Address correspondence and reprint requests to Michael O. Poulter, Director Graduate Program in Neuroscience UWO, Molecular BrainResearch Group, Robarts Research Institute, Dept of Physiology & Pharmacology, Faculty of Medicine, University of Western Ontario, 100 Perth Drive, London Ontario, Canada N6A 5K8. E-mail: email@example.com
Here, we explore the mechanism of action of isoxylitone (ISOX), a molecule discovered in the plant Delphinium denudatum, which has been shown to have anticonvulsant properties. Patch-clamp electrophysiology assayed the activity of ISOX on voltage-gated sodium channels (VGSCs) in both cultured neurons and brain slices isolated from controls and rats with experimental epilepsy (kindling model). Quantitative transcription polymerase chain reaction (qRT-PCR) (QPCR) assessed brain-derived neurotrophic factor (BDNF) mRNA expression in kindled rats, and kindled rats treated with ISOX. ISOX suppressed sodium current (INa) showing an IC50 value of 185 nM in cultured neurons. ISOX significantly slowed the recovery from inactivation (ISOX τ = 18.7 ms; Control τ =9.4 ms; p < 0.001). ISOX also enhanced the development of inactivation by shifting the Boltzmann curve to more hyperpolarized potentials by −11.2 mV (p < 0.05). In naive and electrically kindled cortical neurons, the IC50 for sodium current block was identical to that found in cultured neurons. ISOX prevented kindled stage 5 seizures and decreased the enhanced BDNF mRNA expression that is normally associated with kindling (p <0.05). Overall, our data show that ISOX is a potent inhibitor of VGSCs that stabilizes steady-state inactivation while slowing recovery and enhancing inactivation development. Like many other sodium channel blocker anti-epileptic drugs, the suppression of BDNF mRNA expression that usually occurs with kindling is likely a secondary outcome that nevertheless would suppress epileptogenesis. These data show a new class of anti-seizure compound that inhibits sodium channel function and prevents the development of epileptic seizures.
Isoxylitones, isolated from Delphinium denudatum root prevent seizure induction. Activity was tested on voltage-gated sodium channels (VGSCs) in both cultured neurons and brain slices isolated from controls and rats with experimental epilepsy (kindling model). Isoxylitones enhanced sodium channel inactivation with an IC50 of about 200 nM. Our findings suggest isoxylitones as a potential novel anticonvulsant agent.
Epilepsy remains a major medical challenge. To date, it is not completely curable and more than 30% of patients worldwide are living with refractory epilepsy. Extracts of plants have been used for many years to treat a variety of neurological disorders. The roots of the plant named Delphinium denudatum (locally called Jadwar), which is indigenous to the western Himalayas and Kashmir, have been used to treat seizures by local folk medicine practitioners. Chemical isolation of a number of aqueous and organic extracts has been reported previously and these extracts were shown to contain compounds termed isoxylitones. We reported previously that the highly purified, novel anticonvulsant isoxylitone ([E/Z]-2-propanone-1,3,5,5-trimethyl2-cyclohexen-1-ylidine) suppressed c-Fos protein and mRNA expression in various brain regions following PTZ-induced kindling in mice (Simjee et al. 2012). Previous findings also showed that aqueous extracts of Delphinium denudatum inhibited sustained repetitive firing of hippocampal pyramidal neurons (Raza et al. 2002, 2003, 2004), suggesting that sodium channel inhibition may be the mode of action of these extracts. Sodium currents (INa) are vital for the initiation and propagation of neuronal cell firing and inhibition of their activity is an established anticonvulsant mechanism, having efficacy across a broad range of seizure types (Rogawski and Loscher 2004; White et al. 2007). Most antiepileptic drugs stabilize the inactivated state of sodium channels, thereby decreasing the ability of neurons to generate repetitive action potentials (Lampl et al. 1998; Uebachs et al. 2010). Therefore, we carried out both in vivo and in vitro studies to evaluate the effects of an E/Z isomeric mixture of isoxylitones (ISOX) on voltage-gated sodium channels to determine its mechanism of action.
We also assayed whether ISOX could reduce kindled seizures and attenuate kindling-induced increases in BDNF mRNA expression in rat because of the important role of BDNF in regulating hyperexcitability during epileptogenesis (Kokaia et al. 1995; Xu et al. 2004) as well as the induction of abnormal electrical activity and seizures in the brain (Dugich-Djordjevic et al. 1992; Mhyre and Applegate 2003; Scharfman 2005; Hidaka et al. 2011). Thus, the goal of this study was to investigate the hypothesis that ISOX reduces voltage-gated sodium channel function and to determine whether ISOX could prevent kindled seizures and the associated increase in BDNF expression.
