Brain Stimulation xxx (2014) 1e8

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Original Research

Anti-epileptogenic Effect of High-frequency Stimulation in the Thalamic Reticular Nucleus on PTZ-induced Seizures C.R. Pantoja-Jiménez a, b, V.M. Magdaleno-Madrigal a, b, *, S. Almazán-Alvarado a, R. Fernández-Mas a a b

Laboratorio de Neurofisiología del Control y la Regulación, Dirección de Investigaciones en Neurociencias, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz, Mexico Carrera de Psicología, Facultad de Estudios Superiores Zaragoza-UNAM, Ciudad de México, Mexico

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 April 2013 Received in revised form 31 January 2014 Accepted 27 March 2014 Available online xxx

Background: Deep brain stimulation, specifically high-frequency stimulation (HFS), is an alternative and promising treatment for intractable epilepsies; however, the optimal targets are still unknown. The thalamic reticular nucleus (TRN) occupies a key position in the modulation of the cortico-thalamic and thalamo-cortical pathways. Objective: We determined the efficacy of HFS in the TRN against tonic-clonic generalized seizures (TCGS) and status epilepticus (SE), which were induced by scheduled pentylenetetrazole (PTZ) injections. Methods: Male Wistar rats were stereotactically implanted and assigned to three experimental groups: Control group, which received only PTZ injections; HFS-TRN group, which received HFS in the left TRN prior to PTZ injections; and HFS-Adj group, which received HFS in the left adjacent nuclei prior to PTZ injections. Results: The HFS-TRN group reported a significant increase in the latency for development of TCGS and SE compared with the HFS-Adj and Control groups (P < 0.009). The number of PTZ-doses required for SE was also significantly increased (P < 0.001). Spectral analysis revealed a significant decrease in the frequency band from 0.5 Hz to 4.5 Hz of the left motor cortex in the HFS-TRN and HFS-Adj groups, compared to the Control group. Conversely, HFS-TRN provoked a significant increase in all frequency bands in the TRN. EEG asynchrony was observed during spike-wave discharges by HFS-TRN. Conclusion: These data indicate that HFS-TRN has an anti-epileptogenic effect and is able to modify seizure synchrony and interrupt abnormal EEG recruitment of thalamo-cortical and, indirectly, corticothalamic pathways. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: Thalamic reticular nucleus Deep brain stimulation Spike-wave discharges Neuromodulation Experimental epileptic seizures

Introduction Epilepsy is a neurological disorder that affects 1e2% of the population. Epilepsy treatment is normally initiated after two or more unprovoked epileptic seizures. One-third of epilepsy patients do not respond effectively to anti-epileptic drugs and approximately 30e40% of adult patients remain refractory [1,2]. Surgical resection of the epileptogenic zone is only an option for a minority

This study was supported by the Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz (INP 123240.1). Conflicts of interest: The authors have no known conflicts of interest associated with the material contained in this manuscript. * Corresponding author. Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz, Calz México-Xochimilco No. 101, Col. San Lorenzo Huipulco Del. Tlalpan, Ciudad de México, Mexico CP 14370. Tel.: þ52 55 41605088; fax: þ52 55 56559980. E-mail addresses: [email protected], [email protected] (V.M. MagdalenoMadrigal). 1935-861X/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brs.2014.03.012

of patients [3]. Thus, an alternative treatment is needed for the remaining refractory patients. Recent studies have found that deep brain stimulation (DBS) is a safe and beneficial technique to treat medically intractable epilepsies [4]. The goal of DBS in epilepsy patients is to modify the excitability of a variety of structures involved in the initiation, propagation and maintenance of epileptic activity. Experimental studies of DBS in the cerebellum [5], thalamus [6], hippocampus [7], basal ganglia [8] and nucleus tractus solitarii [9,10], have been used to delay or abolish the secondary generalization of seizures. Human DBS studies have been performed in the cerebellum [11], the anterior and centromedian nuclei of the thalamus [12,13], the hippocampus [14,15], the locus coeruleus [16] and the subthalamic nucleus [17]. However, there are no current studies that have favored one target over another [18]. Although the optimal stimulation parameters are undetermined [19]; experimental [6,20] and clinical [21] studies suggest that highfrequency stimulation (HFS)
100 Hz produces a palliative effect

