PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Epilepsia. Author manuscript; available in PMC 2013 February 9.
Published in final edited form as:
PMCID: PMC3568386
NIHMSID: NIHMS438770

Low Frequency Stimulation of Hippocampal Commissures Reduces Seizures in Chronic Rat Model of Temporal Lobe Epilepsy

SUMMARY

Purpose

To investigate the effects of low frequency stimulation (LFS) of a fiber track for the suppression of spontaneous seizures described by Nissinen in a rat model of human temporal lobe epilepsy.

Methods

Stimulation electrodes were implanted into the ventral hippocampal commissure (VHC) in a rat post-status epilepticus (SE) model of human temporal lobe epilepsy (n = 7). Two recordings electrodes were placed in the CA3 regions bilaterally and neural data was recorded for a minimum of six weeks. LFS (60 minute train of 1Hz biphasic square wave pulses, each 0.1ms in duration and 200μA in amplitude, followed by 15 minutes of rest) was applied to the VHC for, two weeks, 24 hours a day.

Key Findings

The baseline mean seizure frequency of the study animals was 3.7 seizures per day. The seizures were significantly reduced by the application of LFS in every animal (n=7). By the end of the two-week period of stimulation, there was a significant 90% (<1 seizure/day) reduction of seizure frequencies (p < 0.05) and a 57% reduction during the period following LFS (p < 0.05) when compared to baseline. LFS also resulted in a significant reduction of hippocampal interictal spike frequency (71%, p < 0.05), during two weeks LFS session. The hippocampal histological analysis showed no significant difference between rats that received LFS and SE-induction and those that had only received SE-induction. None of the animals showed any symptomatic hemorrhage, infection or complication.

Significance

LFS applied at a frequency of 1Hz significantly reduced both the excitability of the neural tissue as well as the seizure frequency in a rat model of human temporal lobe epilepsy. The results support the hypothesis that LFS of fiber tracts can be an effective method for the suppression of spontaneous seizures in a temporal lobe model of epilepsy in rats and could be lead to the development of the new therapeutic modality for human patients with temporal lobe epilepsy.

Keywords: DBS, LFS, VHC, TLE, temporal lobe epilepsy, brain stimulation, amygdala

INTRODUCTION

Temporal lobe epilepsy (TLE) is the most common form of partial intractable epilepsy, as well as the most refractory. Only 11% to 25% of patients suffering from TLE are seizure free with current antiepileptic drug (AED) treatments(Zumsteg et al. 2006). The alternative for these patients is surgical intervention. However, studies have shown that only 65% to 70% of selected TLE patients that undergo appropriate surgical resection of the epileptic focus become seizure free and usually require additional aid of an AED treatment (Zumsteg et al. 2006). However, many patients diagnosed with bilateral or multiple epileptic foci are generally not considered candidates for surgery (Mogilner and Rezai 2003; Concha et al. 2006; Zumsteg et al. 2006). Temporal lobectomy, is a therapeutically effective treatment option for TLE, but it is an invasive and irreversible procedure that can have detrimental side effects, such as memory loss, speech deficits, hemiparesis, dysphasia, and hemianopsia (Spuck et al. 2010). Deep brain stimulation (DBS) is a promising alternative, which is already FDA approved for the treatment of Parkinson’s disease (2001) essential tremor (Benabid et al. 1996), and dystonia (Vidailhet et al. 2005), while currently being investigated for the treatment of depression (Holtzheimer and Mayberg 2010), obsessive-compulsive disorder (Gabriels et al. 2003), and epilepsy (Hodaie et al. 2002). Clinical trials using DBS for epilepsy treatment has shown varied success, with some trials showing reduction, but usually not elimination of clinical symptoms (Velasco et al. 1995; Velasco et al. 2001; Velasco et al. 2001; Hodaie et al. 2002; Nail-Boucherie et al. 2002). A trial of bilateral stimulation of the anterior nuclei of the thalamus showed a 56% median reduction in seizure frequency (Fisher et al. 2010). Results of a pivotal investigation for treatment of intractable partial epilepsy (Heck 2010) showed that a median % seizure reduction of 37.3% in 140 subjects 2 - 2.5 yrs post implant period.DBS is a flexible paradigm, and it has been applied with a range of frequencies to a number of targets (subthalamic nucleus, anterior nucleus of the thalamus, cerebellum, caudate nucleus, and hippocampus) with varied success. Optimization of the treatment paradigm is difficult because the therapeutic mechanisms of DBS are unclear (Dostrovsky and Lozano 2002; Vitek 2002; McIntyre et al. 2004). Although high frequency stimulation is preferentially used in DBS therapies, low frequency stimulation (LFS) may be advantageous since it inherently requires less current and thus could potentially cause less tissue or electrode damage. Additionally, previous experimental results using LFS have shown antiepileptic effects in clinical studies(Jerger and Schiff 1995; Albensi et al. 2004; Morrell 2009) and in animal studies (Velisek et al. 2002; Goodman et al. 2005; Schrader et al. 2006). However, the stimulation frequency and target capable of producing complete or near-complete suppression of seizure has not yet been found.