All experimental protocols were approved by the Animal Care Committee of University of Western Ontario, Canada and were carried out in compliance with the guidelines of the Canadian Council on Animal Care (CCAC).
Cell culture for electrophysiology (in vitro) and qRT-PCR
Cryopreserved, ready to use primary neuronal cells (CryoCells, Rat Brain Cortex) were a generous gift of QBM Cell Science, Ottawa, Canada. Cells were cultured in 35 mm cell culture dishes coated with poly-d-Lysine. Cells were grown at 37°C in 5%/95% CO2/O2 in Neurobasal medium (Invitrogen Life Technologies, Burlington, ON, Canada) supplemented with 2% B27 supplement, 2 mM l-Glutamine (Invitrogen), and 100 U/mL Penicillin–Streptomycin (Invitrogen). Media were replenished twice per week. To increase cell survival, 5% fetal bovine serum was added to the media for the first 4 h.
Animal surgery for electrode implantation
Male Sprague–Dawley rats (Charles River, Montreal, QC, Canada) weighing 200–250 g at the time of surgery were used. The animals were housed individually in specialized cages with free access to food and water and 12:12 h light/ dark cycle was maintained. Details of the kindling procedure have been described elsewhere (Gavrilovici et al. 2006).
Electrical kindling and slice preparation for electrophysiology in vivo
The rats were allowed 10–12 days post-operative recovery before kindling stimulation. The electrical kindling studies were repeated twice, first for electrophysiological studies and second for qRT-PCR analysis of BDNF mRNA. For electrophysiology studies, the animals were divided into two groups, that is, sham (control) and kindled group.
For BDNF mRNA studies, the animals in the second set of experiments were divided into three groups, that is, the control group (n =5) received vehicle (Intralipid) only, a positive control ‘Kindled Group’ (n =6) in which the animals were injected with vehicle (i.p.) 30–40 min before kindling, and a third ‘Treated Group” (n =5) which received isoxylitones 30 mg/kg (i.p.) 30–40 min prior to giving electrical stimulation for kindling.
The after discharge threshold (ADT) was determined in each amygdala by delivering a 2–3 s 60-Hz sine wave stimulus of progressively increasing intensity (15, 25, 35, 50, 75, 100, 150, 200, 250, 350, and 500 μA) until an ADT was triggered (McIntyre and Plant 1993). During recording the rats were housed in a clear Plexiglas chamber and the electrodes were connected to a Physiodata Amplifier System (Grass Instruments, Astro-Med Inc., Warwick, RI, USA). The rats were stimulated once a day every day until generalized stage 5 convulsions on the Racine's scale were elicited in the controls. After three consecutive daily stage 5 seizures occurred, the animals were considered fully kindled.
Brain samples for BDNF qRT-PCR analysis were collected at the end of the second set of experiments. The rats were killed 2 weeks after the last stage 5 seizures, and brain samples were collected after perfusion as described above. The samples were flash frozen in pre-cooled isopentane and then stored at −80°C for further processing.
To study the Na+ channel inhibition and dose–response curve of ISOX in vitro, whole cell patch-clamp recordings were performed using an Axopatch-200B amplifier (Axon Instruments, Foster City, CA, USA). Cells were grown for 10–14 days before being placed on the stage of an inverted microscope (Olympus IX70; Olympus Canada Inc., Richmond Hill, ON, Canada). Online and offline acquisition and analysis were performed using the pClamp suites of programs (Molecular Devices Inc., Sunnyvale, CA, USA).
Patch pipettes had a resistance between 5 and 8 MΩ and were filled with CsCl pipette (internal) solution containing (in mM) CsCl 145, CaCl2 0.3, Na2-EDTA 3, HEPES 10, and Mg-ATP 1 (pH = 7.3). The external solution contained (in mM) NaCl 145, KCl 5, CaCl2 2, MgCl2 2, Na-HEPES 10, glucose 10. The osmolarity of all the solutions was kept in the range of 295–305 mOsm/kg. Currents were sampled at 10 KHz and filtered at 5 KHz for dose–response and all subsequent electrophysiology studies in vitro. The access resistance (Ra) ranged from 8 to 20 MΩ and recordings with Ra more than 20 MΩ were not used. Series resistance was compensated up to 80% using a lag of 100 μs. However, as clamping errors would be less when the sodium current is suppressed in the presence of the compound, the % block was probably underestimated. All recordings were performed at room temperature (23 ± 2°C).
