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Keywords:

  • Synaptic transmission;
  • Glutamate release;
  • NMDA;
  • Non-NMDA;
  • Fenamates;
  • NSAIDs

Summary

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Purpose: In this study, we explore the antiepileptic effects of flufenamic acid (FFA) in order to identify the cellular mechanisms that underlie the potential anticonvulsant properties of this nonsteroidal antiinflammatory compound.

Methods: The mechanisms of FFA action were analyzed using an in vitro model in which epileptiform activity was induced in hippocampal slices by perfusion with 100 μm 4-aminopyridine (4-AP) added to a modified Mg2+-free solution. The activity of CA1 pyramidal neurons as well as the synaptic connection between CA3 and CA1 was monitored using extracellular and patch-clamp recordings.

Results: Epileptiform activity was suppressed in hippocampal neurons by FFA at concentrations between 50 and 200 μm. Glutamatergic excitatory synaptic transmission was diminished by FFA without modifying recurrent γ-aminobutyric acid (GABA)ergic synaptic inhibition. Several lines of evidence indicated that FFA did not decrease neurotransmitter release probability, implicating a postsynaptic mechanism of action. FFA also potently reduced neuronal excitability, but did not alter the amplitude, duration, or undershoot of action potentials.

Conclusions: Our results suggest that FFA exerts an anticonvulsive effect on hippocampal pyramidal neurons by simultaneously decreasing glutamatergic excitatory synaptic activity and reducing neuronal excitability. Therefore, our study provides experimental evidence that FFA may represent an effective pharmacologic agent in the treatment of epilepsy in the mammalian central nervous system.

Epilepsy is a chronic brain disorder characterized by recurrent seizures that occurs with a prevalence of about 0.5% and a cumulative lifetime incidence of 3% (Shorvon, 1996; Jallon, 1997; Brodie & French, 2000). Although all patients with epilepsy experience seizures, not all individuals with seizures have epilepsy. The incidence of single acute symptomatic seizures tends to vary across studies, but is generally lower than that of epilepsy (Hauser & Beghi, 2008).

Epilepsy is the second leading neurologic disorder, exceeded only by stroke. Despite the availability of more than two dozen approved antiepileptic drugs (AEDs) and several nonpharmacologic options, up to 30% of patients are refractory to treatment (Shorvon, 1996; Bauer & Burr, 2001; Elger, 2003; Rogawski & Loscher, 2004). The therapeutic aim in many of these patients is achieved by combination therapy which, due to serious side effects and drug interactions, decreases quality of life (Tanaka, 1999). Therefore, the development of novel anticonvulsant therapies is necessary for this large population of treatment-resistant patients.

Previous studies have indicated that pretreatment with nonsteroidal antiinflammatory drugs (NSAIDs) attenuates convulsive activity in some animal models (Steinhauer & Hertting, 1981; Wallenstein & Mauss, 1984; Ikonomidou-Turski et al., 1988; Wallenstein, 1991). Although the anticonvulsive action of NSAIDs has been attributed to the modulation of brain cyclooxygenase (COX)/prostaglandin pathways (Ikonomidou-Turski et al., 1988; Wallenstein, 1991; Vezzani & Granata, 2005), NSAIDs exert a variety of other effects in the central nervous system and peripheral tissues that are distinct from the classical inhibition of inflammatory pathways. For example, fenamates, a family of NSAIDs,, regulate various ion channels (Lee & Wang, 1999; Partridge & Valenzuela, 2000; Lee et al., 2003) and N-methyl-d-aspartate (NMDA)–gated cation channels (Lerma & Martin del Rio, 1992), interact with γ-aminobutyric acid (GABA)A receptors (Maksay et al., 1998), and modulate gap junction activity (Srinivas & Spray, 2003). Therefore, the present study was designed to investigate the potential therapeutic role of the fenamate flufenamic acid (FFA) in epilepsy and to elucidate its mechanism of action. To address these questions, we used an electrophysiologic approach to evaluate epileptogenesis in an in vitro model based on hippocampal slices. Our study demonstrates that FFA exerts anticonvulsive effects by decreasing excitatory glutamatergic transmission combined with a reduction in neuronal excitability.

