate boxes (four rats per box). They were maintained in a controlled atmosphere with a 12 h dark/light cycle (lights off at 7:00 PM), a temperature of 22 ± 2°C and 50–70% humidity with free access to pelleted feed and fresh tap water. The animals were decapitated under deep anaesthesia with halothane inhalation and the brains quickly moved to ice-cold oxygenated cutting solution containing (in mM): 125 NaCl, 2.5 KCl, 0.5 CaCl2, 5 MgCl2, 26 NaHCO3, 15 glucose and aerated with 95% O2–5% CO2 (to adjust pH value to 7.4 by 1N HCl and NaOH). Coronal brain slices (300 µm) containing DG were cut by a microslicer (DTK-1000, Dosaka, Kyoto, Japan) in cutting solution and then transferred to a holding chamber with artificial cerebrospinal fluid (ACSF) containing (in mM) 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 26 NaHCO3 and 15 glucose and aerated with 95% O2–5%CO2 (to adjust pH value to 7.4 by 1N HCl and NaOH). Brain slices were then maintained at room temperature (23 ± 2°C) for at least 1 h before recording.

Electrophysiological recordings

The brain slices were transferred to the recording chamber, held submerged and superfused continuously with ACSF at a flow rate of 1–2 mL·min−1 for recording at room temperature (23 ± 2°C). Evoked AMPA receptor-mediated EPSC (eEPSCAMPA) were recorded in the ACSF containing bicuculline methobromide (BMB, 50 µM) and DL-aminophosphonovaleric acid (DL-APV, 50 µM) at a holding potential of −70 mV. Evoked NMDA receptor-mediated EPSC (eEPSCNMDA) were recorded in Mg2+-free ACSF in the presence of BMB (50 µM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM) and D-serine (10 µM) at a holding potential of −70 mV. Patch electrodes were pulled from standard-walled borosilicate glass capillaries (CSF-150, Warner Instrument, Hamden, CT, USA) by micropipette puller (P97, Sutter Instrument, Novato, CA, USA) with a resistance of 3–8 MΩ, then filled with a CsCl-based internal solution containing (in mM): 140 CsCl, 9 NaCl, 1 MgCl2, 1 EDTA, 10 HEPES, 5 QX-314 (N-(2,6-Dimethylphenylcarbamoylmethyl) triethylammonium bromide), 2 Mg-ATP and 0.3 Na-GTP (pH was adjusted to 7.3 with 1N CsOH). Granule cells in the DG were visually identified by using an upright infra-red microscope fitted with a water-immersion lens (Olympus, Tokyo, Japan). Whole-cell patch-clamp recording was made from these granule cells with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA). Series resistance and whole-cell capacitance were monitored continuously during all experiments and estimated by compensating (70–80%) for the fast transients evoked at the onset and offset of 8 ms, −5 mV voltage-command steps. The data were discarded if series resistance changed by more than 20%. To evoke EPSC, a stimulation glass pipette filled with 3 M NaCl solution was placed on the inner molecular layer of DG to elicit EPSC with a stimulator (S-48, Grass-Telefactor, W. Warwick, RI, USA) and isolation unit (A.M.P.I., Jerusalem, Israel). The stimulus duration was between 10 and 50 µs and the strength was between 5 and 20 V, with a frequency of 0.2 Hz. To avoid interference due to synaptic fatigue induced from low-frequency stimulation, we delivered continuous stimulation for more than 5 min (>60 stimuli) after accessing the whole-cell configuration to achieve stable eEPSC amplitudes before starting the recording. eEPSC were quantified by measuring peak amplitudes of average responses. At least 3 min (about 36 events) were recorded to determine the average peak amplitudes in control and drug experiments. The responses of the drug were collected and analysed by stable eEPSC amplitudes after more than 5 min application of the drug (Lee et al., 2008b).