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on seizures. Anatomical structures critical for arousal and EEG desynchronization could be potential targets for antiepileptic DBS [13,22]. However, there are few data available regarding the effect of thalamic reticular nucleus (TRN) electrical stimulation on epileptic seizures. The TRN is composed of GABAergic cells [23,24] and occupies a key position in the cortico-thalamic (CT) and thalamo-cortical (TC) pathways [25,26]. The TRN receives monosynaptic glutamatergic inputs from the cerebral cortex and the thalamus and sends GABAergic projections to the thalamus [25,27,28]. The reticular neurons operate with two different firing modes [29]. The relay mode is associated with the processing of sensory information, corresponding to a desynchronized EEG state. The oscillatory mode is involved in cortical synchronization through the generation of rhythmic discharges, which are associated with rhythmic bursts of high frequency action potentials separated by silent periods that filter the incoming sensory information [30]. These oscillatory rhythmic properties may lead to a hypersynchronous activity pattern and generate spike-wave discharges (SWD) [31]. Chemical stimulation [32] and lesion studies [33] have demonstrated that the TRN generates and modulates the occurrence of SWD in different rat strains [34]. In hippocampal kindling, electrical stimulation of the TRN at 60 Hz induces an EEG desynchronization and can act to suppress limbic motor seizures [35]. Pentylenetetrazole (PTZ) is a model of generalized seizures, displaying typical absence seizures, generalized myoclonic seizures and tonic-clonic generalized seizures (TCGS). This model represents a routine test for screening anticonvulsants [36] and may be used to induce status epilepticus (SE) in immature, adult and old rats [37]. The current study analyzes the efficacy of HFS in the TRN on the development of SWD, TCGS and SE provoked by gradual PTZ-doses. Materials and methods Animals Sixteen Wistar male rats (280e320 g) were used in this study. They were born and raised in the vivarium of the Neurosciences Research Division at Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz, México, D.F. The experiments were performed in accordance with the technical guidelines for the production, care and use of animals in the laboratory issued by SAGARPA (NOM-062 ZOO-1999) and approved by the ethics committee of the Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz. Surgical procedure Surgery was performed using ketamine hydrochloride (50 mg/ kg) and xylazine hydrochloride (5 mg/kg) i.m. All animals were placed in a stereotactic apparatus (Mod. 1430, David Kopf Instruments, Tujunga Ca) to expose the cranium and to determine the electrode positions. Stainless steel tripolar electrodes were implanted into the left TRN at the following dimensions, according to Paxinos and Watson [38]: AP, 1.4; L, 1.7; H, 6.0. Epidural EEG recording screws were implanted in the motor cortices (MCx) for EEG recordings and another was connected as a ground in the parietal bone. The electrodes were welded into a mini-connector and fixed to the skull with dental acrylic. The animals were treated with an analgesic (Butorphanol 0.4 mg/kg) and an antibiotic (Amoxicillin, 0.6 mg/kg) after the surgical procedures. After surgery, the animals were individually housed in cages (50  27  30 cm) with an ambient temperature of 23e25  C and a 12:12-h lightedark cycle. All animals were allowed to recover for 7 days and were provided with food and water ad libitum.