Many stimulation targets have been tried but the hippocampus in particular is an obvious option for TLE. The hippocampus is actively involved in seizures among a substantial proportion of the epilepsy population and has the lowest seizure threshold of any brain region (Durand and Bikson 2001; Theodore and Fisher 2004; Morrell 2006; Parrent and Almeida 2006). However, direct stimulation of the focus has been tested before with limited success (Jobst et al. 2010). One possibility to explain the lack of success is the fact that the seizures occur in large areas and cannot be suppressed before they propagate. Therefore, a potential target of stimulation would be a fiber tract that is connected to a large part of the hippocampus. For the treatment of seizures in the rat hippocampus, an ideal target would be the ventral commissural (VHC) track which connects both hippocampi and does work as a functional pathway for seizure propagation. These white matter fibers innervate a large portion of the bilateral hippocampi and, thus, stimulating them could provide a significant therapeutic effect. Previous work from our laboratory using LFS of the VHC (Kile et al. 2010) in a genetic mouse model showed some suppression of seizure frequency; however baseline seizure frequency was unrealistically high (14 seizures/hour).

Therefore, in this study, we used a drug-free spontaneous seizure rat model that more closely mimics human TLE (Nissinen et al. 2000) to test the hypothesis that LFS of the VHC can reduce seizure frequency and tissue excitability. In addition to monitoring seizures, we also quantified changes in interictal spikes during LFS. Interictal spike activity is routinely recorded clinically for localizing the epileptogenic area during intracortical EEG monitoring. Interictal and ictal discharges in animal models of epileptiform activity consist of similar neuronal depolarization, leading to sustained action potential firing(Dichter and Ayala 1987; Jefferys 1990; de Curtis and Avanzini 2001; Avoli et al. 2002), suggesting that interictal and ictal events may reflect synchronous discharge of an underlying neuronal population. A similar relationship was also suggested by a human study demonstrating a high correlation with seizure onset zone without any correlation with histopathology (Hufnagel et al. 2000). Therefore, we also analyzed the effect of LFS on interictal spike activity. The results indicate that LFS of the VHC can generate a significant decrease in the frequency of both interictal and ictal activity.

METHODS AND MATERIALS

2.1 Animals

Adult male Harlan Sprague–Dawley rats (320–390g) were used in this study. 33 rats developed SE during amygdala stimulation, 10 rats were euthanized within 24-48 hours of SE-induction due to severe violent continuous seizures. Also 10 rats were excluded due to irregular and unstable seizure development (<1seizure/day), as well as 6 rats were excluded due to unexpected head cap loss, leaving 7 rats for the study. All animal procedures were conducted in accordance with the guidelines, reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Case Western Reserve University.

2.2 Electrode implantation

Animals were anesthetized using an intraperitoneal injection of a rat cocktail (ketamine 80mg/kg & xylazine 10mg/kg). A total of four depth electrodes were implanted into the brain using stereotactic coordinates (see Figure 1). For amygdala stimulation, a stainless steel bipolar electrode (diameter 0.125mm, dorso-ventral distance between the tips 0.4mm; Plastics One Inc., Roanoke, VA) was implanted into the lateral nucleus of the left amygdala (3.6mm posterior to bregma, 5.0mm lateral to bregma and 6.5mm ventral to the surface of the brain)(Paxinos 2007). Another bipolar electrode was implanted in the VHC (bregma −1.30mm, lateral 0.1mm, depth 3.5mm) for LFS stimulation. To record the spread of epileptiform activity from the ipsilateral hippocampus to the contralateral hippocampus, two stainless steel monopolar electrodes (diameter 0.25mm, Plastics One, Inc., 29±1K Ohm at 1KHz, Protek z580 meter) were implanted into the CA3 regions of the bilateral hippocampi (bregma −4.0mm, lateral ± 3.0mm, depth 3.4mm) since the CA3 regions is known to be epileptogenic.

Figure 1
Schematic (coronal view) illustrating target of stimulation and recording electrodes. Bipolar stimulation electrodes were implanted into the lateral nucleus of the left amygdala, and the ventral hippocampal commissures. Monopolar recording electrodes ...

Two monopolar stainless steel screw-electrodes, which were fixed to the skull symmetrically over the cerebellum, served as ground and reference electrodes. All electrode leads were connected to a pedestal that was fixed to the screws and the skull with special dental cement Geristore (DenMat, LLC).

2.3 Induction of Status epilepticus (SE)

Status epilepticus was induced by stimulating the lateral nucleus of the left amygdala 1 to 2 weeks after the electrode implantation according to the protocol provided by Nissinen (Nissinen et al. 2000; Nissinen and Pitkanen 2007). The stimulation consisted of a 100ms train of 1ms biphasic square wave pulses (400μA peak-to-peak) that were delivered at 60Hz every 0.5s using a DS8000 Digital Stimulator (World Precision Instruments) connected to a DLS100 Stimulus Isolator (World Precision Instruments). Rats were connected to the system via a 6-channel commutator (Plastics One Inc.) and a 6-channel shielded cable. For induction of status epilepticus (SE), a 2-channel shielded cable was temporarily added. The development and duration of SE in freely moving animals was monitored continuously for 48hrs via the two hippocampal electrodes using an M-series NI-6221 device (National Instruments Inc.), custom-developed LABVIEW® software and P511 Amplifiers (Grass Technologies).

Each rat was stimulated continuously for 20 minutes. The stimulation period was extended if status epilepticus was not reached, however the stimulation period never exceeded 40 minutes. The stimulation time varied as follows: 20 min (n=3), 30 min (n=2) and 40 min (n=2). The first SE episode was observed within 5 min after the cessation of amygdala stimulation and the mean duration of the SE-Like Episodes (SE-LE) were 12±6 min, mean number was 15±6. Rats showed frequent SE-LE episodes and inter-ictal spikes up to 8±2 hours after the beginning of SE. There was no difference in the number of SE-LEs between the animals that developed spontaneous seizures.