To measure paired-pulse ratio (PPR), test and conditioned pulses were delivered at 50 ms intervals. The PPR was then calculated by dividing the second EPSC by the first EPSC. To record AMPA receptor-mediated miniature EPSC (mEPSCAMPA) in isolation, tetrodotoxin (TTX, 1 µM), BMB (50 µM) and DL-APV (50 µM) were added to the bath solution. Spontaneous synaptic currents were filtered at 5k Hz, digitized at 10k Hz and recorded directly to a computer hard disk using AxoScope 9.0 software (Axon Instruments). Access resistance was monitored at regular intervals throughout each study and recordings were discarded if it changed by >20%. The amplitude, inter-event interval (IEI) and frequency after application of drugs were then analysed. After an evoked stimulation, large amounts of Ca2+ flow through VDCCs into the presynaptic nerve terminals, increasing the probability of synaptic vesicle release. Therefore, after the first stimulation of eEPSCAMPA, there were subsequently several small asynchronous EPSC (aEPSC). The aEPSC were collected in the period between 0.15 and 1 s after the first stimulation artefact of eEPSCAMPA and analysed to identify whether levetiracetam acted on presynaptic or postsynaptic sites (Lee et al., 2008b).

Data analysis and statistical procedures

Data were acquired by clampex 9.0 software (Axon Instruments) and stored in a hard disk. eEPSC were analysed by clampfit 9.0 (Axon Instruments). mEPSCAMPA were analysed by mini-analysis programme 6.0 (Synaptosoft Inc., Fort Lee, NJ, USA). Statistical differences were established by Student's t-test and Kolmogorov–Smirnov's test (K-S test). Data were expressed as mean ± SEM, and P < 0.05 was taken to indicate statistical significance.

Materials

All drugs used were bath applied. BMB, calphostin C (Cal-C), D-serine and TTX were purchased from Sigma (St. Louis, MO, USA). CNQX, chelerythrine chloride (CTC), DL-APV, H89 and nimodipine were purchased from Tocris (Bristol, UK). ω-Conotoxin GVIA (ω-CTX) and ω-agatoxin TK (ω-AGX) were purchased from Alomone Labs (Jerusalem, Israel). Levetiracetam was kindly supplied by UCB Pharma (Braine l'Alleud, Belgium). All drugs were dissolved in double distilled water, except CNQX, CTC, H89 and nimodipine, which were dissolved in 100% DMSO (the final concentration of DMSO in ACSF was less than 0.1%).

Results

Levetiracetam inhibited both evoked EPSCAMPA and EPSCNMDA

Evoked EPSCAMPA and EPSCNMDA were obtained by whole-cell patch-clamp recordings from the granule cells in DG. The experimental traces show that levetiracetam significantly reduced the amplitude of eEPSCAMPA (Figure 1A) or of eEPSCNMDA (Figure 1B). The mean amplitudes of eEPSCAMPA and of eEPSCNMDA after application of levetiracetam were reduced equally over the concentration range used (10–500 µM, n= 6, P < 0.01) respectively (Student's t-test, Figure 1C). However, there was no significant difference in the effect of levetiracetam concentrations from 100 to 500 µM. Inhibition by levetiracetam was rapid and readily reversible (Figure 1D), with inhibition or recovery taking about 5 min. Series resistance was continuously monitored during the whole experiment to show the stability of the recording (Figure 1D).

Figure 1.

Figure 1

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LEV inhibited the amplitudes of eEPSCAMPA and eEPSCNMDA in DG. (A) LEV inhibited the amplitudes of eEPSCAMPA in a concentration-dependent manner. The recording was performed in the presence of BMB (50 µM) and DL-APV (50 µM) with holding potential at −70 mV. (B) LEV inhibited the amplitudes of eEPSCNMDA in a concentration-dependent manner. The recording was performed in the presence of BMB (50 µM), CNQX (10 µM) and D-serine (10 µM) with holding potential at −70 mV in Mg2+-free ACSF. (C) Summary data showing a concentration–response curve obtained at various concentration of LEV on the amplitude of eEPSCAMPA and eEPSCNMDA. (**P < 0.01, ***P < 0.001 for eEPSCAMPA and #P < 0.05, ##P < 0.01 for eEPSCNMDA, Student's t-test). (D) A continuous recording from a DG granule cell, illustrating onset of and recovery from, the action of LEV. The series resistance was continuously monitored during the whole experiment. ACSF, artificial cerebrospinal fluid; BMB, bicuculline methobromide; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DG, dentate gyrus; DL-APV, DL-aminophosphonovaleric acid; eEPSCAMPA; evoked AMPA receptor-mediated EPSC; eEPSCNMDA, evoked NMDA receptor-mediated EPSC; EPSC, excitatory postsynaptic current; LEV, levetiracetam.

