[1] 14w Low-frequency stimulation of bilateral anterior nucleus of thalamus inhibits amygdale-kindled seizures in rats ଝ

[1] 214w Brain stimulation with low-frequency is emerging as an alternative treatment for refractory epilepsy. The anterior nucleus thalamus (ANT) is thought to be a key structure in the circuits of seizure generation and propagation. The present study aimed to investigate the effects of low frequency stimulation (LFS) targeting ANT on amygdala-kindled seizures in Sprague-Dawley rats. Electrodes were implanted into the right basolateral amygdala and the right or bilateral ANT of Sprague-Dawley rats. When fully kindled seizures were achieved by daily electrical stimulation of the amygdala, LFS (15 min train of 0.1 ms pulses at 1 Hz and 200-500 A) was applied to the unilateral or bilateral ANT immediately before the kindling stimulation (pre-treatment). Our study showed that LFS of the bilateral ANT significantly decreased the incidence of generalized seizures (GS) and seizure stage, as well as shortened duration of afterdischarge and GS demonstrating an inhibition of the severity of seizures. Moreover, LFS elevated the afterdischarge threshold (ADT) and GS threshold indicating an inhibition of susceptibility to seizures. On the other hand, LFS of the unilateral ANT failed to show any significance in inhibiting seizures. Our study demonstrated that bilateral LFS in ANT could significantly inhibit amygdala-kindled seizures by preventing both afterdischarge generation and propagation. It provided further evidence for clinical use of LFS in ANT.

[1] 53w Given the tremendous success of deep brain stimulation (DBS) for the treatment of movement disorders [6,33,37], clinicians have begun to open up to the possible use of electrical stimulation for the treatment of patients with pharmaco-resistant epilepsy [2,4,10,20,24,26,32]. However, stimulus parameters and the optimal targets for stimulation are two fundamental questions remain unresolved.

[2] 36w Stimulus frequency has been shown to an important factor in optimizing stimulation parameters to achieve efficacy of DBS [30]. Previous experimental and clinical studies mainly focused on the efficacy of high frequency stimulation (>5 Hz, HFS)

[3] 57w ଝ This work was supported by grants from the National Natural Science Foundation of China (30870884) and the Shandong Provincial Outstanding Medical Academic Professional Program. * Corresponding authors at: Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, No. 5 Donghai Middle Road, Qingdao 266071, China. Tel.: +86 532 8890 5659; fax: +86 532 85968434.

[4] 455w E-mail addresses: yu-jintai@163.com (J.-T. Yu), dr.tanlan@163.com (L. Tan). [2,4,10,20,30,36,44], however, evidence showed that repeated and chronic HFS is ineffective and could even aggregate seizures [9,25]; moreover, prolonged HFS may interrupt the normal function of the targets [16]; in addition, the intensity that evoked clinical and afterdischarge in HFS treated rats is lower than the one determined in LFS treated rats [45], which means that LFS is unlikely to induce a seizure compared with HFS under similar conditions. On the other hand, cumulating data suggests that low frequency stimulation (1 Hz, LFS), delivered to either the epileptic focus or other brain regions is found to be antiepileptic and antiepileptogenic [5,12,14,15,31,[41][42][43]48]. Gaito reported that LFS (1 Hz) of kindling stimulus results in a significant increase of seizure threshold and suppression of behavioral seizures [12,13]. More recently, a dramatic decrease in the incidence of generalized seizures in fully kindled animals is observed after preemptive LFS at the kindling focus [15]. In addition, the effectiveness of LFS is reported to be time-dependent on amygdaloid-kindling seizures and modedependent in amygdala-kindled seizures [38,42,43]. Clinically, LFS has been reported to decrease interictal spiking in patients with temporal lobe epilepsy [46]. Anterior nucleus of the thalamus (ANT) is a promising and preferable target in seizure control for intractable epilepsy in animal models and clinical trials [30,47]. It is highly correlated with the hippocampus via the Papez circuit, and hippocampus is supposed to be one of the most epilepsy-susceptible areas and the origin of temporal lobe epilepsy. Moreover, the ANT projects largely to the cingulated gyrus, whence it further projects to limbic structures and wide regions of neocortex to influence physiologic activity throughout the areas of cortex, thus the ANT is thought to function as a relay structure to amplify and synchronize seizure activities in these circuits [40]. In addition, the increased metabolic activity in the central thalamus and the entire posterior thalamus suggests that the ANT cooperates with the central thalamus and posterior thalamus to amplify and synchronize seizure afterdischarges [40]. Therefore, the ANT may be a key structure not only in Papez circuit, but also in the intrathalamic pathways [22]. Furthermore, ANT is relatively distant from the sensory and motor specific thalamic nucleus, thus it bears higher intensity current and acts as a gate keeper of signal propagation in epileptogenic circuitry [3]. Recently, a large and multicenter trial (SANTE: stimulation of the anterior nucleus of the thalamus for epilepsy) was conducted and subsequently with encouraging results, making ANT the most wellestablished target for DBS in the treatment of epilepsy to date [10]. However, previous human and animal studies exploring the efficacy of DBS of ANT mainly employed HFS, and less is known about whether LFS (1 Hz) can inhibit seizures in rats.

