Effective inhibition of substantia nigra by deep brain stimulation fails to suppress tonic epileptic seizures

Effective inhibition of substantia nigra by deep brain stimulation fails to suppress tonic epileptic seizures

Neurobiology of Disease 43 (2011) 725–735 Contents lists available at ScienceDirect Neurobiology of Disease j o u r n a l h o m e p a g e : w w w. e...

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Neurobiology of Disease 43 (2011) 725–735

Contents lists available at ScienceDirect

Neurobiology of Disease j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y n b d i

Effective inhibition of substantia nigra by deep brain stimulation fails to suppress tonic epileptic seizures Safa Shehab a,⁎, Arwa Al-Nahdi a, Fatema Al-Zaabi a, Fadwa Al-Mugaddam a, Mahmood Al-Sultan b, Milos Ljubisavljevic c a b c

Department of Anatomy, Faculty of Medicine and Health Sciences, UAE University, Al-Ain, PO BOX 17666, UAE Department of Pharmacology, Faculty of Medicine and Health Sciences, UAE University, Al-Ain, PO BOX 17666, United Arab Emirates Department of Physiology, Faculty of Medicine and Health Sciences, UAE University, Al-Ain, PO BOX 17666, United Arab Emirates

a r t i c l e

i n f o

Article history: Received 24 March 2011 Revised 22 May 2011 Accepted 6 June 2011 Available online 14 June 2011 Keywords: Deep brain stimulation Substantia nigra c-fos Tonic seizures MES Rat

a b s t r a c t Experimental and clinical data suggest that high-frequency deep brain stimulation (DBS) of different subcortical structures can be used to control or modulate epileptic seizures. Recent studies showed that DBS of the substantia nigra reticulata (SNr) in rats has an anticonvulsant effect on forebrain clonic seizures. The aim of this study was to determine whether DBS of SNr could also suppress tonic epileptic seizures evoked in hindbrain structures. DBS with high frequency often mimics the effects of surgical ablation of a particular area of the brain. However, the optimal parameters of DBS stimulation to induce ablation-like effects on seizures are not well defined. Consequently, in the first experiment we examined the effects of different stimulation frequencies (80, 130, 260 and 390 Hz) on neuronal activation induced in SNr, using c-fos immunocytochemistry. The results showed that the stimulation of the SNr with 80 Hz has no inhibitory effect while stimulation with 130, 260 and 390 Hz produced a remarkable suppressive effect compared with the control unstimulated side. The aim of the second experiment was to determine whether bilateral inhibition of SNr with DBS could suppress tonic seizures induced by electric shock. Statistical analysis showed that the mean tonic seizure scores following SNr stimulation with either 130 or 260 Hz were not significantly different from scores following the application of the electrode without current. The data suggest that DBS of the SNr produces neuronal inhibition but fails to suppress tonic seizures. We conclude, therefore, that DBS of SNr with frequencies used in this study might not be effective for treatment of patients who suffer from tonic epileptic seizures. © 2011 Elsevier Inc. All rights reserved.

Introduction Most epilepsy patients can be successfully treated with the different available antiepileptic drugs. However, many patients continue to have seizures despite the correct diagnosis and appropriate pharmacologic treatment either because, after a while, they stop responding to drug therapy or have related side-effects that preclude their continued use (Kwan and Brodie, 2000, 2004; Löscher and Schmidt, 2006). Over the past decade the use of high-frequency deep brain stimulation (DBS) to control several movement disorders has renewed the interest for use of this technique as an alternative treatment for epilepsy in patients resistant to drug treatment or who cannot benefit from the resective surgery. Since the pioneering work of Cooper et al. (1976) to influence epilepsy by cerebellar stimulation, a number of the subcortical brain structures have been explored as potential targets for DBS. So far, DBS of several brain areas including the subthalamus, thalamus and caudate nucleus has been shown to evoke anticonvulsant effect in experimental ⁎ Corresponding author. Fax: + 971 3 7672033. E-mail address: [email protected] (S. Shehab). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2011.06.002