Effects of levetiracetam on PPR indicated a presynaptic action

Paired-pulse ratio is a form of short-term plasticity, reflecting presynaptic release probability (Zucker, 1989). To determine the synaptic site of levetiracetam action, we investigated the effect of this compound on the PPR in the granule cells of DG. A typical example of eEPSCs in Figure 2A shows a change in the PPR resulting from the application of levetiracetam. Before levetiracetam perfusion, the PPR was 1.39 ± 0.05 at an interval of 50 ms. After application of levetiracetam (100 µM), the PPR was significantly decreased (Figure 2B, n= 7, P < 0.01 with Student's t-test).

Figure 2.

Figure 2

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LEV altered the PPR of eEPSCAMPA. (A) LEV (100 µM) altered the PPR of eEPSCAMPA at 50 ms interval. The recording was performed in the presence of BMB (50 µM) and DL-APV (50 µM) with holding potential at −70 mV. (B) Summary data showing the average PPR of eEPSCAMPA in control and LEV (100 µM) conditions (**P < 0.01, Student's t-test). The PPR was decreased after application of LEV (100 µM) (n= 7, **P < 0.01, Student's t-test). BMB, bicuculline methobromide; DL-APV, DL-aminophosphonovaleric acid; eEPSCAMPA; evoked AMPA receptor-mediated EPSC; EPSC, excitatory postsynaptic current; LEV, levetiracetam; PPR, paired-pulse ratio.

The effect of levetiracetam on the IEI and amplitude of mEPSCAMPA

We then examined the effects of levetiracetam (100 µM) on action potential-independent spontaneous mEPSCAMPA, recorded in the presence of TTX (1 µM), BMB (50 µM) and DL-APV (50 µM) in DG (Figure 3A). As shown in Figure 3B, the mean IEI of mEPSCAMPA was significantly increased by levetiracetam (100 µM) treatment (Figure 3B, P < 0.001 with K-S test, n= 9). However, the mean amplitude of mEPSCAMPA was not changed (Figure 3C, P > 0.05 with Student's t-test, n= 9) after levetiracetam (100 µM).

Figure 3.

Figure 3

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The effect of LEV (100 µM) on mEPSCAMPA. (A) LEV (100 µM) inhibited the frequency but not the amplitude of mEPSCAMPA. The recording was performed in the presence of BMB (50 µM), DL-APV (50 µM) and TTX (1 µM) with holding potential at −70 mV. (B) Summary data showing the mean IEI of mEPSCAMPA before and after LEV (100 µM) perfusion. LEV (100 µM) shifted the cumulative probability plot of mEPSCAMPA IEI to the right (***P < 0.001, K-S test). The inset plot summarizes the mean IEI of mEPSCAMPA before and after LEV (100 µM) perfusion. The mean IEI of mEPSCAMPA was significantly increased by LEV (100 µM, ***P < 0.001, Student's t-test). (C) Summary data showing the mean amplitude of mEPSCAMPA before and after LEV (100 µM) perfusion. The mean amplitude of mEPSCAMPA was not significantly affected by LEV (100 µM, P > 0.05, Student's t-test). BMB, bicuculline methobromide; DL-APV, DL-aminophosphonovaleric acid; EPSC, excitatory postsynaptic current; mEPSCAMPA, AMPA receptor-mediated miniature EPSC; IEI, inter-event interval; LEV, levetiracetam; TTX, tetrodotoxin.