[5] 75w Kindling produced by repeated application of low-intensity electrical stimulation to amygdala resulting in secondary generalized seizures has been considered an ideal model of temporal lobe epilepsy. Previous studies mainly employed chemical convulsantkindled models (i.e. pentylenetetrazol, pilocarpine, kainic acid) to investigate the efficacy of ANT DBS [18,25,30,40]. It is necessary to use an amygdaloid-kindled seizure model, which mimic the seizure attack of human temporal lobe epilepsy, to explore the effectiveness of LFS (1 Hz) of ANT.

[1] 232w In this study, LFS of the bilateral ANT significantly decreased the incidence of GS to 36% (29 times) compared with that of the control group (100%, P < 0.001), while GS incidence of the unilateral ANT group was 94% (75 times) and reached no significance compared with the control group (P > 0.05) (Fig. 1B). Additionally, LFS of the bilateral ANT also significantly decreased the expression of seizure stage, shortened average ADD and average GSD compared with that of the control group (2.23 ± 0.2 vs. 4.96 ± 0.02, P < 0.001; 38.3 ± 2.6 vs. 67.2 ± 3.9, P < 0.001; 18.7 ± 1.2 vs. 33.5 ± 1.9, P < 0.001) (Fig. 1). As for LFS of the unilateral ANT, no significance was observed in average ADD and average GSD compared with that of the control group (58.0 ± 3.8 vs. 67.2 ± 3.9, P > 0.05; 28.9 ± 1.9 vs. 33.5 ± 1.9, P > 0.05) and the decrease of seizure stage failed to reach significance (4.64 ± 0.1 vs. 4.96 ± 0.1, P < 0.001), either (Fig. 1). It is worth mentioning that statistical significance of generalized seizure incidence, seizure stage, average ADD and average GSD were also observed between the unilateral group and bilateral group of LFS (data not shown) (Fig. 1). Representative EEGs recorded from the right amygdala during an experimental session are shown in Fig. 2.

[2] 358w There were no differences in ADT and GST among groups on the day before LFS. ADT and GST both remained stable on day 5 (P > 0.05), but declined to 78.5% of pre-LFS ADT (P < 0.01) and to 84.8% of pre-LFS GST (P < 0.05) respectively on day 10 in the control group. Bilateral LFS of ANT significantly elevated ADT to 148.4% and 166.4% of pre-LFS ADT on days 5 and 10, respectively (P < 0.05 and P < 0.01, respectively). Moreover, this treatment not only prevented the decline of GST, but elevated it to 150.3% on day 5 and to 175.9% on day 10 compared with pre-LFS GST (P < 0.05 and P < 0.01, respectively). ADT and GST both remained unchanged on day 5, as well *** P < 0.001 represents statistically significant differences as compared with the control group. ## P < 0.01 and ### P < 0.001 represent statistically significant differences as compared with the bilateral group. One-way ANOVA was used for statistical analysis, followed by the Dunnett's t-test. In the case of comparing generalized seizure incidence, Fisher exact test was used. as on day 10 for the rats received unilateral stimulation (P > 0.05 for all of them). It is worth to mention that no statistically significant differences were observed in ADT and GST both on days 5 and 10 between the control group and unilateral LFS group (P > 0.05 for all of them) (Fig. 3). During days 1-5, bilateral ANT stimulation has been shown effective in suppressing the severity of seizures (Table 1). During days 6-10, bilateral ANT procedure resulted in similar inhibitory effects on seizures as did during days 1-5. Still, Data are shown as mean ± S.E.M. One-way ANOVA was used for statistical analysis, followed by the Dunnett's t-test. ** P < 0.01 represents statistically significant difference as compared with the control group. *** P < 0.001 represents statistically significant difference as compared with the control group. ## P < 0.01 represents statistically significant difference as compared with days 1-5. the seizure stage and ADD during 6-10 were further reduced compared with those during days 1-5 (Table 1).