Electrical stimulation and recording Electrical stimulation was delivered using a Grass Instruments S88 stimulator (Grass, Massachusetts) controlled by a device designed in our laboratory that allows the automatic firing of the stimulator [10]. Stimulation parameters consisted of a 10 min sequence of biphasic square wave pulses at the following levels: frequency, 100 Hz; pulse width, 0.5 ms; and intensity, 200 mA. Brain electrical activity recordings were collected using polygraphic equipment model 78-E (Grass, Massachusetts). Experimental procedure The animals were assigned to three experimental groups: the Control group (n ¼ 6), which received only PTZ injections; the HFSTRN group (n ¼ 5), which received HFS in the left TRN prior to PTZ injections; and the HFS-Adj group (n ¼ 5), which received HFS in the left adjacent nuclei prior to PTZ injections. A baseline EEG recording was performed for 15 min. To gradually increase the intensity of induced seizures, we chose the systemic PTZ injection schedule employed by Lüttjohann et al. [39]. Rats received an initial dose of 20 mg/kg PTZ i.p. Every 15 min additional dose of 10 mg/kg were administered until a TCGS lasting 5 min (SE) was observed. Sessions were conducted between 14:00 and 18:00. Behavioral and EEG analysis All animal behavior was videotaped during the basal EEG and behavioral seizure stages. The videos were analyzed off-line using a double-blind technique, following the classification scheme proposed by Lüttjohann et al. [39]: Stage 1, sudden behavioral arrest and/or motionless staring; Stage 2, facial jerking with muzzle or muzzle and eye; Stage, 3 neck jerks; Stage 4, clonic seizure in a sitting position; Stage 5, convulsions including clonic and/or tonicclonic seizures while lying on the belly and/or pure tonic seizures; and Stage 6, convulsions, including clonic and/or tonic-clonic seizures, while lying on the side and/or wild jumping. The highest score after each PTZ injection was assigned for each animal. The mean values obtained from each group were quantified to determine seizure intensity stages. The EEG signals were amplified and the band-pass was filtered between 3 and 60 Hz. Then, the signals were digitized at 300 samples/second and stored on a hard disk. Off-line spectral analyses by the Fast Fourier Transform (FFT) and wavelets methods at 1 min periods after PTZ injections were performed with computer software developed in our laboratory [40,41]. The power spectrum of both the left- and right-motor cortices (L-MCx; R-MCx, respectively) and the left TRN recordings, showing the evolution of the EEG in the frequency domain, was computed. The variables quantified in the experiments were the latency and duration of the SWD, TCGS and SE. Histology The animals were sacrificed after status epilepticus was observed. A transcardial perfusion with saline solution (0.9%) and paraformaldehyde (10%) was performed. Brains were removed and cut into coronal slices of 60 mm. The histological verification of the placement of the stimulation and recording electrodes was conducted using the rapid procedure method [42]. Statistical analysis Seizure intensity stage changes induced by PTZ, duration, latency, cumulative doses of PTZ to SE, the SWD frequency and the

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Figure 1. Representative illustrations of the locations of the tips of the stimulating electrodes based on histological verification. White circles, Control group; gray squares, HFS-TRN group; black triangles, HFS-Adj group. TRN, thalamic reticular nucleus; AVVL, anteroventral thalamic nucleus, ventrolateral part; AVDL, anteroventral thalamic nucleus, dorsomedial part; B, basal nucleus (Meynert); LV, lateral ventricle; D3V, dorsal 3rd ventricle. Modified from Ref. [38].

spectrum power of baseline and SWD epochs (Fig. 6) were evaluated with a one-way ANOVA followed by the post hoc Bonferroni test to determine differences among groups. EEG abnormalities observed with wavelets analysis were evaluated with Kruskalle Wallis test (Fig. 7). The data are expressed as the mean  the standard error of the mean. Differences were considered statistically significant for P < 0.05. Results Histological verification Histological examination showed that the electrodes were located in the TRN for the Control (n ¼ 6) and HFS-TRN groups (n ¼ 5). Electrodes located in the nucleus anteroventrale ventrolateral (AVVL) and the nucleus anteroventraledorsomedial of the thalamus (AVDM) were assigned to HFS-Adj group (n ¼ 5) (Fig. 1). Additionally, no apparent injuries were observed after HFS or insertion of the electrodes.