The compound was prepared in the external solution which was constantly perfused through the culture dish. At least 5 min were allowed for equilibrium prior to recordings. All control measurements were performed in the same cell prior to the addition of the compound. The compound effects were calculated by normalizing the peak current amplitude during a brief depolarization in the presence of the compound with the largest inward current in the control response. The averaged data were curve fitted to the standard dose–response equation to determine the dose–response curve and IC50 of ISOX using Prism 5 (GraphPad Software Inc., La Jolla, CA, USA) in the cultured cortical neurons.
To study the recovery of sodium channels from the inactivated state, we blocked the calcium currents by using an external solution having the same composition as described above except that 2 mM CaCl2 was replaced by 0.5 mM NiCl2. A concentration of 200 nM isoxylitones (IC50 value in cultured neurons) was used in these experiments. Two-week-old cultured cells were patched in the voltage clamp mode as described above. The inactivation-recovery pulse protocol was used for recordings which consisted of step depolarization (3 ms) from −100 mV to −40 mV to obtain the maximum peak INa current (Ampo) and next another test depolarization step was done at intervals of 2, 4, 8, 16, 20, 30, 40, 50, and 110 ms after the initial step (Ampo). The fraction of INa recovered at various time points (Ampt) was calculated as the ratio of Ampt/Ampo before and after the compound application. This ratio was converted to percentage using the following formula:
The data were averaged and % recovery was plotted on the y-axis against the respective time on the x-axis and curve fitted with a single exponential equation as follows:
The fitting was done using the software package called Origin 6.0 (OriginLab, Northhampton, MA, USA).
To study the effect of ISOX on the development of inactivation, a voltage ramp protocol was used. Cells were initially held at −60 mV and then the holding potential was slowly changed by a series of voltage ramps (lasting 240 ms) ranging from −100 mV to −25 mV. At the end of each ramp, a test pulse of 3 ms to 0 mV was used to determine the amplitude of the sodium current that could be activated. The recordings were performed before (control) and after perfusing the cells with 200 nM ISOX. The peak inward sodium current (Po) was measured both for control and treated recordings, independently, and inward INa for each step (Pa) was normalized to the respective peak current amplitude Po, that is, control current amplitudes Pa were normalized to the respective Po for control, and treated currents Pa were normalized to the respective Po for treated cell recordings. The percent Pa/Po was calculated, averaged, and data were curve fitted to a Boltzmann equation showing the relationship between voltage (independent variable) and current (dependent variable) using the Microcal Origin 6.0 software as follows:
In this case;
Po = Initial amplitude of the sodium current
Pa = amplitude for each Vm
V50 = Center of the curve
k = slope of the fit
The current amplitude values Pa for each post-drug treated cell recording were also normalized to the respective pre-drug maximum peak current amplitude Po to show the % inhibition of INa compared to control, and the data were subjected to the same sigmoidal Boltzmann fit to observe the effects of isoxylitones on the development of inactivation.
Dose–response assays of isoxylitones in isolated brain slices prepared from sham and kindled rats were done in the same manner as in the isolated cultured neurons. For these electrophysiology studies, rats were deeply anaesthetized with Ketamine-domitor on the day of recording and then perfused through the heart with 60 mL of ice-cold artificial CSF (ACSF) solution containing (in mM): choline Cl, 110; NaH2PO4, 1.2; KCl, 2.5; NaHCO3, 25; MgCl2, 7; CaCl2, 0.5; Na pyruvate, 2.4; d-Glucose, 20; l-ascorbate, 1.3. After perfusion, the brain was rapidly removed and sliced coronally using a Vibratome (400-μm-thick sections). Slicing, incubation, and storage were all performed in the choline-ACSF solution. The external Ringer's solution used during electrical recordings was similar to the choline-ASCF solution except that ascorbate and pyruvate were removed, equimolar NaCl replaced the choline Cl, and MgSO4 was included at a concentration of 1 mM. These solutions were maintained at pH 7.4 and bubbled with 5% CO2 ⁄95% O2 (carbogen) at all times during the experiments. A more detailed description has been previously published (McIntyre et al. 2002).