[1] 126w Recently, a large and multi-center trial (SANTE, stimulation of the anterior nucleus of the thalamus for epilepsy) demonstrated the effectiveness of bilateral stimulation of ANT, thus making ANT the most well-established target for DBS in the treatment of epilepsy to date [10]. In this study, we provided the first evidence that bilateral pre-treatment of LFS application of ANT significantly inhibited amygdala-kindled seizures in rats. Reduced behavioral seizures and shortened ADD and GSD were observed in rats with bilateral LFS. In addition, ADT and GST which represent the susceptibility of the animal to initiation and propagation of the seizure activity, respectively, dramatically elevated in rats from the bilateral LFS group. These obtained data support the protective role of LFS of ANT in the treatment of refractory epilepsy.

[2] 355w There is a concept that disorder of treated with DBS (i.e. epilepsy) is fundamentally disorder of a specific brain network, as opposed to a specific neuron type, ion channel, or molecule [8,27]. Radiologic imaging and animal studies have shown that ANT mediates cortical-subcortical interactions between the limbic system and brainstem through several neuroanatomic circuits (including the Papez circuit, the corticothalamic circuit, the mammallary circuit), which are involved in the propagation of seizures [30,40], thus making ANT a promising target for brain stimulation in refractory epilepsy. All of the previous clinical studies and most animal studies exploring the efficacy of DBS in ANT have focused on bilateral stimulation and the effects of unilateral stimulation in ANT is seldomly studied so far. In our study, we are interested in the issue that if unilateral stimulation in ANT would be enough to prevent seizures or bilateral stimulation are necessitated. It would be of great benefit for patients receiving DBS treatment because unilateral stimulation lowered the risks of operation as well as the costs compared with bilateral ANT procedure. However, our study showed that only bilateral ANT stimulation is effective in inhibiting seizures. Unilateral AN procedure is totally useless. However, a study employed kainic acid (KA)-induced focal cortical seizure model in rats demonstrated that both unilateral and bilateral DBS of ANT were effective in suppressing seizure activity and the authors failed to give a proper explanation about the results [40]. It might due to the different seizure models and stimulation parameters in the two experiments. Recently, Hamani reported that only bilateral ANT stimulation had a protective role against seizures induced by an intraperitoneal pilocarpine injection [18], which is consistent with our findings. They also noted that because of various pathways involved in generalization of seizures and only part of the ANT efferents have bilateral projections, it is not surprising that only bilateral ANT procedures were effective [18]. Even though we have no direct data to explain it, our results at least suggest that LFS of the ANT mainly recruits fibers projecting to the ipsilateral hemisphere rather than fibers projecting bilaterally, therefore, unilateral stimulation is not enough in suppressing seeizures.

[3] 167w Previous animal and clinical studies about DBS of ANT have mainly focused on HFS [2,10,[18][19][20][24][25][26]30,32]. Mirski et al. reported an elevation in pentylenetetrazole (PTZ)-induced clonic seizure threshold after 100 Hz stimulation of the ANT [30]. Many other animal studies employed HFS (ranging from 100 to 130 Hz) of ANT also observed effectiveness in seizure suppression employing HFS of ANT [18,19,40]. Human studies mainly used HFS ranging from 100 to 185 Hz to examine the utility of DBS of ANT and proved to be effective [2,10,20,24,26,32]. In contrast, Lado et al. performed ANT stimulation in rats with chronic epilepsy after KAinduced status epilepticus and reported a significant increase in seizure frequency with 100 Hz stimulation [25]. The mechanism of action of HFS in reducing seizures is still not fully understood. It is suggested that DBS-induced attenuation of seizure activity probably mimics that of high frequency DBS for movement disorders. DBS invokes a mixture of excitatory and inhibitory effects, ultimately resulting in disruption of the pathologic neuronal networks [29].