of PTZ (20e30 mg/kg) provoked sudden behavioral arrest and/or motionless staring as well as facial jerking of the muzzle or the muzzle and the eye (stages 1 and 2, respectively), which was accompanied by widespread symmetric bilateral SWD in both the TRN and MCx. The cumulative PTZ doses of 40 and 50 mg/kg provoked neck jerks accompanied by single high amplitude spikes (stage 3). Cumulative doses higher than 50 mg/kg provoked clonic and/or tonic-clonic seizures accompanied by faster, high-voltage EEG spikes and generalized spikes (stages 4 and 5). Clonic and/or TCGS were separated by at least 15 min. Within this separation period a gradual decline in seizure intensity was observed (stages 1, 2 or 3). Finally, doses of 90 and 100 mg/kg intensified the severity and duration of seizures until the induction of SE and death (Fig. 2).

Effects of HFS on behavior During HFS delivery for 10 min, 80% of the animals in the HFSTRN group and 40% of the animals in the HFS-Adj group exhibited compulsive-like behavior, which is characterized by repetitive grooming episodes. These behavioral changes were observed within the first 2 min of the initiation of HFS. Seizure intensity development from scheduled injections of PTZ in the control conditions PTZ induced a sustained increase in seizure severity, as well as stage-dependent EEG changes in the control conditions. Low doses

Figure 2. Comparison of seizure intensity stages using the scheduled PTZ injections between groups. A significant increase in PTZ cumulative doses was necessary to reach a TCGS and/or SE in the HFS-TRN group. A significant decrease in seizure severity at doses of 40, 50 and 90 mg/kg of PTZ was observed. *P < 0.016, **P < 0.030, #P < 0.006. Statistical analysis was performed until 110 mg/kg because the HFS-Adj and Control groups reached SE at this dose.

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Figure 3. First clonic or tonic-clonic generalized seizures (TCGS) corresponding to stages 4, 5, and 6. A) Latency to reach TCGS. A significant increase in the HFS-TRN group was observed (**P < 0.009). B) Mean duration of TCGS. No significant changes were observed (P < 0.821). White bar, Control group; Gray bar, HFS-TRN group; Black bar, HFS-Adj group.

Effects of HFS-TRN on PTZ seizures During the first four PTZ-doses (from 20 until 50 mg/kg), the animals that were subjected to HFS in the TRN stayed in stages 1 and 2. Significant differences were observed in the latency of the development of first clonic or tonic-clonic generalized seizures (stages 4, 5, and 6). Latency to TCGS in the HFS-TRN group was 97.98  7.56 min, while latency was 60.83  3.71 min in the HFSAdj group and 62.03  6.76 min in the Control group (F2,13 ¼ 8.693, P < 0.009 HFS-TRN vs HFS-Adj and Control) (Fig. 3A). We also observed a decrease in duration of the first generalized seizure (stages 4, 5, and 6) in the HFS-TRN and HFS-Adj groups (Fig. 3B). This decrease was not significant (F2,13 ¼ 0.200, P < 0.821). HFS prior to the first PTZ dose (20 mg/kg) in both HFS-TRN (85.2  13.4 s) and HFS-Adj (110.6  12.8 s) groups reduced the latency to the appearance of SWD compared to the Control group (181.2  37.3 s) (F2,13 ¼ 4.348, P < 0.044, HFS-TRN vs Control; F2,13 ¼ 4.348, P < 0.178, HFS-Adj vs Control; F2,13 ¼ 4.348, P ¼ 1, HFSTRN vs HFS-Adj) (Fig. 4). Effect of HFS on status epilepticus HFS in the TRN induced a delay in the latency to SE (227.74  18.45 min) compared to both the HFS-Adj (133.55  1.61 min) and Control (133.29  7.37 min) groups (F2,13 ¼ 18.856, P < 0.001) (Fig. 5A). The PTZ amount required to

reach SE showed a significant increase in the HFS-TRN group (166  15.03 mg/kg) compared to both the HFS-Adj (106  2.4 mg/ kg) and the Control (95  3.4 mg/kg) groups (F2,13 ¼ 19.935, P < 0.001) (Fig. 5B). One out of five HFS-TRN animals required more PTZ-doses (200 mg/kg) to reach SE (see Fig. 2).