The effect of levetiracetam on the frequency and amplitude of aEPSC

To further confirm the presynaptic effect of levetiracetam, we collected and analysed the aEPSC following the eEPSCAMPA (Figure 4A). Compared with mEPSCAMPA, which arises randomly from synapses all over the cell, aEPSC comes from the evoked synapses in distal dendrites, in which most AMPA receptors are located. This characteristic of aEPSC helps to identify the presynaptic effects of levetiracetam (Bekkers and Clements, 1999). The amplitudes of these small EPSC were similar to mEPSCAMPA acquired under TTX perfusion and were analysed in the same way as mEPSCAMPA to identify the pre- or postsynaptic mechanisms. The mean frequency of aEPSC was significantly suppressed after levetiracetam (100 µM) treatment (Figure 4B, P < 0.05, Student's t-test, n= 5), although the mean amplitude of aEPSC was not changed (Figure 4C and D, P > 0.05 with K-S test, n= 5). This result was compatible with the effect of levetiracetam on mEPSCAMPA, suggesting a presynaptic site of the action of levetiracetam.

Figure 4.

Figure 4

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LEV inhibited the frequency of asynchronous excitatory postsynaptic currents (aEPSC). (A) aEPSC (indicated by the black arrows) was recorded between 0.15 and 1 s after the first stimulation artefact of evoked AMPA receptor-mediated EPSC in controlled conditions and during LEV (100 µM) perfusion. The recording was performed in the presence of bicuculline methobromide (50 µM) and DL-aminophosphonovaleric acid (50 µM) with the holding potential at −70 mV. (B) Summary data showing that the average frequency of aEPSC was reduced by LEV (100 µM, *P < 0.05, Student's t-test). (C) Summary data showing that the average amplitude of aEPSC was not significantly affected by LEV (P > 0.05, Student's t-test). (D) LEV (100 µM) did not significantly shift the cumulative probability plot of aEPSC amplitude, showing that the amplitude of aEPSC was not reduced by LEV (P > 0.05, K-S test).

Only ω-AGX but neither nimodipine nor ω-CTX prevented the effects of levetiracetam

Glutamate release from presynaptic nerve terminals is elicited by Ca2+ influx through VDCCs (Wu and Saggau, 1997). To clarify the presynaptic mechanisms of the reduction of glutamate release by levetiracetam, in terms of the VDCC involved, we used selective VDCC blockers: nimodipine (for L-type), ω-CTX (for N-type) and ω-AGX (for P/Q-type) (Cunningham et al., 2004; Pisani et al., 2004). Figure 5A shows that application of nimodipine (10 µM) caused a decrease in the eEPSCAMPA to 85 ± 5% of the control value (P < 0.05 with Student's t-test). The combination of levetiracetam (100 µM) and nimodipine (10 µM) further reduced the eEPSCAMPA amplitude (Figure 5A and B; n= 6, P < 0.01 to control and P < 0.01 to nimodipine alone, Student's t-test,). Similarly, when N-type VDCCs were blocked by ω-CTX (400 nM), eEPSCAMPA was reduced and combination with levetiracetam (100 µM) further reduced the amplitude of eEPSCAMPA (Figure 5C and D; n= 6, P < 0.001 to control and P < 0.01 to ω-CTX with Student's t-test,). Figure 5E shows that ω-AGX (100 nM) blocked the total eEPSCAMPA in DG granule cells (P < 0.01, Student's t-test), but now the addition of levetiracetam (100 µM) did not further inhibit the amplitude of eEPSCAMPA (Figure 5E and F; n= 4, P < 0.05 to control, P > 0.05 to ω-AGX, Student's t-test).

Figure 5.