Methods

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

All experiments were performed in accordance with guidelines of the European Union (86/609/EEC) for the use of laboratory animals, and every effort was made to minimize the number of animals used.

Details for most of the procedures have been described previously (Martin & Buño, 2003). Briefly, transverse slices (400 μm) of the dorsal hippocampus from Wistar rats (13–17 days old) were prepared using conventional methods, and incubated (≈1 h at 20–22°C) in gassed (95% O2, 5% CO2 mixture) artificial cerebrospinal fluid (aCSF). The aCSF contained (in mm): NaCl 124, KCl 2.69, KH2PO4 1.25, MgSO4 2, NaHCO3 26, CaCl2 2, and glucose 10, with a pH of 7.4. Slices were transferred to an immersion recording chamber, and infused (1.5 ml/min) with equilibrated aCSF. Recordings from CA1 hippocampal pyramidal neurons were made using the whole-cell configuration of the “blind” patch-clamp technique, as described previously (Martin & Buño, 2003). Patch electrodes had a resistance of 4–6 MΩ when filled with the internal solution that contained (in mm): K-gluconate 97.5, KCl 32.5, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) 5, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) 10, MgCl2 1, and ATP 4, at a pH of 7.2–7.3, and osmolarities between 280 and 290 mOsm/L. Whole-cell recordings in the current- or voltage-clamp modes were performed with a 2400 patch amplifier (A-M Systems Inc., Carlsborg, WA, U.S.A.). Fast and slow capacitances were neutralized, and series resistance was always compensated (about 70%). Cells were used only when the series resistance (6–14 MΩ) did not change >20% throughout the experiment. The membrane potential (Vm) was held at −70 mV in voltage-clamp experiments. Data were filtered at 2 KHz and transferred to the hard disk of a Pentium-based computer using a DigiData 1440A interface and the pCLAMP 10.0 software (Molecular Devices, Union City, CA, U.S.A.). Synaptic responses were evoked by bipolar Schaffer collaterals stimulation, through a pair of Elgiloy electrodes (SSM33A05, WPI, Hertfordshire, United Kingdom) placed in the stratum radiatum near the border of CA1 pyramidal layer. Stimuli were single or paired pulses (50–100 ms delay) delivered at 0.033 Hz via a 2100 isolated pulse stimulator (A-M Systems); adjusted in intensity to evoke excitatory postsynaptic currents (EPSCs) in voltage-clamp experiments, or EPSPs in current-clamp experiments. Stimulation intensity was adjusted to evoke EPSCs or EPSP amplitudes that were the maximal responses. Extracellular population spikes were recorded with a glass microelectrode (impedances 2–3 MΩ; filled with 1 m NaCl) positioned in pyramidal layer area CA1. Evoked population spikes were elicited by Schaffer collaterals stimulation as described previously for the EPSC recording. The amplitude of the population spike was measured as follows: (1) a line was drawn at the base of the population spike connecting the first and the second peaks of the field response, (2) a second line was drawn at the peak of the downward deflection of the population spike, and (3) at the peak of the spike, a line was drawn vertically between these two lines, thus, giving the amplitude of the population spike. To analyze the effects of FFA on inhibitory synaptic transmission, pairs of stimuli with an interstimulus interval ranging from 15 to 200 ms were applied to the Schaffer collaterals, and evoked population responses were recorded in the CA1 cell layer (see Fig. 3A). Data were compared using the Student’s t-test and values are presented as the mean ± standard error of the mean (SEM). p-values less than 0.05 were considered to be statistically significant.

image

Figure 3.   Flufenamic acid (FFA) did not modified γ-aminobutyric acid (GABA)ergic inhibition in the hippocampus. (A) Representative population responses recorded in the CA1 cell layer in control condition (top) and during perfusion with 100 μm FFA (bottom) at different interstimulus intervals. (B) Paired-pulse depression of the population spike amplitude in control conditions (green-blue circle; n = 6) were not significantly different from the ratio of amplitudes in the presence of 100 μm FFA (red circle; n = 6) or 100 μm FFA plus 50 μm bicuculline plus 200 μm saclofen (green circle; n = 6).