Sodium channel α subunit mRNA analysis by qRT-PCR
qRT-PCR analysis for the expression and relative abundance of various Na+ channel alpha subunits was performed on mRNA from the cultured cortical neurons used for the electrophysiology studies in vitro. Four different batches of cultured dishes (and four dishes from each batch) were selected for RNA isolation and stored at −80°C. Trizol® (Invitrogen) was employed for RNA isolation, and the total RNA concentration was determined using a Nano-Drop Spectrophotometer ND-1000 (Nanodrop Technologies Inc., Wilmington, DE, USA). The Optical Density ratio 260/280 of all samples was greater than 1.9.
For cDNA synthesis, 2 μg RNA from each sample was subjected to reverse transcription (RT) using a Superscript II® RT kit from Invitrogen (Life Technologies, Burlington, ON, Canada). Negative control (No RT) was also prepared in which RNA was treated according to the same protocol with the addition of water instead of the RT enzyme.
For quantitative PCR, the primer sets were designed to produce 80–110 bp amplicons using the Primer-BLAST primer designing tool (NCBI). The gene-specific primers used were synthesized by Sigma-Genosys (Sigma Life Sciences, Oakville, ON, Canada). Only primers with efficiencies between 90 and 100% were used for quantitative analysis. The PCR mix contained MyiQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA), 20 ng of cDNA and 200 nM of each primer. cDNA was amplified as previously described (Anisman et al. 2008; Mylvaganam et al. 2010). To confirm that only one product had formed, a dissociation profile of the PCR products was obtained by a temperature gradient running from 60°C to 95°C. Data are expressed as the normalized cycle threshold (Ctn) of the target mRNA with respect to a reference gene (synaptophysin). Positive Ctn indicates higher abundance than the reference gene whereas negative Ctn indicates less abundance than the reference gene.
BDNF mRNA expression analysis by qRT-PCR
The cortical region of the brain was collected after the second set of electrical kindling experiments, and samples were processed for RNA isolation and qRT-PCR as described above but with some modifications. Following treatment with Trizol® (1 mL per 50–100 mg of tissue), RNA was isolated with RNeasy® Spin columns (Qiagen, Mississauga, ON, Canada) as specified by the manufacturer and included on-column DNase treatment using the RNase-Free DNase set® from Qiagen.
The isolated RNA (1 μg) was reverse transcribed using the protocol and reagents for SuperScript™ III RT (Life Technologies). A ‘no-RT control’ was also prepared as described above. Real-time PCR for total BDNF and individual BDNF transcripts was performed in the Stratagene MX3000P (La Jolla, CA, USA) using the DNA binding dye SYBR Green (Platinum SYBR Green qPCR SuperMix-UDG®; Invitrogen). The primers for total BDNF analysis were designed using Primer 3 software (NCBI) from the rat BDNF coding sequence (exon IX; Accession Numbers NM_012513.3). Similarly, for amplification of individual rat BDNF transcripts, the reverse primer (common for all the individual transcripts examined) from the BDNF coding region was used, and, for forward primers, the sequence was taken from the upstream corresponding exon of the transcript. The primers were checked for any cross-reactivity prior to use by using the BLAST tool of NCBI. Rat β-actin was used as a reference standard to normalize the BDNF values in all samples, as its levels do not change with seizures (Ullal et al. 2005).
All unknown samples, ‘no RT’, and ‘no template’ controls (the latter with water instead of cDNA to control for reagent contamination) were run in triplicate. Standards were also run in triplicate with six 10-fold dilutions, that is, starting from 1 pg to 1 ag. For analysis, only experiments with R2 > 0.990 and a PCR efficiency between 90 and 100% were used. The following thermal profile was used for BDNF and β-actin: 2 min at 50°C, 2 min at 95°C followed by 40 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 45 s. A dissociation curve after PCR was added to verify that no secondary products had formed. Results were expressed as copies/50 ng of total RNA.
Copy number of each sample was calculated according to the standard curve using Stratagene Mx3000p software.
BDNF mRNA analyses were done by one-way anova with post hoc Tukey tests using SPSS version 15 (SPSS Inc., Chicago, IL, USA). Differences among the means were significant at the level of p <0.05. All data were expressed as mean ± SEM.
All procedures in this manuscript conform to the ARRIVE guidelines.