Figure 5

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The effect of LEV (100 µM) in the presence of VDCC blockers. (A) Experimental traces showing nimodipine (Nimo; 10 µM) inhibited eEPSCAMPA, and LEV (100 µM) further inhibited eEPSCAMPA. The recording was performed in the presence of BMB (50 µM) and DL-APV (50 µM) with holding potential at −70 mV. (B) Nimodipine (10 µM) decreased the amplitude of eEPSCAMPA (*P < 0.05 to control). In the presence of nimodipine, LEV (100 µM) further decreased the amplitude of eEPSCAMPA (n= 6, **P < 0.01 to control and ##P < 0.01 to nimodipine treatment, Student's t-test). (C) Experimental traces showing ω-CTX (400 nM) inhibited eEPSCAMPA, and LEV (100 µM) further inhibited eEPSCAMPA. The recording was performed in the presence of BMB (50 µM) and DL-APV (50 µM) with holding potential at −70 mV. (D) ω-CTX (400 nM) alone decreased the amplitude of eEPSCAMPA (**P < 0.01, Student's t-test). In the presence of ω-CTX, LEV (100 µM) further decreased the amplitude of eEPSCAMPA (n= 6, ***P < 0.001 to control and ##P < 0.01 to ω-CTX treatment, Student's t-test). (E) Experimental traces showing ω-AGX (100 nM) inhibited eEPSCAMPA, and LEV (100 µM) did not further inhibit eEPSCAMPA. The recording was performed in the presence of BMB (50 µM) and DL-APV (50 µM) with holding potential at −70 mV. (F) ω-AGX (100 nM) decreased the amplitude of eEPSCAMPA (**P < 0.01, Student's t-test). In the presence of ω-AGX, the amplitude of eEPSCAMPA after LEV treatment was not further decreased (n= 4, *P < 0.05 to control, P > 0.05 to ω-AGX treatment, Student's t-test). (G) Experimental traces showing LEV (100 µM) inhibited eEPSCAMPA, and ω-AGX (100 nM) did not further inhibit eEPSCAMPA. The recording was performed in the presence of BMB (50 µM) and DL-APV (50 µM) with holding potential at −70 mV. (H) Summary data showing that LEV (100 µM) alone decreased the amplitude of eEPSCAMPA (*P < 0.05 with Student's t-test) and, after adding ω-AGX, the amplitude of eEPSCAMPA was not further decreased (n= 6, *P < 0.05 to control, P > 0.05 to LEV treatment, Student's t-test). (I) Experimental traces showing that combining three VDCC blockers (nimodipine 10 µM, ω-CTX 400 nM, ω-AGX 100 nM) inhibited eEPSCAMPA. The recording was performed in the presence of BMB (50 µM) and DL-APV (50 µM) with holding potential at −70 mV. (J) Summary data showing the effects of combining three VDCC blockers (nimodipine 10 µM, ω-CTX 400 nM, ω-AGX 100 nM) on the amplitude of eEPSCAMPA (n= 3, **P < 0.01 to control, Student's t-test). BMB, bicuculline methobromide; DL-APV, DL-aminophosphonovaleric acid; eEPSCAMPA, evoked AMPA receptor-mediated EPSC; EPSC, excitatory postsynaptic current; LEV, levetiracetam; VDCC, voltage-dependent Ca2+ channel; ω-AGX, ω-agatoxin TK; ω-CTX, ω-conotoxin GVIA.

Another set of experiments used these compounds in reverse order, that is, levetiracetam (100 µM) first and then adding the selective VDCC blocker. As shown in Figure 5G, levetiracetam (100 µM) blocked the total eEPSCAMPA in DG granule cells (P < 0.05 with Student's t-test); adding ω-AGX (100 nM) did not further inhibit the amplitude of eEPSCAMPA (Figure 5G and H; n= 6, P < 0.05 to control, P > 0.05 to levetiracetam, Student's t-test,). Combining all three selective VDCC blockers (nimodipine 10 µM, ω-CTX 400 nM and ω-AGX 100 nM) markedly reduced the amplitude of eEPSCAMPA but did not totally block the response (Figure 5I and J; n= 3, P < 0.05 to control, Student's t-test). The remaining eEPSCAMPA might be due to the presence of other subtypes of VDCC (Evans and Zamponi, 2006).

We also tested the effects of ω-AGX on the PPR as this point is crucial for the hypothesis that levetiracetam selectively blocks P/Q-type VDCC. In contrast to the effects of levetiracetam, application of ω-AGX (100 nM) significantly increased the PPR over control values (Figure 6, n= 4, P < 0.05, Student's t-test).