2.4 Video-EEG recording and analysis

Depth intracortical EEG was recorded from the both CA3 regions of the bilateral hippocampi using a National Instruments M-series NI-6221 device (sampling rate 2000Hz, high-pass filter 0.3Hz, and low-pass filter 300Hz), custom-developed LABVIEW software and P511 Amplifiers (Grass Technologies, RI). The animals were connected to the system with a 6-channel commutator and shielded cables, allowing the animals to move freely without twisting the cables. The behavior of the animals was recorded using Webcam Live Ultra (Creative Labs, Inc.) cameras that were positioned above each cage. A red light bulb was used at night to allow for video monitoring the animals’ behavior. Video and electrographic monitoring of the animals began 3 months after SE.

Each EEG file was analyzed manually with Custom LabVIEW® software (see Figure 2) designed to display the channels. An electrographic seizure was defined (described by Nissinen) as a high-frequency (>5Hz), high-amplitude (>2x baseline) discharge, originating either in the left CA3 or in the right CA3 (or both), that lasted for at least 5 seconds, and with corresponding video to eliminate artifacts. Interictal spikes were counted according to the criterion provided by Adjouadi, high amplitude(1.6X the background noise), frequency < 5Hz, duration < 100ms, single peak with sharp rise and fall time(Adjouadi et al. 2004). All EEG data as well as behavioral observations were randomly verified with the member of the study team to minimize inter rater variability for the visual analysis.

Figure 2
Demonstrating the process by which EEG seizure confirmation was performed, using a custom seizure detection and analysis LabVIEW software (see criteria in methods section). The top two traces show bilateral EEG data marked as a seizure. The seizure event ...

2.5 LFS stimulation

LFS was applied (n=7), via the bipolar VHC electrode, using a DS8000 Digital Stimulator connected to a DLS100 Stimulus Isolator. LFS was composed of a 60 minute train of 1Hz biphasic square waves, each 0.1ms in duration at amplitude of 200μA, and followed by 15 minutes of rest. Stimulation continued for two weeks, 24 hours a day, 60 minutes on and 15 minutes off. Another 14 days of video-EEG monitoring was done after the cessation of LFS, and then the animals were sacrificed and perfused.

2.6 Histology

Prior to perfusion, 50μA DC-current for 10 seconds was injected in the tissue to mark the location of the electrode tips. Frozen coronal sections of 20μm were stained with cresyl violet and examined with a light microscope to verify electrode location (Figure3A and 3B).

Figure3
Shortly after receiving LFS treatment, DC current was applied to the electrode tips and the rat brains were perfused for histological analysis. The top image (3A) shows a coronal section of a rat brain stained with cresyl violet, showing the tracks formed ...

In order to quantify neural damage to the surrounding tissue, 8 rat brains (4 that received both SE-induction and LFS; and 4 that had only received SE-induction were fixed, sliced at 20μm thick into coronal sections and stained with cresyl violet to mark Nissel bodies. From the temporal part of the hippocampus, 10 slices per track marking were mounted into slides. 3 of the clearest slides from each set were then imaged and used for analysis. The width of the CA3, CA1 and Dentate Gyrus (DG) for each rat was measured at three locations and averaged (see Fig 6). Analyses of the mean pyramidal cell layer width in the CA3, CA1 and DG region were performed using a Model MA664 stage micrometer (Swift, CT).

Figure 6
Effect of LFS on CA3, CA1 and Dentate Gyrus (DG) morphology in SE plus LFS (6A) and SE only (6B) rats. Nissl bodies stained with cresyl violet exhibit regular neuronal development of the CA3, CA1 and DG regions in a rat. The black lines indicate the locations ...

2.7 Statistical analysis

The rats that exhibited more than one seizure per day and received LFS was included (<1 seizure/day considered irregular and unstable seizure) for analysis (n=7). Seizure and spike frequency differences between baseline, LFS, and the post-LFS periods (each 14 days long) were evaluated for statistical significance using a one-way repeated measure ANOVA with Greenhouse-Geisser correction. To determine the significance between the various group means, a Fisher’s Least Significant Difference (LSD) test was then applied. A p value of less than 0.05 was considered statistically significant. Interictal spike analysis was also done in the same way for the baseline, LFS and the post-LFS periods. Neural damage quantification analysis was done with unpaired t-test based on SE-induced and SE-induced/LFS rats.

RESULTS

Seizure activity during baseline recording

All animals were stimulated one time via the amygdala electrode to produce a long lasting seizure (status epilepticus in scale 5 on the Racine scale (Racine, 1972). Three months following SE-induction, EEG recordings were obtained and spontaneous seizures appeared in most animals. Seizures for all of the rats were analyzed in an identical manner, via both manual EEG marking and video confirmation. No study animal died during the LFS or post LFS period.

Figure 2(top) shows an example of electrographic activity that met the duration (>5s), frequency (>5Hz), and amplitude (>2x baseline) criteria and was marked as a seizure. Figure 2(bottom) also shows large amplitude activity that resembles a seizure. However, the frequency spectrum (bottom right) shows that the frequency content is too low therefore this activity is more likely due to movement. This was confirmed with video analysis. There was no significant variation of seizure frequency between day and night and these data were pooled together.