Change in power spectra (FFT) A significant decrease in the frequency band from 0.5 Hz to 4.5 Hz in both the HFS-TRN and HFS-Adj groups, compared to the Control group (F2,111 ¼ 8.448, P < 0.007), was observed in L-MCx. A significant increase in the frequency band from 13.1 Hz to 30 Hz was observed in the HFS-Adj group compared to both the HFS-TRN and Control groups (F2,383 ¼ 9.946, P < 0.001, HFS-Adj vs HFS-TRN; F2,383 ¼ 9.946, P < 0.034, HFS-Adj vs Control). In the R-MCx, we observed a significant increase in all frequency bands in the HFS-Adj group (0.5e4.5 Hz, F2,109 ¼ 6.150, P < 0.007; 4.6e7.9 Hz, F2,67 ¼ 4.218, P < 0.025, HFS-Adj vs HFS-TRN; 8e13 Hz, F2,123 ¼ 4.270, P < 0.025, HFS-Adj vs Control; 13.1e30 Hz, F2,381 ¼ 29.342, P < 0.001, HFS-Adj vs Control and HFS-TRN). HFS-TRN also provoked a significant increase in all frequency bands in the TRN (0.5e4.5 Hz, F2,93 ¼ 5.117, P < 0.025; 8e13 Hz, F2,105 ¼ 6.435, P < 0.007; 13.1e30 Hz F2,345 ¼ 20.411, P < 0.001), except from 4.6 Hz to 7.9 Hz (F2,72 ¼ 2.522, P < 0.123 and P < 0.241 respectively) (Fig. 6).

Abnormalities observed by HFS-TRN with wavelets analysis

Figure 4. Latency to the occurrence of spike-wave discharges (SWD). Significant differences were only observed between the HFS-TRN and Control groups (*P < 0.044). No significant changes were observed between Control and HFS-Adj groups (P < 0.178) and between HFS-TRN and HFS-Adj groups (P ¼ 1). White bar, Control group; Gray bar, HFS-TRN group; Black bar, HFS-Adj group.

During the control recordings, SWD were observed following the first two doses of PTZ (20 and 30 mg/kg). The discharges were bilaterally symmetric and seemed to be synchronous on visual inspection in the TRN, L- and R-MCx recordings. These discharges correspond to stages 1 and 2. An increase in spectrum power, with peaks that were within the 6e9 Hz range and a mean duration of 2 s (Fig. 7A), were observed (P < 0.001). Single high amplitude spikes, which were sometimes embedded in some irregular spikes of smaller amplitude with short duration, corresponded to stage 3. In contrast, HFS in the TRN induced asynchrony between the left TRN and left MCx during SWD (stage 1 and 2). The SWD were asymmetric and seemed to be asynchronous in the time-domain, in addition a significant increase was observed in the frequency band from 3 to 4 Hz (P < 0.001) (Fig. 7B). Abnormalities observed in the HFS-TRN group were the morphological and duration changes of SWD. The SWD initiated with a mean onset power spectrum in the frequency band of 7 Hz, with a sudden waning of the power spectrum in the frequency band of 4.5 Hz, which finally returned to its starting frequency band of 7 Hz (P < 0.001). The duration increased between 2 and 4 s (Fig. 7C).

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Figure 5. Quantitative analysis for status epilepticus (SE) occurrence between groups. A) Latency to reach SE in all animals. A significant increase in the HFS-TRN group was observed. B) Cumulative doses of PTZ to reach SE. Note, a significant increase in the HFS-TRN group. *P < 0.001. White bar, Control group; Gray bar, HFS-TRN group; Black bar, HFS-Adj group.