[4] 260w Although many studies have proved the effectiveness of HFS of ANT, increasing experimental and clinical evidence now appears that LFS can reduce or prevent seizure activity [5,12,14,15,31,[41][42][43]48]. Our findings that LFS (sine wave pulses, 1 Hz, 0.1 ms per pulse, 15 min) significantly decreased the severity of and the susceptibility to seizures provided further evidence that LFS of appropriate structures in the brain is anti-epileptic in a preemptive way. Usually, the frequency range between 10 and 70 Hz is generally avoided as it is known to elicit seizures and is commonly used to induce kindling. This leaves a low frequency range (<10 Hz) to be explored. Most of the studies exploring the efficacy of LFS focused on 1 Hz frequency and achieved inspiring results. In addition, 5 Hz frequency stimulation of the hippocampus was also found to be anti-epileptic [45]. In contrast with our findings, a previous study found that LFS at a frequency of 8 Hz was proconvulsant in the ANT [30]. The different stimulation parameters (i.e. frequency, intensity, duration) and seizure models (pentylenetetrazol-induced vs. amygdala-kindled) may contribute to the discrepancy. It is worthy to note that the current used by Mirski et al. ranged between 350 and 1000 A and were much larger than ours (200-500 A). Their current intensity could induce seizures in our experiment. Furthermore, high current intensity in the ANT could impair memory in rats [17]. Moreover, our observation supported the study of J.H. Goodman et al. [15], reporting that the sine wave pulse may be alternative for LFS therapy besides monophasic square-wave pulse [38].

[5] 140w Our data supported the effectiveness of LFS in ANT, however, we failed to give a proper explanation for the mechanisms of action of LFS. It is demonstrated that LFS at 1 Hz can induce long term depression (LTD) and depotentiation [1,7,11,39]. In addition, other changes induced by LFS, including modification of GABA and mu receptor binding [28], activation of galanin [35] and adenosine receptors [31], may also contribute to the inhibition of synaptic transmission. We speculated that it is possible that LFS of ANT may transmit low frequency activity along the seizure-propagating pathways to the structures involved in epileptogenesis, causing LTD and depotentiation, as well as decreased synaptic transmission of the ictogenic foci in the circuits, thus suppressing the seizure activity. More studies are needed to clarify the exact mechanism of LFS of the ATN in decreasing the seizure activity.

[6] 114w To date, two modes of DBS, scheduled and responsive brain stimulation are in clinical use for the treatment of refractory epilepsy. The former mode refers to the stimulation delivered according to a predefined schedule, independent of physiological activity in brain. In contrast, the latter one refers to the stimulation delivered in response to electrographic activity [21]. Both of the two modes have their pros and cons [23]. In our experiment, we employed pre-treatment LFS which mimics the scheduled brain stimulation in clinical use to some extent. In future, we would explore the efficacy of post-treatment LFS and further make comparison of both pre-treatment and post-treatment LFS in ANT to provide evidence for clinical use.

[7] 34w In summary, our study demonstrated that bilateral LFS in ANT can significantly inhibit amygdala-kindled seizures. Further studies are needed to elucidate the underlying mechanisms of action of LFS to lay foundation for clinical use.

[1] 100w We used male Sprague-Dawley rats (280-300 g, provided by the Experimental Animal Center, Qingdao University, China), maintained in individual cages with a 12-h light-dark cycle (lights on from 8:00 to 20:00). Water and food were given ad libitum. Experiments were carried out between 10:00 and 17:00. All experiments were approved by the Qingdao University Animal Experimentation Committee and were in complete compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23) revised in 1996. Efforts were made to minimize the number of animals used in the study and their suffering.