Sodium channel inhibition and dose–response to Isoxylitones in vitro
Previous studies have shown that a fraction of the plant extract (isolated from Delphinium denudatum) reduced the number of action potentials that occurred in response to a current pulse (Raza et al. 2004). These previous data suggested that the purified compound (isoxylitone; ISOX) used here may suppress activity by reducing the magnitude of voltage-gated sodium currents (VGSCs). To test this, experiments were conducted in vitro on cultured rat cortical neurons that were patch clamped in voltage clamp mode at a holding potential of −60 mV and depolarized by steps of 10 mV (see 'Methods' for a complete description). Figure 1a shows that 100 nM ISOX suppressed the inward sodium current by about 50%. To better define the IC50 of ISOX we constructed a dose-inhibition curve ranging from 1 nM to 100 μM ISOX. This was done by perfusing the dish for 10 min and then assaying the magnitude of the sodium current because of a single step from −60 mV to 0 mV. In Fig. 1b, we show representative traces of the inhibition of the sodium currents at 10 nM and 1 μM where ISOX suppressed the VGSC by 36 ± 1% and 59.3 ± 1.5%, respectively. At the highest concentration used (100 μM), the current was suppressed by 73 ± 5.6% (see Fig. 1b right panel, c). Partial reversibility of the INa amplitude was also observed both at high and low concentrations of ISOX used after washout with the external solution (Fig. 1b, light gray traces). Fitting the curve to a standard dose-inhibition curve showed the calculated IC50 to be 185 nM (Fig. 1c).
ISOX effects on sodium channel inactivation
To assess the effects of the ISOX on sodium channel recovery from inactivation, we used the IC50 concentration of ISOX in cultured neurons. In Fig. 2a and b, we show the representative traces of the sodium current activated again by a voltage step from −60 to 0 mV. At various times (ranging from 2 to 110 ms as described in the 'Methods'), we again stepped the voltage to 0 mV to assay the rate of recovery from inactivation. As can been seen in Fig. 2, ISOX significantly slowed the recovery from activation. While control responses were almost completely recovered by 40 ms, the recovery was only about 70% complete in the presence of 200 nM ISOX at 40 ms. A plot of the percent recovery versus the time of the test pulse from 14 separate recordings is shown in Fig. 2c. A single exponential fit was sufficient to describe the rate of recovery before and after the application of ISOX. We found that ISOX slowed the rate of recovery by about 100% (Control 9.4 vs. Treated 18.7 ms; p <0.001).
The fact that ISOX slowed inactivation raised the possibility that it may reduce sodium current activity by enhancing inactivation at higher membrane potentials (essentially creating a steady-state inactivation at resting membrane potential). To test this possibility and see if ISOX alters the entry into the inactivated state, we used a series of voltage ramps (see inset in Fig. 3a) that induced inactivation and then gave a test pulse to 0 mV. In Fig. 3, we show representative recordings from the same cell before and after perfusion with 200 nM ISOX. First, it is clear that no level of hyperpolarization could prevent the ISOX from reducing the sodium current. So, the tonic block is not voltage dependent. However, we found when we scaled the fitted Boltzman curve for the treated recordings that there was about a −10 mV shift in the voltage dependence for the development of inactivation. The voltage required for 50% development of inactivation (V50) for post-drug treatment recordings was calculated to be −59.2 ± 0.5 mV (Fig. 3b) as compared to the control recordings where V50 was −48.0 ± 0.5 mV (n =13, Fig. 3b). Thus, ISOX does enhance entry into the inactivated state once the channels are activated. These findings demonstrate that ISOX significantly shifted the steady-state inactivation of sodium channels by −11.15 mV (p < 0.05). Thus, ISOX produces block at steady state but also enhances channel inactivation by shifting the voltage dependence to more hyperpolarized potentials.
Sodium channel α subunit mRNA analysis by qRT-PCR
To determine which type of VGSCs in cultured cortical neurons were inhibited by the ISOX, we performed qRT-PCR analysis of the mRNA extracted from cells from which the recordings were made. We assayed five differing α subunit isoforms that are known to be expressed in mouse brain; Nav1.1, Nav1.2, Nav1.3, Nav1.6, and Nav1.7. Figure 4 shows the expression levels of these sodium channel subtypes relative to a common neuronal reference gene (synaptophysin). Sodium channel alpha subunit 1.3 (Nav1.3) mRNA was the most abundant, being expressed is, about 12-fold higher than the reference gene (Ctn = +3.7 ± 0.6). The next most abundant isoforms present in these cells were 1.7 and 1.6 subtypes (Nav1.7 and Nav1.6) which were about half as abundant as NaV 1.3 (Ctn values 2.2 ± 0.4 and 1.8 ± 0.4, respectively). NaV 1.2 and Nav1.1, which are abundant in adult brain (Catterall et al. 2005), were expressed in very low levels in comparison to the other subtypes and would, therefore, not significantly contribute to the overall sodium currents found in these cells. The order of abundance was 1.3 > 1.7 = 1.6 >> 1.2 >> 1.1.