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Drugs were stored as frozen concentrated stock solutions and dissolved in oxygenated aCSF at the desired concentration immediately before use. Bicuculline methochloride, saclofen, flurbiprofen, 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), and D (-)-2-amino-5-phosphonopentanoic acid (AP5) were purchased from Tocris Cookson (Bristol, United Kingdom). All other drugs were purchased from Sigma (St. Louis, MO, U.S.A.). FFA was prepared as a stock solution in dimethyl sulfoxide (DMSO) each day and diluted to the required concentrations in the extracellular solution. Epileptiform activity was induced with 100 μm 4-aminopyridine (4-AP) added to a modified Mg2+-free aCSF solution. The effectiveness of FFA in inhibiting epileptiform activity was estimated by constructing a dose–response relationship. The percentage of slices showing complete seizure cessation after the onset of the perfusion of FFA at different concentrations was averaged and plotted as a function of drug concentration (0.1–1mm). The plot was fitted to the formula:

  • image

derived from the Hill equation, where c is the half maximal inhibitory concentration (IC50) and b is the Hill coefficient.

Results

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

FFA suppresses epileptiform activity

After baseline recordings were obtained, slices were perfused with 100 μm 4-AP + Mg2+-free aCSF while the activity was continuously monitored under current-clamp conditions. Epileptiform activity was characterized in current-clamp recordings by spontaneous depolarizations and bursts with multiple action potentials (Fig. 1A), and appeared 673 ± 62 s (n = 14 slices) after the onset of infusion with 4-AP. The spontaneous bursts that occurred at a frequency of 0.35 ± 0.05 Hz had durations of 17.4 ± 4.1 s, with 23 ± 7 spikes/burst (mean spike amplitude during the burst was 58.1 ± 1.2 mV; n = 14). These characteristics are in agreement with the in vitro epileptiform discharges reported previously for CA1 pyramidal neurons of rat hippocampal slices (e.g., Jones & Heinemann, 1988; Perreault & Avoli, 1991; Martin et al., 2001; Martin & Pozo, 2004). The addition of FFA (100 μm) to the 4-AP + Mg2+-free aCSF gradually abolished epileptiform activity (1,920 ± 360 s after onset of FFA perfusion Fig. 1A) in all slices tested (n = 7). Moreover, the potentiation of the evoked EPSPs induced by prior application of 4-AP + Mg2+-free aCSF, was abolished by FFA (100 μm) perfusion (Fig. 1B; n = 7). We next estimated the efficacy of FFA at inhibiting epileptiform activity by constructing a dose–response relationship (Fig. 1C). The plot fit well (r2 = 0.99) to the formula derived from the Hill equation (see Methods), yielding an IC50 of 60.9 μm and a Hill coefficient of 7.7 (Fig. 1C; n = 7). These results demonstrate that FFA suppresses the epileptiform activity induced in vitro by 4-AP + Mg2+-free aCSF and abolishes the increase of evoked excitatory synaptic transmission, thereby suggesting the potential use of FFA as an anticonvulsive drug for the treatment of epilepsy.

image

Figure 1.   Flufenamic acid (FFA) blocks epileptiform activity. (A) Representative current clamp traces showing that the abnormal epileptiform activity induced by 4-aminopyridine (4-AP) + Mg2+-free challenge was suppressed by adding 100 μm FFA. Expanded traces showing CA1 discharges in hippocampal slices. (B) Superimposed excitatory postsynaptic potentials (EPSPs) evoked by Schaffer collateral-commissural stimulation in control, 4-AP + Mg2+-free aCSF, and 100 μm FFA + 4-AP in Mg2+-free aCSF. Note the abolition of evoked burst induced in the presence of 4-AP + Mg2+-free by FFA. (C) Normalized dose–response relationship of the effect of FFA (see Methods) on epileptiform activity induced by 4-AP + Mg2+-free solution (n = 7). Data were fitted to an equation derived from the Hill equation, yielding an IC50 = 60.9 μm. (D) Representative current traces showing CA1 activity in control, after epileptiform activity induced by 4-AP + Mg2+-free aCSF, and after addition of 1 μm flurbiprofen (FBPF) (n = 6).