[2] 109w Under deep chloral hydrate anesthesia (400 mg/kg, i.p.), rats were mounted in a stereotaxic apparatus (Stoelting, USA). Electrodes were implanted into the right basolateral amygdala (AP: -2.4 mm; L: -4.8 mm; V: -8.8 mm) and the right or bilateral ANT (AP: -2.0 cm; L: -1.8 mm; V: -6.4 mm). The electrodes were made of twisted stainless steel wires with a diameter of 0.2 mm (A.M. Systems, USA) and teflon-coated except for 0.5 mm at the tip. The tip separation was 0.7-0.8 mm. The electrodes were connected to a miniature receptacle, which was embedded in the skull with dental cement. Animals were allowed to recover from surgery for 10 days.

[3] 193w Kindling stimulation of the amygdala consisted of 1 s trains of monophasic, 1 ms square-wave pulses at 60 Hz delivered by a constant current stimulator (AD Instrument, USA). Electroencephalograms (EEGs) of the right amygdala were recorded with a digital amplifier (AD Instrument, Bio Amp, USA). According to the ascending method to measure afterdischarge threshold (ADT), the stimulus intensity was initiated at 50 A and increased in increments of 20 A until at least 5 s of afterdischarge was observed in the EEG, and this current intensity was defined as the ADT. Consecutive trials were separated by at least 30 min in order to avoid the influence of previous stimulation. Then all animals were subjected to kindling stimulation with the same current intensity as their own ADT once daily until they were fully kindled, i.e., the animal exhibited three consecutive stage 5 seizures. Seizure severity was classified according to a modification of Racine [34]: (1) facial movement; (2) head nodding; (3) unilateral forelimb clonus; (4) bilateral forelimb clonus and rearing; and (5) bilateral forelimb clonus and rearing and falling. Stages1-3 were considered focal seizures, while stages 4 and 5 were considered generalized seizures (GS).

[4] 118w To assess the location of the electrodes, animals were sacrificed at the end of the experiments by deep anesthesia with chloral hydrate (400 mg/kg, i.p.) and transcardiac perfusion with 0.9% saline followed by a 10% formaldehyde fixative solution. The brains were removed and stored in a 10% formalin solution for a period of at least 3 days. They were then frozen at -20 • C, sliced into 12 m sections and stained with toluidine blue O. The stained slices were qualitatively analyzed for electrode position using a light microscope. Only animals with electrodes correctly implanted both in the basolateral amygdale and ANT were included in the statistical analysis. In our experiments, 24 of 29 rats fulfilled this criterion.

[5] 58w All data are presented as mean ± S.E.M. Statistical analysis was carried out by SPSS11.5 for Windows. One-way analysis of variance (ANOVA) followed by Dunnett's test was used to calculate statistical significance. Fisher exact test was used to compare the incidence of GS. For all analyses, the tests were two-sided and a P < 0.05 was considered significant.

[1] 237w The fully kindled animals were randomly assigned to one of three groups: control (n = 9), unilateral (n = 10) and bilateral (n = 10) groups. The pre-LFS ADT and generalized seizure threshold (GST) were determined by the same procedure used for kindling ADT determination. The method for ADT measurement was as same as that for initial ADT. Then the current intensity was increased in increments of 20 A until a generalized seizure was evoked and this current intensity was defined as GST. On the next day, immediately before the kindling stimulation (using pre-kindling ADT), the experimental rats (n = 20, 10 for unilateral and 10 for bilateral ANT stimulation) were subjected to LFS (sine wave pulses, 1 Hz, 0.1 ms per pulse, 15 min). Control rats were also connected to the low-frequency stimulator for 15 min, but no current was delivered. The current intensity of DBS in the ANT was pre-determined in each animal before the kindling session. The current was gradually increased from 100 A until an increase in motor and abnormal behavior in each animal was observed; after this point, it was decreased to 70% of the behavioral threshold, within the range of 200-500 A. All rats were stimulated daily for 10 days and the afterdischarge duration (ADD), generalized seizure duration (GSD), seizure stage, and GS incidence were recorded. In addition, both ADT and GST were determined again at the fifth and tenth days.