LFS reduces seizure frequency

To determine the effect of LFS on seizure activity, the mean seizure frequency (seizures/day) across animals was calculated for the baseline, LFS, and post-LFS periods. Figure 4A shows an example of the onset of a seizure, which begins in the left CA3 and follows the right CA3 with increased amplitude. Seven animals were entered into the study with a mean frequency of 3.7 seizures per day). The number of seizures remained somewhat stable during the baseline period but decreased substantially during the application of LFS to less than one per day with every animal showing this trend (Fig. 3B). During the post-LFS period, the number of seizures increased but remained below the original baseline. The mean seizure frequency over time in each period was calculated for each rat. A repeated measures ANOVA with a Greenhouse-Geisser correction for three groups:“before”, “during” and “post-LFS” determined that mean seizure frequency differed statistically significantly between time points with a p value of 0.011. Post hoc tests using the Fisher’s Least Significant Difference (LSD) test revealed that LFS treatment elicited a reduction in seizure frequency from pre-LFS to 2-weeks of LFS (3.7 ± 2.4 vs. 0.34 ± 0.19, respectively) which was statistically significant (P = .008). Also, post-LFS treatment had been reduced to 1.3 ± 0.88 seizure which was significantly different to pre-LFS (P =.03) and 2-weeks LFS (P = .01). Therefore, it is possible to conclude that LFS (2 weeks) elicits a statistically significant reduction in seizure frequency both during and after LFS treatment. Overall, animals exhibited a significant (90%) average decrease in mean seizure frequency from baseline to LFS and 57% post-LFS (Figure 4C). Seizure duration was also evaluated during all three experimental periods. However, LFS had little or no effect on the duration of the seizures (mean seizure duration in seconds 28 ± 12 before and 34 ± 10 during and 38 ± 12 post LFS).

Figure 4
Effect of LFS on seizure frequency

LFS decreases interictal spike frequency

LFS was applied 1 hour ON and 15 minutes OFF throughout each day (24 hrs) for a period of 2 weeks (1Hz). At the end of 2 weeks, the stimulation was turned off and followed by another 2 weeks recording segment without LFS application. To determine the effect of LFS on neural excitability, interictal spike activity was analyzed as an average over animals across time, as well as an average over time for each animal. Spikes were counted (see Methods) as described by Adjouadi (Adjouadi et al. 2004). Examples of spikes that satisfy these criteria recorded during the baseline are shown in Figure 5A. The mean duration (±SD) of high amplitude single peak (1.6 times baseline) with sharp rise and fall time spike was 84±6ms. Individually, all seven animals showed a decrease in mean spike frequency during 1 Hz LFS from baseline, as well as during post-LFS period and all animals spike reductions were statistically significant (p-value <0.05). The mean spike frequency ± SEM was, on average, 150 ± 72 spikes/day before treatment, 46 ± 28 spike/day during treatment, and about 92 ± 55 spikes/day after stimulation was terminated (n = 7) (see Figure5B). A one-way repeated measures ANOVA with a Greenhouse-Geisser correction determined that mean inter ictal frequency differed statistically significantly between time points (p value < 0.0005). Post hoc tests using the Fisher’s Least Significant Difference (LSD) test showed that LFS treatment elicited a reduction in spike frequency from pre-LFS to 2-weeks of LFS (150 ± 68 vs. 46 ± 27, respectively) which was statistically significant (P = .001). Also, post-LFS treatment was reduced to 92 ± 58 spike and was statistically significantly different to pre-LFS (p =.001) and 2-weeks LFS (p = .01). Therefore, LFS (2 weeks) elicits a statistically significant reduction in spike frequency both during and after LFS treatment. The mean reduction rate was 71% and 42%, respectively (see Figure 5C).

Figure 5
Effect of LFS on interictal spike count: A) Inter ictal spikes from right CA3 (bottom trace) only and base line trace from left CA3 (upper trace). B) Mean interictal spike frequencies plot per day before, during and after LFS treatment. C) Mean seizure ...

LFS does not cause obvious damage in the hippocampus

To determine if LFS of SE-induced animal generated neural damage in the hippocampus, an additional animal group was evaluated with SE induction but without LFS (SE/NoLFS). These animals were compared to age-matched animals with both SE-induction and LFS (SE/LFS). The mean width of the pyramidal cell layer in the CA3, CA1 and DG regions of the both groups of animals were analyzed (see Table 1 and Figure 6). Visual inspection of cresyl violet stained sections did not indicate any obvious differences between control and the Rats that received LFS. The mean width and SEM for the SE/NoLFS group (n = 4) and the SE/LFS group (n = 4), were (mean ± SEM) 0.59 μm ± 0.01 and 0 .597 μm ± 0.01 for CA3; 0.52±0.01 and 0.53±0.00 for CA1; and 0.63 μm ± 0.01 and 0 .57 μm ± 0.04 for DG and the differences were not significant.

Table 1
Quantitative histology: Quantitative measurement of cresyl violet stained temporal part of hippocampal slices (10 slices per track marking were mounted into slides. 3 of the best slides from each set of ten were then imaged and used for analysis) demonstrates ...

DISCUSSION

A previous study in a genetic model of epilepsy supported the hypothesis that fiber track stimulation could decrease seizure frequency (Kile et al, 2010). Here we tested a similar paradigm on a temporal lobe model of epilepsy. There are many well-characterized and widely used models of TLE in adult rats where epileptogenesis triggered by intra-peritoneal injection of kainic acid (Stafstrom et al. 1992), intraperitoneal injection of lithium-pilocarpine (Leite et al. 1990; Cavalheiro et al. 1991), or by 90-min electrical stimulation of the ventral hippocampus(Lothman et al. 1989; Lothman et al. 1990). For this study we chose amygdala stimulation, a non-chemical spontaneous seizure model where epileptogenesis and spontaneous seizures are somewhat similar to human TLE (Nissinen et al. 2000). The amygdala has a low threshold for electrically induced seizure (Goddard et al. 1969) and stimulation applied to the lateral nucleus facilitates the recruitment of parallel amygdaloidal circuitries into seizure activity (Pitkanen et al. 1997). Finally, The stimulation region (the amygdala) is distant from the hippocampus proper and the stimulation is less likely to cause neural damage to the hippocampus.