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Because FFA is a nonsteroidal antiinflammatory compound, we next analyzed whether inhibition of COX pathways might underlie the effects of FFA on epileptiform activity. To test this possibility, we used flurbiprofen (FBPF), a potent inhibitor of COX (the IC50 values are 0.1 and 0.4 μm for inhibition of COX-1 and COX-2, respectively; Gierse et al., 1995). The addition of FBPF (1 μm) to the 4-AP + Mg2+-free aCSF did not modify the epileptiform activity in any of the slices tested (Fig. 1D; n = 6), suggesting that FFA suppresses epileptiform activity by COX-independent mechanisms.

FFA reduces EPSC at the postsynaptic level

To identify the mechanisms that mediate the antiepileptic effect of FFA, we first tested whether FFA could reduce synaptic excitation or increase inhibitory synaptic activity. Therefore, to investigate the regulation of excitatory synaptic transmission, we examined the effects of bath-applied FFA under more controlled experimental conditions. Perfusion with aCSF containing 100 μm FFA induced a significant decrease of EPSC amplitudes (49.5 ± 4.3% of controls) approximately 20 min after the onset of the experiment (Fig. 2A,B left; n = 7; p < 0.001). To determine whether the effects of FFA on synaptic transmission were at the presynaptic or postsynaptic level, we estimated changes in the paired-pulse facilitation (PPF), which is considered of presynaptic origin (Creager et al., 1980; Clark et al., 1994; Martin & Buño, 2003). Pairs of stimuli with an interstimulus interval of 100 ms were applied to the Schaffer collaterals, and evoked EPSCs were recorded in the CA1 pyramidal neurons. The PPF index measured ≈20 min after the onset of the experiment and was not significantly different (Fig. 2A,B right; 0.41 ± 0.12 in control sections and 0.45 ± 0.14 in those treated with FFA; n = 7). These results suggest that FFA acts postsynaptically to reduce EPSCs without modifying neurotransmitter release parameters.

image

Figure 2.   Flufenamic acid (FFA) reduced both the non–N-methyl-d-aspartate (NMDA) and NMDA excitatory postsynaptic current (EPSC) components through a postsynaptic mechanism. (A) Average EPSCs (n = 20) evoked by paired-pulse stimulation (100 ms delay) in control conditions and in the presence of FFA (100 μm). In right traces (scaled), EPSCs under FFA were rescaled so that the first EPSC had the same size in control and FFA conditions. (B) Summary data depicting mean EPSC peak amplitudes (left) and averaged paired-pulse facilitation (PPF) index [(R2–R1)/R1] (right) in control conditions and in the presence of 100 μm FFA (n = 7). Holding potential was −70 mV. Significant differences with respect to control were established at ***p < 0.001. (C) Summary data showing the time-course of FFA effects on normalized (to control values) mean EPSC amplitudes in the presence of either AP5 (red circle, n = 6), or CNQX in Mg2+-free artificial cerebrospinal fluid (aCSF) (orange circle, n = 6). Holding potential was −70 mV. Experiments were performed in the presence of 50 μm bicuculline to isolate EPSCs from γ-aminobutyric acid (GABA)A-mediated inhibitory synaptic transmission.

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FFA lowers both non-NMDA and NMDA-receptor–mediated EPSCs

The results described in the preceding text indicate that FFA reduced glutamatergic EPSCs at the postsynaptic level. We further tested whether non-NMDA and NMDA-receptor–mediated EPSC components (non-NMDAEPSC and NMDAEPSC, respectively) displayed different sensitivities to FFA. We first isolated pharmacologically the non-NMDAEPSC component with AP5 (50 μm) in normal aCSF and the NMDAEPSC component with CNQX (20 μm) in an Mg2+-free external solution (Hestrin et al., 1990). To isolate EPSCs from GABAA-mediated inhibitory synaptic transmission, these experiments were performed in the presence of 50 μm bicuculline. Perfusion with 100 μm FFA in the presence of AP5 (to isolate the non-NMDAEPSC) induced a progressive reduction (48.8 ± 13%; 35 min; n = 6; p < 0.001) of the EPSC amplitude that was fully reversed after washout (Fig. 2D, red circle). Similarly, when the NMDAEPSC was isolated by adding CNQX to the Mg2+-free aCSF, FFA (100 μm) also induced a progressive reduction (42.7 ± 12%; 35 min; n = 6; p < 0.001) of the NMDA-mediated EPSC component amplitude (Fig. 2D, orange circle). Therefore, FFA inhibited both the non-NMDA and NMDA-mediated glutamatergic excitatory synaptic transmission between CA3 and CA1 pyramidal neurons.