Effect of ISOX sodium channel activity and BDNF mRNA expression after kindling-induced seizures
To see if ISOX could prevent the development of kindled seizures, we treated rats with ISOX at a dosage of 30 mg/kg (i.p.) 30–40 min prior to stimulations. This dose produced no obvious behavioral changes (ataxia, lethargy, etc.) in the rats. The control animals were injected with vehicle (intralipid) 30–40 min before they were stimulated (see 'Methods' for details). In all the animals, the stimulus was increased until an after discharge was produced. On the first day of stimulation the after discharge threshold was significantly higher for the treated animals (Control 122 ± 54 vs. Treated 275 ± 27 μA p <0.05) and remained so for the entire procedure. For the untreated rats (n = 5), stage 5 seizures were induced after 11–14 daily stimulations. After 14 stimulations (done in parallel), no treated rats (n = 5) had stage 5 seizures.
We found that four out of five rats were experiencing stage 3 seizures, while one rat was at stage 4
Furthermore, all the treated rats were injected and stimulated for another three days along with controls who had already produced stage 5 seizures (this is standard to confirm that the response to the stimulation is stable). The further three stimulations did not progress any of the ISOX treated rats to higher levels of seizure activity. As these rats were needed for electrophysiological and/or molecular analyses, the kindling procedure was terminated and both groups were killed. Thus, ISOX is able to prevent the induction of stage 5 seizures by stimulations that produced seizures in untreated rats.
Next, we wished to see if ISOX had similar effects on sodium channel activity in adult brain slices and to determine if their activity was significantly altered by kindling-induced seizures. The reasons for these experiments were twofold. First, as adult brain expresses differing sodium channel α subunits, we wanted to determine if potency was different. Second, as kindling has been shown to induce drug resistance to some sodium channel blockers (Loscher and Schmidt 2006), we wished to determine if ISOX maintained its activity after the induction of kindled seizures. The effects of ISOX on INa inhibition in individual neurons were studied using the voltage clamp protocol in both control and kindled rat brain slices in piriform cortex (a region that is well known to be highly epileptogenic, see review (Loscher and Ebert 1996). Patch clamp recordings were performed in an identical manner as in cultured neurons. We also included 0.2% biocytin in the patch electrode to be able to determine the location of patched neurons and reconstruct the morphology of the cells after recordings were completed. We observed that ISOX inhibited INa both in pyramidal cells and in interneurons. ISOX exhibited concentration-dependent suppression of INa that was not statistically different from cultured neurons (Fig. 5). The fitted IC50 was 185 nM (as compared to 180 nM in cultured cells). After kindling, we found that the IC50 was 200 nM. Thus, there was no difference in the IC50 after the induction of seizures.
It is well documented that seizures increase BDNF mRNA expression and that BDNF expression enhances kindling development (Dugich-Djordjevic et al. 1992; Mhyre and Applegate 2003; Scharfman 2005; Xu et al. 2011). Thus, it was of interest to see if ISOX treatment reduced BDNF expression. These data would indicate whether ISOX blocks epileptogenesis independent of an increase in BDNF expression or whether the reduced epileptogenesis also decreased BDNF expression, perhaps compounding the anti-seizure activity of ISOX. To do this, we isolated total cellular RNA from the piriform cortex of sham (n =5), kindled (n =6) and ISOX treated (n =5) rats. We found a significant difference in total BDNF levels between the three groups, (F =5.82, p <0.02). A post hoc Tukey's test showed that kindled animals exhibited higher total BDNF levels than controls (p < 0.03), whereas BDNF levels in the treated animals were not different from controls (p =0.996). To further explore the changes in BDNF expression, we also assayed individual BDNF transcripts (III, IV, and VI). In Fig. 6, we show that all transcripts were significantly increased in kindled untreated groups, but these levels were normalized by ISOX treatment. Therefore, ISOX treatment is associated with normal levels of BDNF expression.