Finding the appropriate target for seizure control with electrical stimulation has been difficult but falls into two categories, gray and white matter. Gray matter targets such as entorhinal cortex (Xu et al. 2010), hippocampus subfields (Zhang et al. 2009), cerebellum (Yang et al. 2006) have been effective. However, white matter tract stimulation has also been effective: VHC (Kile et al. 2010) and corpus callosum (Ozen et al. 2008). Both animal and human studies showed that LFS can successfully suppress seizure activity (Jerger and Schiff 1995). LFS stimulation of the VHC was also recently successfully applied to control seizures in an SCN2A sodium channel mutation model (Kile et al. 2010). The seizure reduction generated by LFS in that model was significant but modest. Possible causes for this difference include the use of a genetic model with a much higher seizure frequency, shorter duration of LFS application (4 days) and the difficulty of VHC electrode implantation in a mouse compared to a rat model. However, these studies show that LFS applied to the VHC is effective in two different animal models of epilepsy.

An LFS study in the rat brain suggested involvement of GABA-benzodiazepine and endogenous opioid systems (Lopez-Meraz et al. 2004), perhaps, those mechanisms may work to decrease neural excitability.

In human patients with epilepsy, LFS at 0.5 Hz delivered to different ictal onset zones each day in 30 min intervals, was able to reduce seizure frequency (Schrader et al. 2006). Stimulation of the caudate nucleus at 4-8 Hz in 57 patients suffering from intractable epilepsy also reduced the seizure frequency. The mechanism of the suppression was attributed to the possible activation of inhibitory responses in the network (Chkhenkeli et al. 2004). Activation of the ictal zones in patients with intractable epilepsy showed a small but detectable decrease in seizure frequency (Kinoshita et al. 2005). Possible mechanisms suggested by the authors include the induction of LTD and activation of GABA-benzodiazepine and local opioid systems (Kinoshita et al. 2005). Study based on adenosine receptors blockers (Mohammad-Zadeh et al. 2009) showed that adenosine could be responsible for the suppressive effect of LFS.

It has also been shown that LFS could suppress the after-discharge duration of seizures generated through the kindling process (Velisek et al. 2002; Goodman et al. 2005) suggesting discharge threshold is raised following LFS. 1Hz stimulation to the Schaeffer collaterals in rat hippocampal slices treated with 200μM bicuculline showed strong suppressive effect and attributed to the effect of long-term depression (LTD). Another study also showed that LFS could suppress seizure activity in in-vitro brain slices and attributed the effect to excitatory synaptic depression (Schiller and Bankirer 2007). The results reported here suggest that neural plasticity might be partially involved since a significant amount of residual suppression was observed one week following the termination of the stimulation. Another LFS study in the rat brain suggested the involvement of GABA-benzodiazepine and endogenous opioid systems (Lopez-Meraz et al. 2004), in raising seizure threshold.

Taken together the results presented above support our hypothesis previously stated that LFS of white matter tracts can effectively suppress temporal lobe seizure episodes but the mechanisms for the effect are still quite unclear. Yet this methodology could be deployed for therapy provided that a white matter tract similar to the VHC could be identified in humans. In order to develop the therapeutic role of LFS on fiber tract stimulation in the human brain for temporal lobe epilepsy control, potential pathways need to be identified. In addition of commissural fiber tract, another potential white matter target would be fornix, which connects the hippocampus to the mammillary body (Luders 2004). Because of the anatomical differences between rat and human commissural fiber systems, it is unclear whether hippocampal commissural tract would work in human patients. The rat as well as other non-primate mammals, VHC pathway carries numerous fibers and provides a powerful commissural connection between the two hippocampi which originates along the full length of the hippocampal axis (Blackstad 1956; Gottlieb and Cowan 1973; Swanson et al. 1978; Laurberg 1979). In the human brain, the ventral hippocampal commissural tract is non-existent but there is a sizable dorsal commissural tract which has been documented histologically and more recently using diffusion tensor imaging technique (Gotman 1987; Gloor et al. 1993; Colnat-Coulbois et al. 2010). However the physiological function of this pathway is still in debate. One study suggests that human DHC have little functional value (Gotman 1987; Lieb et al. 1987), whereas other studies suggest that human dorsal hippocampal commissure is a functional pathway in humans and monkeys as well as actively involved in the propagation of seizure activity between the hippocampi (Spencer et al. 1987; Gloor et al. 1993). Despite the controversy of the functional pathway of DHC in human, a preliminary study has shown that dorsal commissural fiber track connects and activates the hippocampi directly in patients with depth electrodes implanted for seizure monitoring (Koubeissi MZ 2009). As part of this study, preliminary unpublished results of LFS stimulation of the DHC pathway and the fornix in humans patients show similar results to those obtained from rats, namely, decrease in interictal and ictal activity with no observable effect on memory.

In conclusion, the LFS paradigm of the VHC, a potential DBS target for the treatment of temporal lobe epilepsy was tested in a rat model and generated four key findings. First, analyses revealed a spontaneous stable seizure and interictal spike frequency over several weeks. Second, LFS at 1 Hz significantly reduces seizures by 90% and interictal spike frequency in TLE animal model. Third, the LFS applied to the VHC was a well tolerated effect on seizure and interictal spike frequency reduction. Lastly, although the sample size was small LFS did not show any significant damage of hippocampus between the control and LFS groups. Additional studies are planned to assess significant damage within the tract by the electrode, as well as to the hippocampus and/or surrounding sub-regions of brain. Further studies and in particular additional ongoing clinical work will allow us to determine whether LFS of white matter tracts will be effective to control seizure in patients with mesial temporal lobe epilepsy.