Figure 6.

Figure 6

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ω-AGX altered the PPR of eEPSCAMPA. (A) Experimental traces showing that ω-AGX (100 nM) altered the PPR of eEPSCAMPA at 50 ms interval. The recording was performed in the presence of BMB (50 µM) and DL-APV (50 µM) with holding potential at −70 mV. (B) Summary data showing the average PPR of eEPSCAMPA in control conditions and after adding ω-AGX (100 nM). The PPR was clearly increased after ω-AGX (100 nM) application (n= 4, *P < 0.05, Student's t-test). BMB, bicuculline methobromide; DL-APV, DL-aminophosphonovaleric acid; eEPSCAMPA, evoked AMPA receptor-mediated EPSC; EPSC, excitatory postsynaptic current; PPR, paired-pulse ratio; ω-AGX, ω-agatoxin TK.

Discussion and conclusions

Levetiracetam is a new anti-epileptic drug and its synaptic mechanism(s) are not known. In the present study, we addressed this issue by using whole-cell patch-clamp recordings. Our data demonstrated a presynaptic action of levetiracetam, based on the as following findings. Firstly, levetiracetam (100 µM) inhibited both eEPSCAMPA and eEPSCNMDA with similar concentration–response patterns indicating a presynaptic inhibition of glutamate release (Perkel and Nicoll, 1993). Then, PPR facilitation is an enhancement of the synaptic release caused by presynaptic activation of VDCC (Inchauspe et al., 2007), Ca2+ current facilitation (Müller et al., 2008), accumulation of intracellular Ca2+ and other mechanisms (Zucker, 1989). The levetiracetam-induced reduction of eEPSCAMPA was associated with a modification of the PPR, demonstrating that the action of levetiracetam was located at presynaptic sites to regulate transmitter release. Also, the frequency and amplitude of mEPSCAMPA reflects the changes in postsynaptic AMPA receptor-mediated responsiveness to presynaptic glutamate release (Redman, 1990). Levetiracetam reduced the frequency of mEPSCAMPA significantly, supporting a presynaptic action. Finally, levetiracetam induced the same pattern of amplitude and frequency in aEPSC as in mEPSCAMPA, further confirming its presynaptic action. The strongest effect of levetiracetam appeared to be on the mEPSCAMPA frequency, consistent with the results of Carunchio et al. (2007). Thus, it appears that this parameter would be the most sensitive and appropriate to assess the mechanisms of levetiracetam. Taken together, we concluded that levetiracetam inhibited glutamate release, presynaptically, to suppress synaptic transmission between the perforant pathway and the granule cells in the DG.

Levetiracetam binds specifically to the vesicle protein, SV2A, located in a presynaptic site and involved in neurotransmitter release (Lynch et al., 2004). An in vitro extracellular recording has reported the site of action of levetiracetam to be located on the presynaptic nerve terminal in the CA1 region of the hippocampus (Yang et al., 2007). Although earlier work suggested that the site of action of levetiracetam is the presynaptic terminal, the evidence for this suggestion is indirect. To further investigate the presynaptic mechanism of levetiracetam in the DG, the involvement of three types of HVA-VDCCs was assessed with specific channel blockers. Nimodipine, ω-CTX and ω-AGX inhibited the amplitude of eEPSCAMPA, indicating that L-, N- and P/Q-type VDCCs all contributed to the Ca2+ transient in the presynaptic nerve terminal. Among three HVA-VDCC antagonists, only ω-AGX blocked the inhibitory action of levetiracetam, demonstrating that levetiracetam inhibited glutamate release mainly through affecting P/Q-type VDCCs in the DG (Figure 5). Similar results were also obtained by using these compounds in reversed order (Figure 5G and H), strengthening the hypothesis that the effects of levetiracetam were exerted via the presynaptic nerve terminal and the P/Q-type VDCCs.