ISOX has been previously reported to have anti-epileptogenic characteristics in a pentylenetetrazole-induced kindling model (Simjee et al. 2012), while the aqueous extract of Delphinium denudatum – from which ISOX is isolated – was previously shown to inhibit sustained repetitive firing of sodium action potentials in cultured hippocampal pyramidal neurons (Raza et al. 2003). In an effort to understand the mechanism of ISOX activity and to determine if it may work in another model of epilepsy, we have conducted electrophysiological studies on the effects of ISOX on VGSCs and determined its ability to prevent electrically kindled seizures in rats. We have shown that ISOX inhibits VGSC channel function with an IC50 in all the preparations used here of about 200 nM. It appears to have two actions on VGSCs. The first action is that it produces a tonic block that is not altered by voltage. The second activity is that it alters the inactivation kinetics of the VGSCs by slowing recovery from inactivation and causing a hyperpolarizing shift in the voltage dependence of the entry into the inactivated state. Thus, ISOX does not appear to simply act as a compound that shifts the inactivation producing tonic block, as hyperpolarizing the cells did not reverse the attenuation of the VGSC function. We also found that ISOX treatment of rats prevented both the induction of generalized tonic clonic (stage 5) seizures, within the number of stimulations done here, and a reduction in the after discharge threshold. We also found that the IC50 was not different in any of the neuronal preparations used (cultured, sham, and kindled). So, while sodium channel subunit expression varies widely between these preparations, there is no evidence that ISOX is selective for one subtype of VGSC over another. However, this would need to be confirmed by the construction of dose-inhibition curves using recombinant VGSCs. Overall, our data show that ISOX is a potent VGSC modulator that is effective in preventing kindling-induced seizures.
Many antiepileptic drugs (AEDs) exert their effects by impairing the permeation of Na+ through VGSCs, including local anesthetics, class I anti-arrhythmic agents, and some anticonvulsants (Cosford et al. 2002; Liu et al. 2003; Rogawski and Loscher 2004; Fukuda et al. 2011). Many antiepileptic drugs have been developed in the past two decades that modulate the activity of these channels (Rogawski and Loscher 2004), as there is growing evidence that suggests the involvement of abnormal VGSC activity in the pathophysiology of both familial and acquired epilepsy (Meldrum and Rogawski 2007; Zuliani et al. 2012). Here, we have a new class of VSCG modifier, isoxylitones, that blocks sodium current (INa) in a concentration-dependent manner with an IC50 of 185 nM. However, we could only partially reverse the effects of ISOX within the time frame that the patch recordings could be maintained. This perhaps is not surprising, as the IC50 values of many sodium channel modifiers have been shown to linearly correlate with their lipophilicity (i.e., the more lipophilic the drug is, the more it blocks the INa (Lenkey et al. 2011; Desaphy et al. 2012). Thus, the lipophilicity of ISOX may be one of the underlying factors associated with its small IC50 value and with the partial/slow reversibility of INa. This strongly suggests that the site of action on the sodium channel is within the lipid membrane and that, once at its site of action, this would impede a complete washout from this site. Moreover, ISOX is more potent than other VGSC blockers and AEDs including phenytoin, carbamazepine, oxcarbazepine, and lamotrigine which have IC50 values in the range of micromoles (Lees and Leach 1993; Huang et al. 2008; Sheets et al. 2008; Fantini et al. 2009). It will be important to determine in future studies whether this higher potency will result in fewer off site target activities which may cause unwanted side effects. It will also be interesting to directly compare ISOX with conventional sodium channel AEDs.
Genetic diseases altering VGSC inactivation may result in epileptic seizures because of a so-called window current during which VGSCs are tonically active, causing nerve cells to become hyper-excited. Carbamazepine has been shown to stabilize the inactivated state of VGSCs which is associated with its anti-epileptic activity (Willow et al. 1985). ISOX may work in a similar manner in this regard since it significantly delayed the recovery from inactivation by almost twofold. Our results also suggest that ISOX preferably binds to and promotes the inactivated state, hence reducing neuronal hyper-excitability. These characteristics are also similar to carbamazepine, phenytoin, and lamotrigine (Czapinski et al. 2005). We also demonstrated that ISOX significantly shifted the steady-state inactivation curve to more hyperpolarized potentials, showing an enhancement of the steady-state inactivation. Thus, the suppression of INa amplitude at −60 mV may in part be because of this enhancement of the number of channels that are in the inactivated state. However, this steady-state attenuation of activity was not reversed by hyperpolarization of the cells. Thus, shifting of the voltage dependence of inactivation is not the only mechanism of action. It seems that ISOX is able to prevent gating of the channel by an allosteric interaction, that is still not clear at this time. It remains to be determined whether the combination of these two attributes is an important determinant in the potential therapeutic use of ISOX to treat epilepsies.