ACKNOWLEDGMENTS

This work is supported by a Grant from the Walter H Coulter Foundation and NIH grant 3RO1NS060757. Thanks are due to Kara Kile, David Tang, Nan Tian, Sheela Toprani, Thomas Ladas, Luis Gonzalez, Yazan Dweiri, Nicholas Couturier, Tina Goetz and all our lab members for their generous time and assistance.

Footnotes

DISCLOSURE 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. None of the authors has any conflict of interest to disclose.

REFERENCES

  • Deep-brain stimulation of the subthalamic nucleus or the pars interna of the globus pallidus in Parkinson’s disease. N. Engl. J. Med. 2001;345:956–963. [PubMed]
  • Adjouadi MD, Sanchez M, Cabrerizo M, Ayala P, Jayakar I, Yaylali A, Barreto A. Interictal spike detection using the Walsh transform. IEEE Trans. Biomed. Eng. 2004;51:868–872. [PubMed]
  • Albensi BC, Ata G, Schmidt E, Waterman JD, Janigro D. Activation of long-term synaptic plasticity causes suppression of epileptiform activity in rat hippocampal slices. Brain Res. 2004;998:56–64. [PubMed]
  • Avoli M, D’Antuono M, Louvel J, Kohling R, Biagini G, Pumain R, D’Arcangelo G, Tancredi V. Network and pharmacological mechanisms leading to epileptiform synchronization in the limbic system in vitro. Prog. Neurobiol. 2002;68:167–207. [PubMed]
  • Benabid AL, Pollak P, Gao D, Hoffmann D, Limousin P, Gay E, Payen I, Benazzouz A. Chronic electrical stimulation of the ventralis intermedius nucleus of the thalamus as a treatment of movement disorders. J. Neurosurg. 1996;84:203–214. [PubMed]
  • Blackstad TW. Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination. J. Comp. Neurol. 1956;105:417–537. [PubMed]
  • Cavalheiro EA, Leite JP, Bortolotto ZA, Turski WA, Ikonomidou C, Turski L. Long-term effects of pilocarpine in rats: structural damage of the brain triggers kindling and spontaneous recurrent seizures. Epilepsia. 1991;32:778–782. [PubMed]
  • Chkhenkeli SA, Sramka M, Lortkipanidze GS, Rakviashvili TN, Bregvadze ESh, Magalashvili GE, Gagoshidze TSh, Chkhenkeli IS. Electrophysiological effects and clinical results of direct brain stimulation for intractable epilepsy. Clin. Neurol. Neurosurg. 2004;106:318–329. [PubMed]
  • Colnat-Coulbois S, Mok K, Klein D, Pénicaud S, Tanriverdi T, Olivier A. Tractography of the amygdala and hippocampus: anatomical study and application to selective amygdalohippocampectomy. J. Neurosurg. 2010;113:1135–1143. [PubMed]
  • Concha L, Gross DW, Wheatley BM, Beaulieu C. Diffusion tensor imaging of time-dependent axonal and myelin degradation after corpus callosotomy in epilepsy patients. Neuroimage. 2006;32:1090–1099. [PubMed]
  • de Curtis M, Avanzini G. Interictal spikes in focal epileptogenesis. Prog. Neurobiol. 2001;63:541–567. [PubMed]
  • Dichter MA, Ayala GF. Cellular mechanisms of epilepsy: a status report. Science. 1987;237:157–164. [PubMed]
  • Dostrovsky JO, Lozano AM. Mechanisms of deep brain stimulation. Mov. Disord. 2002;17(Suppl 3):S63–68. [PubMed]
  • Durand DM, Bikson M. Suppression and control of epileptiform activity by electrical stimulation: a review. Proceedings of the IEEE. 2001;89:1065–1082.
  • Fisher R, Salanova V, Witt T, Worth R, Henry T, Gross R, Oommen K, Osorio I, Nazzaro J, Labar D, Kaplitt M, Sperling M, Sandok E, Neal J, Handforth A, Stern J, DeSalles A, Chung S, Shetter A, Bergen D, Bakay R, Henderson J, French J, Baltuch G, Rosenfeld W, Youkilis A, Marks W, Garcia P, Barbaro N, Fountain N, Bazil C, Goodman R, McKhann G, Babu Krishnamurthy K, Papavassiliou S, Epstein C, Pollard J, Tonder L, Grebin J, Coffey R, Graves N, SANTE Study Group Electrical stimulation of the anterior nucleus of thalamus for treatment of refractory epilepsy. Epilepsia. 2010;51:899–908. [PubMed]
  • Gabriëls L, Cosyns P, Nuttin B, Demeulemeester H, Gybels J. Deep brain stimulation for treatment-refractory obsessive-compulsive disorder: psychopathological and neuropsychological outcome in three cases. Acta Psychiatr. Scand. 2003;107:275–282. [PubMed]
  • Gloor P, Salanova V, Olivier A, Quesney LF. The human dorsal hippocampal commissure. An anatomically identifiable and functional pathway. Brain. 1993;116:1249–1273. [PubMed]
  • Goddard GV, McIntyre DC, Leech CK. A permanent change in brain function resulting from daily electrical stimulation. Exp. Neurol. 1969;25:295–330. [PubMed]
  • Goodman JH, Berger RE, Tcheng TK. Preemptive low-frequency stimulation decreases the incidence of amygdala-kindled seizures. Epilepsia. 2005;46:1–7. [PubMed]