Discussion The present study was designed to assess the effects of DBS in the TRN on the development of SWD, TCGS and SE provoked by PTZ. HFS in the rostral portion of the TRN decreases the severity, increases the latency of TCGS and SE and decouples the EEG during SWD. These results were site-specific for the TRN, strengthening the importance of site-selection within the TRN. The role of DBS on chronic seizures in animal models suggests an anti-seizure effect [7,20,43]. However, in the current study, the effect of DBS is anti-epileptogenic because it retards the development of generalized secondary seizure activity (from stage 4 to stage 6) over a long period. In our results, all animals developed progressive behavioral changes, and once a more severe seizure stage was reached, the animals did not revert to earlier-stage seizures. In addition, we observed that HFS in the TRN induced a delay in the latency of seizures and SE; we did not quantify the effect of HFS after SE onset because our goal in this study was to examine the effect of HFS on the gradual increase of PTZ-induced seizures. On the other hand, unpublished data from our group on the effects of HFS in the TRN during ongoing tonic-clonic or clonic seizure induced by high doses of PTZ revealed that this experimental approach can also modify the seizure pattern (Magdaleno-Madrigal et al., 2013 unpublished results). The TRN plays a critical role in the generation and synchronization of low-frequency activities in the thalamus, such as sleep spindles and in the development of SWD by generating rhythmic inhibition of the relay neurons [29,31]. The increased delay of the appearance of TCGS and SE and the decreased latency to SWD appearance by HFS in the TRN that was observed in our study could be related to direct modulation of the TC loop, which is responsible for the spread of motor seizures [44]. Although we do not present the direct evidence, we argue that these changes may be due to an enhancement of GABAergic levels in TRN. The GABAergic reticular neurons are crucial in inhibiting external signals through TC neurons, which leads to unconsciousness during absence epilepsy [31]. It is known that intra-TRN GABAergic inhibition modulates output from the TRN [45,46], this inhibition may be sufficient to suppress the “synchronous” spikes in the TC loop provoked by PTZ. Our results could be due to an antidromic activation of TC neurons provoking phasic inhibitory postsynaptic potentials (IPSPs), steadily hyperpolarized, thus the thalamic reticular neurons abolish rhythmic burst firing and the appearance of tonic, single spike activity in TRN neurons may be sufficient to inhibit the synchronization of oscillatory activity in the CT and TC loops and induce EEG desynchronization [47]. Similar results were reported with HFS of the posterior hypothalamus, suggesting that anatomical centers critical for arousal and EEG desynchronization could be the potential targets for antiepileptic treatments [22].

Figure 6. Relative power spectrum values analyzed after PTZ injection of Control (white bar), HFS-TRN (gray bar) and HFS-Adj (black bar) groups using FFT. L-MCx showed a decrease in both the HFS-TRN and HFS-Adj groups, whereas R-MCx showed a significant increase in the HFS-Adj group. Conversely, L-TRN showed a significant increase in the HFS-TRN group while L-MCx and R-MCx showed a decrease. *P < 0.001, **P < 0.007, #P < 0.034,  P < 0.025.

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Figure 7. Simultaneous EEG recordings, wavelets and relative power spectrum values of the left motor cortex (L-MCx) and left thalamic reticular nucleus (L-TRN). A) Recordings during baseline and SWD occurrence in the Control group. The SWD are bilaterally symmetric and seem to be synchronous on visual inspection. Right panel shows the increased power spectrum between baseline (black line) and SWD-Control (blue line). B) Synchrony abnormalities during SWD in the HFS-TRN group between L-MCx and L-TRN. Changes in the power spectrum when comparing SWD-Control (blue line) and SWD-HFS (red line) were also observed. C) Morphological abnormalities during SWD in the HFS-group. The onset power spectrum in SWD starting at a frequency of 7 Hz, with a sudden waning of the power spectrum in the frequency band of 4.5 Hz and immediately returning to its starting frequency band of 7 Hz (white arrows). Vertical color bar: red maximum potency.