Most sodium channels have specific developmental, tissue, or cellular distributions (Catterall et al. 2005). To determine which subtypes of VGSCs were present in cultured (immature) cortical neurons, we compared the mRNA distribution of various sodium channel alpha subunits known to be expressed in rat cortical regions and capable of supporting INa (i.e., Nav1.1, Nav1.2, Nav1.3, Nav1.6, and Nav1.7). Our analysis revealed that Nav1.3 and 1.6 displayed the highest level of expression, whereas mRNA for Nav1.1 (predominant adult subtype) was the least expressed. In adult brain where α subunit expression is substantially different (e.g., low Nav 1.3 and high Nav 1.2), the potency was unchanged. Thus, ISOX does not appear to have any subunit specificity, but this would need to be confirmed using recombinant receptors. It is largely unknown whether subunit selective AEDs directed against differing VGSC subtypes may increase their therapeutic efficacy. Thus, it is difficult to determine whether the apparent lack of selectivity is clinically advantageous. However, it has been known for some time that some sodium channel AEDs are more effective against genetic epilepsy involving mutations of Nav1.1 (SCN1A (Thompson et al. 2011; Zhou et al. 2012), raising the possibility that drug efficacy may vary with the kind of mutation and the functional change (gain or loss) that occurs. In this regard, it would be important to screen ISOX against the various SCN1A mutations that have been identified in human epilepsy to determine if it is effective in attenuating neuronal activity, perhaps even restoring function to a more ‘normal’ range.
Seizure-induced plasticity has been reported to have an enormous impact on regional excitability in brain by causing changes in neurochemistry or cellular morphology (Causing et al. 1997; Verpelli et al. 2010). In particular, it is well documented that BDNF is both regulated by and a regulator of epileptogenesis, being up-regulated in various regions of rodent brain following seizure activity (Ernfors et al. 1991; Bengzon et al. 1993, 1997; Katoh-Semba et al. 1999; Wang et al. 2012). However, many AEDs like phenytoin, valproate, topiramate, lamotrigine, and phenobarbital have been reported to reduce BDNF mRNA levels in various regions of rat brain (Bittigau et al. 2002; Shi et al. 2010). Here, although we compared rats at differing stages of seizures, we have found that ISOX treatment was associated with reduced BDNF expression. Thus, the fact that this occurs across many differing kinds of AEDs makes it highly unlikely that these compounds are directly affecting the transcription of the BDNF gene. It is more likely that decreased spontaneous firing, reduced excitability and decreased activation of neuronal circuits trigger events that reduce the expression of BDNF (Akiyama et al. 1989; McIntyre and Plant 1989, 1993; Loscher and Ebert 1996). Thus, the high electrical activity which triggers BDNF expression may exacerbate the epileptic state but AEDs, which reduce activity, may have the added benefit of reducing BDNF expression. This certainly seems to be the case here as ISOX, which prevented seizures in the kindling model, also reduced BDNF levels as well. So, in common with most AEDs, ISOX may reduce seizures through this indirect effect of reducing excitability.
In conclusion, this study showed that isoxylitones inhibited voltage-gated sodium channels in a concentration-dependent manner. Isoxylitones stabilized sodium channel steady-state inactivation by shifting the voltage dependence to more hyperpolarized potentials as well as by slowing recovery from the inactivated state. Although it is important to note that sodium channel blockers are not considered to be anti-epileptogenic, our observation that ISOX could prevent seizure development, despite the induction of an after discharge during kindling, indicates a novel property of this compound. What aspect of the drug mechanism may confer this property is unknown, but we suggest that the tonic blockade of the sodium current as well as the use-dependence working in concert may be important. However, this mechanism may only partially account for its antiepileptic effects since there was a concomitant suppression of BDNF mRNA expression following kindling, suggesting that it may normalize some of the underlying molecular changes that are induced during seizures. We should also note that our data do not speak to the possibility that ISOX influences VGSC activation, but this should be explored in the future. As well it will be important to see how effective this compound will be in an epilepsy model that produces spontaneous seizures (e.g., pilocarpine model). Nevertheless, thus far our data indicate ISOX as a potential candidate for development of a new anti-epileptic drug.
This study was supported by Higher Education Commission (HEC) of Pakistan through International Research Support Initiative Program (IRSIP), and by operating grants from the Canadian Institutes of Health Research to MOP and MF. The authors declare no conflict of interest exists in this report.
Statement of contribution
MIC, AR, FS Synthesized and performed structure characterization of (E/Z) isoxylitones, SUS helped design experiments. MNA, CG, SUAS performed experiments; MNA co-wrote manuscript. MF helped design experiments and contributed to manuscript writing. MOP designed experiments and co- wrote manuscript.