  • Gotman J. Interhemispheric interactions in seizures of focal onset: data from human intracranial recordings. Electroencephalogr. Clin. Neurophysiol. 1987;67:120–133. [PubMed]
  • Gottlieb DI, Cowan WM. Autoradiographic studies of the commissural and ipsilateral association connection of the hippocampus and detentate gyrus of the rat. I. The commissural connections. J. Comp. Neurol. 1973;149:393–422. [PubMed]
  • Heck C. Long Term Follow-up of the RNS(TM) System in Adults with Medically Intractable Partial Onset Seizures. AES; San antonio: 2010.
  • Hodaie M, Wennberg RA, Dostrovsky JO, Lozano AM. Chronic anterior thalamus stimulation for intractable epilepsy. Epilepsia. 2002;43:603–608. [PubMed]
  • Holtzheimer PE, Mayberg HS. Deep brain stimulation for treatment-resistant depression. A. J. Psychiatry. 2010;167:1437–1444. [PubMed]
  • Hufnagel A, Dümpelmann M, Zentner J, Schijns O, Elger CE. Clinical relevance of quantified intracranial interictal spike activity in presurgical evaluation of epilepsy. Epilepsia. 2000;41:467–478. [PubMed]
  • Jefferys JG. Basic mechanisms of focal epilepsies. Exp. Physiol. 1990;75:127–162. [PubMed]
  • Jerger K, Schiff SJ. Periodic pacing an in vitro epileptic focus. J. Neurophysiol. 1995;73:876–879. [PubMed]
  • Jobst BC, Darcey TM, Thadani VM, Roberts DW. Brain stimulation for the treatment of epilepsy. Epilepsia. 2010;51(Suppl 3):88–92. [PubMed]
  • Kile KB, Tian N, Durand DM. Scn2a sodium channel mutation results in hyperexcitability in the hippocampus in vitro. Epilepsia. 2008;49:488–499. [PMC free article] [PubMed]
  • Kile KB, Tian N, Durand DM. Low frequency stimulation decreases seizure activity in a mutation model of epilepsy. Epilepsia. 2010;51:1745–1753. [PMC free article] [PubMed]
  • Kinoshita M, Ikeda A, Matsuhashi M, Matsumoto R, Hitomi T, Begum T, Usui K, Takayama M, Mikuni N, Miyamoto S, Hashimoto N, Shibasaki H. Electric cortical stimulation suppresses epileptic and background activities in neocortical epilepsy and mesial temporal lobe epilepsy. Clin Neurophysiol. 2005;116:1291–1299. [PubMed]
  • Koubeissi MZ, Durand D, Rashid S, Syed T, Maciunas R, Luders H. In vivo demonstration of connectivity between dorsal hippocampal commissure and bilateral mesial temporal structures. 63rd Annual Meeting of the American Epilepsy Society; Boston. 2009.
  • Laurberg S. Commissural and intrinsic connections of the rat hippocampus. J. Comp. Neurol. 1979;184:685–708. [PubMed]
  • Leite JP, Bortolotto ZA, Cavalheiro EA. Spontaneous recurrent seizures in rats: an experimental model of partial epilepsy. Neurosci. Biobehav. Rev. 1990;14:511–517. [PubMed]
  • Lieb JP, Hoque K, Skomer CE, Song XW. Inter-hemispheric propagation of human mesial temporal lobe seizures: a coherence/phase analysis. Electroencephalogr. Clin. Neurophysiol. 1987;67:101–119. [PubMed]
  • López-Meraz ML, Neri-Bazán L, Rocha L. Low frequency stimulation modifies receptor binding in rat brain. Epilepsy Res. 2004;59:95–105. [PubMed]
  • Lothman EW, Bertram EH, Bekenstein JW, Perlin JB. Self-sustaining limbic status epilepticus induced by ‘continuous’ hippocampal stimulation: electrographic and behavioral characteristics. Epilepsy Res. 1989;3:107–119. [PubMed]
  • Lothman EW, Bertram EH, Kapur J, Stringer JL. Recurrent spontaneous hippocampal seizures in the rat as a chronic sequela to limbic status epilepticus. Epilepsy Res. 1990;6:110–118. [PubMed]
  • Luders H. Brain stimulation and epilepsy: novel approaches for seizure control. Suppl Clin Neurophysiol. 2004;57:379–382. [PubMed]
  • McIntyre CC, Savasta M, Kerkerian-Le Goff L, Vitek JL. Uncovering the mechanism(s) of action of deep brain stimulation: activation, inhibition, or both. Clin. Neurophysiol. 2004;115:1239–1248. [PubMed]
  • Mogilner AY, Rezai AR. Brain stimulation: history, current clinical application, and future prospects. Acta Neurochir Suppl. 2003;87:115–120. [PubMed]
  • Mohammad-Zadeh M, Mirnajafi-Zadeh J, Fathollahi Y, Javan M, Jahanshahi A, Noorbakhsh SM, Motamedi F. The role of adenosine A(1) receptors in mediating the inhibitory effects of low frequency stimulation of perforant path on kindling acquisition in rats. Neuroscience. 2009;158:1632–1643. [PubMed]
  • Morrell M. Brain stimulation for epilepsy: can scheduled or responsive neurostimulation stop seizures? Curr. Opin. Neurol. 2006;19:164–168. [PubMed]
  • Morrell M. The RNS System is safe and effective as adjunctive therapy in medically intractable partial epilepsy. 2009.
  • Nail-Boucherie K, Lê-Pham BT, Marescaux C, Depaulis A. Suppression of absence seizures by electrical and pharmacological activation of the caudal superior colliculus in a genetic model of absence epilepsy in the rat. Exp. Neurol. 2002;177:503–514. [PubMed]