FFA does not modify GABAergic synaptic inhibition in hippocampus

To analyze the effects of FFA on inhibitory synaptic transmission, we studied paired-pulse depression (PPD) of the population spike, which is attributable in large part to feedback inhibition (Barnes, 1979; Adamec et al., 1981). For an interstimulus interval ranging from 15 to 200 ms, the ratio of the amplitudes of the second to the first population spikes (PS2/PS1) in control conditions were not significantly different from the ratio of amplitudes in the presence of 100 μm FFA (Fig. 3A,B, n = 6) or in the presence of 50 μm bicuculline (GABAA antagonist), 200 μm saclofen (GABAB antagonist), and 100 μm FFA (Fig. 3B, n = 6). These results suggest that the antiepileptic effects of FFA observed (Fig. 1A) are not due to alterations of GABAergic inhibition.

FFA reduces neuronal excitability without modifying action potentials

In addition to the effects of FFA on synaptic mechanisms, the disruption of the abnormal firing synchronization between pyramidal neurons induced by FFA could reflect a marked reduction in the excitability of these neurons. To address this issue, we compared the responses evoked by depolarizing pulses in control aCSF with those elicited by identical current pulses in buffer containing 100 μm FFA. The frequency of the action potentials evoked by the depolarizing pulse was significantly decreased by FFA (14.5 ± 0.78 Hz in control vs. 1.03 ± 0.14 Hz in FFA; n = 6; p < 0.001; Fig. 4A,B), whereas the voltage threshold needed to trigger action potentials was not modified. The effects of FFA were completely reversed after a washout in control aCSF (13.4 ± 0.93 Hz; n = 6; Fig. 4A). The significant decrease in the frequency of action potentials produced by FFA was maintained in the presence of AP5 (50 μm) or CNQX (20 μm), thereby confirming that FFA modifies neuronal excitability through glutamatergic receptor–independent mechanisms. In addition, bath application of FFA did not change the amplitude (88.7 ± 3.1 mV in control vs. 83.4 ± 1.9 mV in FFA; n = 6) or duration (3.6 ± 0.09 ms in control vs. 3.9 ± 0.1 mV in FFA; n = 6) of the first action potential evoked by pulse depolarization (Fig. 4C,D). These data are in agreement with previous studies in rat supraoptic neurons (Ghamari-Langroudi & Bourque, 2002). Therefore, these results show that repetitive action potential activity was markedly reduced by FFA, indicating that these NSAIDs diminish the excitability of CA1 pyramidal neurons. In addition, these data suggest that FFA may possibly affect neuronal intrinsic mechanisms.

image

Figure 4.   Flufenamic acid (FFA) reduces neuronal excitability. (A) Responses evoked by depolarizing current pulse in control artificial cerebrospinal fluid (aCSF), after 10 min in 100 μm FFA, and after washout. Note the reduction of neuronal excitability in the presence of FFA. (B) Summary data (n = 6) showing the action potential frequency in control conditions, after 20 min in the presence of FFA, FFA plus AP5 (50 μm), and FFA plus CNQX (20 μm). Significant differences with respect to control were established at ***p < 0.001. (C) Example of first action potential evoked by pulse depolarization under control conditions (left panel) and in the presence of 100 μm FFA (right panel). (D) Summary data (n = 6) showing mean values of action potential amplitude (left panel) and action potential duration (right panel) recorded in the absence (green-blue bar) and presence (green bar) of FFA (100 μm).