Levetiracetam has been found to act on HVA-VDCCs and reduce neuronal excitability in isolated neuronal cells. Niespodziany et al. (2001) found levetiracetam inhibited L- and N-type VDCC in CA1 pyramidal neurons in hippocampal slices. Lukyanetz et al. (2002) reported levetiracetam blocked N-type VDCC in isolated CA1 hippocampal neurons. Pisani et al. (2004) showed levetiracetam modulated epileptiform activity through both N- and P/Q-type VDCC in isolated neocortical pyramidal neurons. However, these findings could not address the question of the synaptic mechanism of levetiracetam. HVA-VDCCs not only regulate membrane potential and neuronal excitability in isolated neurons, but they also augment synaptic activity and neurotransmitter release in synapses. After depolarization of the presynaptic nerve terminal, Ca2+ flows in through HVA-VDCCs, binds to nearby Ca2+-binding proteins, initiates the transmitter releasing machinery and, finally, triggers the release of glutamate (Augustine, 2001). Different subtypes of HVA-VDCCs are co-localized at presynaptic terminals in different proportions in different brain areas. They can coexist at a single release site as a heterogeneous mixture and contribute jointly to the local Ca2+ transient that triggers transmitter release (Reid et al., 2003). The P/Q-type VDCC is reported to be the predominant Ca2+ channel involved in triggering neurotransmitter release in the perforant path from the entorhinal cortex to the DG (Qian and Noebels, 2001), which is compatible with our pharmacological analysis that levetiracetam preferentially acts on P/Q-type VDCCs to decrease the amount of Ca2+ influx, reduce glutamate release and thus modulate synaptic activity in the DG.

Several other anti-epileptic drugs have shown to block P/Q-type VDCCs and reduce glutamate release. Gabapentin and pregabalin block P/Q-type VDCCs to reduce glutamate release, presynaptically, in entorhinal cortical slice preparations (Cunningham et al., 2004). Gabapentin also inhibited synaptic transmission in spinal cord dorsal horn through blockade of P/Q-type VDCCs (Bayer et al., 2004). Lamotrigine reduces Ca2+ influx in cortical neurons by inhibiting P/Q-type VDCC (Stefani et al., 1996). From our investigation, the inhibition of eEPSCAMPA by levetiracetam, through an action on P/Q-type VDCCs, may therefore participate in its anti-epileptic property. Anti-epileptic drugs have different effects, depending on the region of the brain, reflecting the diversity of individual synapses in terms of the ion channels, receptors, pre- and postsynaptic cell types and surrounding environment (Craig and Boudin, 2001). The possibility that levetiracetam could act on different subtypes of HVA-VDCC in different brain regions needs further investigation.

Paired-pulse ratio is a form of short-term plasticity, associated with the probability of presynaptic release. Reduction in presynaptic release is typically correlated with an increase in PPR, while enhancement of release is typically correlated with a reduction in PPR (Zucker, 1989). We found levetiracetam decreased the PPR (Figure 2), which was an ‘atypical’ response, if it reduced presynaptic release. However, Yang et al. (2007) reported that with 10 and 80 Hz stimulation (at a stimulation interval of 100 ms and 12.5 ms), treatment with levetiracetam (10 and 100 µM) reduced the EPSP2/EPSP1 ratio. According to our results and the previous findings, levetiracetam's action on presynaptic VDCCs to regulate the Ca2+ pool and reduce glutamate release may possibly be use-dependent (Yang et al., 2007). This possibility is supported by the finding that levetiracetam blocked VDCCs to attenuate the repetitive firing in cultured cortical neurons (Pisani et al., 2004).

It is relevant to note that ω-AGX decreased the amplitude of eEPSCAMPA concomitant with an increase in PPR (Figure 6), while levetiracetam decreased PPR (Figure 2), implying that they cannot be acting by the same mechanism, despite the apparent block of the levetiracetam effect by ω-AGX. This discrepancy in altered PPR, between levetiracetam and ω-AGX, might be explained as follows. Firstly, levetiracetam could be use-dependently modulating presynaptic VDCC to reduce glutamate release, as mentioned earlier (Yang et al., 2007), although our results at this stage do not provide enough data to support this hypothesis, and further investigations are necessary to prove this point of view. Another explanation is that levetiracetam might act on P/Q-type VDCCs by affecting the channel's biophysical properties (open or closed states), which is distinct from the action of ω-AGX (Mori et al., 1996). Finally, an effect independent of inhibiting VDCC could be involved in the mechanism of action of levetiracetam.