The fact that HFS in the TRN modified the frequency bands may be a marker of long-lasting anti-epileptogenic effects. Epileptic patients who underwent HFS have shown a decrease of 10e32 Hz, suggesting an inhibitory role in epileptogenesis [48]. The decrease from 0.5 to 7.9 Hz bandwidths in the motor cortex and an increase from 0.5 to 30 Hz in the TRN could facilitate inhibition of the TRN and reduce the nonspecific component of this pathway, which has a critical role in TC dysrhythmia and may be responsible of some neurological disorders [49]. These changes in bandwidths in the

cortical EEG were similar when an antiepileptic drug was administered [50]. In the current study, the protective effect was specific to the TRN. HFS in adjacent nuclei (AVVL and AVDL) did not result in a protective effect on PTZ-induced seizure. HFS in the anterior nucleus of the thalamus (ANT) has focused on the potential therapeutic roles for the control of epileptic seizures [12,43,51]. However, reports have shown an apparent contrast with its use. Some studies have shown that HFS in the ANT exacerbates the seizures induced by

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kainic acid [52] and that it does not have an anti-seizure effect on seizures induced by PTZ [22]. In contrast, Mirski et al. [6] demonstrated that HFS for 10 min in the ANT was sufficient to induce a neuroprotective effect on the propagation of seizures provoked by PTZ. The stimulation parameters used in our study and by Mirski et al. [6] were similar, with the exception of the administration protocol. Mirski et al. infused the PTZ (20 mg/ml) at a rate of 5.5 mg/ kg/min i.v. In addition, subspecialization exists among subnuclei of the ANT, which exerts an anticonvulsive effect [53] and there is a difference between stimulating the gray matter of the thalamus and stimulating the white matter tracts [52]. Therefore, the discrepancies in these findings could be related to the stimulation parameters (i.e., pulse duration, frequency and intensity). DBS has produced promising results against pharmacoresistant epilepsy; the frequency may play a critical role in modulating the abnormal recruitment of epileptic activity [20]. A frequency commonly employed to control epilepsy is 130 Hz [12,14,15,17]. Hodaie et al. [54], reported a median reduction in seizure frequency (53.8%) using 100 Hz, similar results were observed with 110 Hz [55] and 100e500 Hz [56,57]. Experimental studies have reported protective effects with 100 Hz [6,22,58]. These observations are comparable to our results, in which 100 Hz in the TRN was able to protect against PTZ-induced seizures. Possible mechanism of action of HFS DBS is now widely utilized as a functional surgical strategy for the treatment of a variety of neurological and psychiatric disorders. Although the mechanism through which the beneficial effects are mediated is unknown, it has been hypothesized that the most potent effect of HFS was mediated by neurotransmitter release [58]. Previous studies indicate that HFS in the hippocampus provokes a decrease in glutamate levels [59] and an increase in GABA levels [44,60] also when was applied in the thalamic nuclei [61]. The inhibitory GABAergic inputs into the thalamus are believed to be from the TRN and local interneurons. The excitatory glutamatergic inputs into the thalamus are thought to originate from the cerebral cortex. These results suggest that the neurotransmitter release abolish spindle oscillations and 3-Hz absence-like seizures [58]. We suggest that HFS appears to reinforce GABA receptors in the TRN, interrupting the abnormal recruitment of TC relay neurons and may reduce axonal burst firing in CT neurons [30,62,63]. Furthermore, it is known that high levels of GABA in the TRN neurons reduce oscillatory activity and absence seizures [64]. Similar increases in GABA-receptor activity in the TRN were associated with the antiepileptic effect of vagus nerve stimulation on seizures induced by PTZ [65]. Conclusion Few studies have focused on the effects of DBS in the TRN on experimental seizures [35]. We found that HFS in the TRN was sufficient to decrease cerebral excitability and delay the appearance of motor seizures. In summary, the TRN may be a promising target to modify abnormal EEG recruitment of thalamo-cortical and cortico-thalamic pathways. HFS-TRN effects are probably complex and could involve multiple mechanisms that remain to be determined. Therefore, additional studies are needed to confirm the safety, efficacy, tolerability and potential role of HFS of the TRN as a target to treat refractory epilepsy. Acknowledgments We thank Psych. Edith López-Ruíz and Mr. Alfredo Martínez for their technical assistance.

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