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Discussion

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Medication is effective at controlling seizures in only 70% of patients with epilepsy. Refractory epilepsy is associated with considerable medical, social, and psychiatric complications and enormous financial costs (Sander, 2003). Therefore, development of innovative and effective approaches is necessary for treating these patients. Consistent with this clinical goal, the identification of new AEDs with novel mechanisms of action would provide much needed tools to the field of epilepsy pharmacotherapy. Because previous reports have indicated that pretreatment with NSAIDs attenuates convulsive activity in some animal models (Steinhauer & Hertting, 1981; Wallenstein & Mauss, 1984; Ikonomidou-Turski et al., 1988; Wallenstein, 1991), we explored the potential antiepileptic effects of FFA, a member of the NSAID fenamate family. With the present study, we provide novel evidence that FFA suppresses abnormal epileptiform activity in the CA1 region by COX-independent mechanisms. This compound exerted inhibitory effects in the 4-AP model of epileptogenesis, where epileptiform activity is induced by cellular and network mechanisms that are consistent with a central origin of abnormal hyperexcitable states (Jones & Heinemann, 1988; Perreault & Avoli, 1991; Traub et al., 1996; Martin et al., 2001).

In addition to its antiinflammatory effects in the central nervous system (Chen et al., 1998), the pharmacologic effects of FFA involve a variety of mechanisms including modulation of Ca2+-permeable nonselective cationic channels and voltage-gated Na+ and K+ channels (Lee & Wang, 1999; Partridge & Valenzuela, 2000; Lee et al., 2003), inhibition of Ca2+-activated chloride currents (Kim et al., 2003), interactions with GABAA receptors (Maksay et al., 1998), inhibition of NMDA-gated cation channels (Lerma & Martin del Rio, 1992), and modulation of gap junction activity (Srinivas & Spray, 2003). Theoretically, FFA could reduce the abnormal network interactions by directly decreasing synaptic excitation or increasing inhibitory synaptic activity. We tested these possibilities and our results suggest that FFA reduces EPSCs at the postsynaptic level by inhibition of NMDA and non-NMDA receptors in hippocampal slices, an action that could explain the antiepileptic effects of the drug, since glutamatergic EPSCs are known to play a role in epilepsy (de Curtis & Avanzini, 2001; McCormick & Contreras, 2001). It is noteworthy that our studies demonstrate that FFA does not modify inhibitory GABAergic synaptic transmission, thereby excluding the participation of GABAergic inhibition in the antiepileptic effects of FFA.

Alterations of intrinsic neuronal properties may also contribute to in vitro models of epileptogenesis because they control cellular excitability (e.g., Traub & Jefferys, 1994; Martin et al., 2001; Yaari & Beck, 2002). We present evidence that FFA also decreases neuronal excitability in CA1 pyramidal neurons, and these data suggest that FFA may possibly affect neuronal intrinsic mechanisms. Previous studies have shown that FFA is a nonspecific drug, which blocks different currents and thereby modifies neuronal excitability, including Ca2+-activated nonselective cation current (CAN), and certain subtypes of K+ channels and Ca2+-activated chloride channels (Lee & Wang, 1999; Kim et al., 2003; Lee et al., 2003; Schiller, 2004). In addition, FFA has been reported to eliminate the CAN-mediated activity that follows paroxysmal depolarization shift (PDS) discharges and probably participates in sustained seizure-like events in neocortical slices (Schiller, 2004). However, the pharmacologic effects of FFA upon intrinsic neuronal properties are complex, and the inhibition of different currents could act synergistically to modulate neuronal excitability.

In summary, our data suggest that flufenamic acid exerts an anticonvulsant effect by a complementary mechanism that involves a reduction of glutamate EPSCs in the CA3–CA1 synapses and a decrease in neuronal excitability. An important therapeutic goal in the treatment in epilepsy is to completely control seizures without causing unacceptable side effects. Further experiments will be required to confirm the efficacy and safety of FFA. Nevertheless, the present results indicate that FFA may be a useful therapeutic agent for the treatment of human epilepsy.

Acknowledgments

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This work was supported, in part, by grants PAI07-0042-1097 from Consejería de Educación y Ciencia, JCCM; PI-2007/49 and G-2007_E/06 from FISCAM, and BFU2008-04196/BFI and Programa Ramón y Cajal from Ministerio de Ciencia e Innovación to E.D.M. M.F. is recipient of a Post-doctoral Fellowship from Fondo de Investigación Sanitaria. C.L-P. is recipient of a Pre-doctoral Fellowship from Consejería de Educación y Ciencia, JCCM. We thank Dr. D. Burks, and Dr. A. Araque for their valuable suggestions and comments on the manuscript.

We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Disclosure: The authors report no conflicts of interest.

References

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References