A recent study found that levetiracetam modulated AMPA receptor-mediated currents on cultured cortical neurons (Carunchio et al., 2007). According to their results, levetiracetam (200 µM) inhibits both the amplitude (to 0.86) and the frequency (to 0.82) of mEPSCAMPA recording on the cultured cortical neurons. Both the previous and our present results showed a decrease in the frequency of mEPSCAMPA, which supported a presynaptic effect. On the other hand, we did not observe any decrease in the amplitude of mEPSCAMPA in our current study. This discrepancy may be due to the heterogeneous composition of AMPA receptors and consequently to the interaction of levetiracetam with some specific receptor subunits. The heterogeneous sensitivity to levetiracetam, observed in different brain areas, indicates that levetiracetam is able to interact with some AMPA subunits that are differently expressed in the brain (Ozawa et al., 1998). Another difference is that we used freshly prepared brain slices with an intact synaptic system, rather than a culture of neuronal cells, in which such synapses might not be found.

Clinical effective doses of levetiracetam (1–3 g per day) result in plasma levels between 35 and 100 µM and peak levels between 90 and 250 µM (Patsalos, 2000). Levetiracetam reached maximum inhibitory effect on eEPSC in the granule cells of the DG at 100 µM, which is close to the plasma levels after clinical effective doses. At these concentrations, levetiracetam may reduce glutamatergic synaptic transmission (to 80%), which is consistent with previous findings that levetiracetam inhibited the calcium current by ∼18% (Lukyanetz et al., 2002). It may be an advantage for levetiracetam to exert its anti-epileptic property without significant overall suppression of the CNS, which would be expected from extensive suppression of glutamatergic transmission.

The DG is considered as a gate of the hippocampus and plays a critical role in regulating the propagation of seizure activities from the entorhinal cortex to the hippocampus (McNaughton and Barnes, 1997). It also acts as a frequency-dependent filter for paroxysmal activity spreading from the entorhinal cortex to the hippocampus (Stringer et al., 1989). A decreased excitability of the DG has proved effective in the treatment of epilepsy (Lee et al., 2008b). Levetiracetam tends to inhibit the neuronal excitability in this brain area, which is crucial to the regulation of seizures, thus preventing the initiation and spread of seizures. The pharmacological profile of levetiracetam in our work is consistent with previous findings that levetiracetam is effective in the chronic animal model of temporal lobe epilepsy in vivo (Loscher et al., 1998; Klitgaard et al., 2003) and in vitro (Gorji et al., 2002). The efficacy of levetiracetam in this DG-based epilepsy model implies that levetiracetam may directly act on the DG to regulate excitatory synaptic transmission in order to exert its anti-epileptic effects.

Acknowledgments

This work was supported by the National Science Council (NSC 96–2323-B-002–014) Taipei, Taiwan. We thank Professor Kazuhiko Yamaguchi (Laboratory for Memory and Learning, Brain Science Institute, RIKEN, Japan) and Dr Kayoko Onodera (Institute of Brain Research, Faculty of Medicine, University of Tokyo, Japan) for their kind suggestions and technical help.

Glossary

Abbreviations:

ACSF

artificial cerebrospinal fluid

BMB

bicuculline methobromide

CNQX

6-cyano-7-nitroquinoxaline-2,3-dione

DG

dentate gyrus

DL-APV

DL-aminophosphonovaleric acid

EPSC

excitatory postsynaptic current

HVA-VDCCs

high-voltage-activated voltage-dependent Ca2+ channels

IEI

inter-event interval

PPR

paired-pulse ratio

QX-314

N-(2,6-Dimethylphenylcarbamoylmethyl) triethylammonium bromide

TTX

tetrodotoxin

ω-AGX

ω-agatoxin TK

ω-CTX

ω-conotoxin GVIA

Conflicts of interest

None.

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