  • Nissinen J, Halonen T, Koivisto E, Pitkänen A. A new model of chronic temporal lobe epilepsy induced by electrical stimulation of the amygdala in rat. Epilepsy Res. 2000;38:177–205. [PubMed]
  • Nissinen J, Pitkanen A. Effect of antiepileptic drugs on spontaneous seizures in epileptic rats. Epilepsy Res. 2007;73:181–191. [PubMed]
  • Ozen LJ, Young NA, Koshimori Y, Teskey GC. Low-frequency stimulation reverses kindling-induced neocortical motor map expansion. Neuroscience. 2008;153:300–307. [PubMed]
  • Parrent A, Almeida CS. Deep brain stimulation and cortical stimulation in the treatment of epilepsy. Adv. Neurol. 2006;97:563–572. [PubMed]
  • Paxinos GW. The rat brain stereotaxic coordinates. 2007.
  • Pitkänen A, Savander V, LeDoux JE. Organization of intra-amygdaloid circuitries in the rat: an emerging framework for understanding functions of the amygdala. Trends Neurosci. 1997;20:517–523. [PubMed]
  • Schiller Y, Bankirer Y. Cellular mechanisms underlying antiepileptic effects of low- and high-frequency electrical stimulation in acute epilepsy in neocortical brain slices in vitro. J. Neurophysiol. 2007;97:1887–1902. [PubMed]
  • Schrader LM, Stern JM, Wilson CL, Fields TA, Salamon N, Nuwer MR, Vespa PM, Fried I. Low frequency electrical stimulation through subdural electrodes in a case of refractory status epilepticus. Clin Neurophysiol. 2006;117:781–788. [PubMed]
  • Spencer SS, Williamson PD, Spencer DD, Mattson RH. Human hippocampal seizure spread studied by depth and subdural recording: the hippocampal commissure. Epilepsia. 1987;28:479–489. [PubMed]
  • Spuck S, Tronnier V, Orosz I, Schönweiler R, Sepehrnia A, Nowak G, Sperner J. Operative and technical complications of vagus nerve stimulator implantation. Neurosurgery. 2010;67:489–494. [PubMed]
  • Stafstrom CE, Thompson JL, Holmes GL. Kainic acid seizures in the developing brain: status epilepticus and spontaneous recurrent seizures. Brain Res. Dev. Brain Res. 1992;65:227–236. [PubMed]
  • Swanson LW, Wyss JM, Cowan WM. An autoradiographic study of the organization of intrahippocampal association pathways in the rat. J. Comp. Neurol. 1978;181:681–715. [PubMed]
  • Theodore WH, Fisher RS. Brain stimulation for epilepsy. Lancet Neurol. 2004;3:111–118. [PubMed]
  • Velasco F, Velasco M, Velasco AL, Jimenez F, Marquez I, Rise M. Electrical stimulation of the centromedian thalamic nucleus in control of seizures: long-term studies. Epilepsia. 1995;36:63–71. [PubMed]
  • Velasco F, Velasco M, Velasco AL, Menez D, Rocha L. Electrical stimulation for epilepsy: stimulation of hippocampal foci. Stereotact. Funct. Neurosurg. 2001;77:223–227. [PubMed]
  • Velasco M, Velasco F, Velasco AL. Centromedian-thalamic and hippocampal electrical stimulation for the control of intractable epileptic seizures. J Clin Neurophysiol. 2001;18:495–513. [PubMed]
  • Velísek L, Velísková J, Stanton PK. Low-frequency stimulation of the kindling focus delays basolateral amygdala kindling in immature rats. Neurosci. Lett. 2002;326:61–63. [PubMed]
  • Vidailhet M, Vercueil L, Houeto JL, Krystkowiak P, Benabid AL, Cornu P, Lagrange C, Tézenas du Montcel S, Dormont D, Grand S, Blond S, Detante O, Pillon B, Ardouin C, Agid Y, Destée A, Pollak P, French Stimulation du Pallidum Interne dans la Dystonie (SPIDY) Study Group Bilateral deep-brain stimulation of the globus pallidus in primary generalized dystonia. N. Engl. J. Med. 2005;352:459–467. V. [PubMed]
  • Vitek JL. Mechanisms of deep brain stimulation: excitation or inhibition. Mov. Disord. 2002;17(Suppl 3):S69–72. [PubMed]
  • Xu ZH, Wu DC, Fang Q, Zhong K, Wang S, Sun HL, Zhang SH, Chen Z. Therapeutic time window of low-frequency stimulation at entorhinal cortex for amygdaloid-kindling seizures in rats. Epilepsia. 2010;51:1861–1864. [PubMed]
  • Yang LX, Jin CL, Zhu-Ge ZB, Wang S, Wei EQ, Bruce IC, Chen Z. Unilateral low-frequency stimulation of central piriform cortex delays seizure development induced by amygdaloid kindling in rats. Neuroscience. 2006;138:1089–1096. [PubMed]
  • Zhang SH, Sun HL, Fang Q, Zhong K, Wu DC, Wang S, Chen Z. Low-frequency stimulation of the hippocampal CA3 subfield is anti-epileptogenic and anti-ictogenic in rat amygdaloid kindling model of epilepsy. Neurosci. Lett. 2009;455:51–55. [PubMed]
  • Zumsteg D, Andrade DM, Wennberg RA. Clinical electrophysiology factors indicative of intractability in patients with temporal lobe epilepsy. Adv. Neurol. 2006;97:45–62. [PubMed]