animals with encouraging results in humans (Chabardes et al., 2002; Kahane and Depaulis, 2010; Kerrigan et al., 2004; Lockman and Fisher, 2009; Loddenkemper et al., 2001; Nagel and Najm, 2009; Saillet et al., 2009; Theodore and Fisher, 2004; Wille et al., 2011). Recently, several attempts have been tried to find out whether DBS of the substantia nigra pars reticulata (SNr) can also produce anticonvulsant effects. Results from different laboratories have shown that HFS of the SNr in rats produce antiepileptic effects on fluorothylinduced clonic seizures (Velisek et al., 2002), kindled seizures (Morimoto and Goddard, 1987; Shi et al., 2006) and spontaneous seizures in a genetic model of absence epilepsy (Feddersen et al., 2007). In all these models of epilepsy, seizures are generally thought to originate from the forebrain. Thus, the primary aim of this study was to elucidate whether DBS of SNr could also suppress tonic seizures that are evoked in the hindbrain (Browning and Nelson, 1986; Gale, 1988; Gale and Browning, 1988) since they entail different mechanism and progression. We used maximal electrical shock (MES) test in which electrical stimulation of sufficient intensity is applied either via the ears or cornea to induce clonic-tonic seizures (Fisher, 1989; Swinyard, 1972). In this model a compound is found to possess anticonvulsant properties if it abolishes or remarkably attenuates the tonic hind limb extension

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phase of the MES. In addition, different types of seizures in different animal's models respond to different types of antiepileptic drugs. For example, antiepileptic drugs against generalized tonic seizures, i.e., phenytoin, phenobarbital, primidone and valproate have high protective indices in the MES model while drugs with efficacy against absence and myoclonic seizures, i.e., valproate, ethosuximide and benzodiazepines have high protective indices in the chemically induced seizures with pentylenetetrazol (Löscher and Nolting, 1991). Despite the clinical success of DBS in treating motor disorders like Parkinson's disease, its mechanism of action is not fully understood and several hypotheses have been proposed in this regards (Benabid et al., 2005; Deniau et al., 2010; Kringelbach et al., 2007; McIntyre et al., 2004a, b; Vitek, 2002). However, current debate is centered around the issue of whether HFS has direct excitatory or inhibitory effects on stimulated brain tissue (Deniau et al., 2010; McIntyre et al., 2004b). Furthermore, although stimulation with HFS often in practice mimics the effects of surgical ablation of particular areas of the brain it has been shown that its effectiveness critically depends on optimal stimulation parameters such as intensity, frequency and pattern of stimulation. Therefore, it was imperative to determine the optimal frequency of HFS capable of producing inhibition of SNr. This was investigated by applying electrical current stimulations of SNr with different frequencies (80, 130, 260 and 390 Hz) in combination with c-fos immunocytochemistry of brain sections. Subsequently, we investigated whether the acute stimulation with the most powerful SNr inhibition producing frequency could suppress tonic seizures using the MES test. Part of this study had appeared as an abstract (Shehab et al., 2010). Materials and methods All experiments carried out in this study were approved by the Animal Ethics Committee of the Faculty of Medicine and Health Science of the United Arab Emirates University.

induced c-fos expression in SNr was examined (see also later control experiments). Experimental rats were left to recover for 90 min (sufficient time for c-fos to be expressed) after 20 min of exposure to ether (Shehab et al., 1992; Sonnenberg et al., 1989). The rats were then sacrificed with an over dose of urethane 25% (1.5 ml/200 g of animal weight) and perfused transcardially with 10% formalin in phosphate buffer. The brains were removed, postfixed in the same fixative solution for 4 h and stored in 0.1 M phosphate buffer (PB) overnight. Coronal sections (70 μm) were cut with vibratome, collected serially and processed for detection of c-fos immunoreactivity using the avidin– biotin-complex (ABC) method. Effects of HFS on c-fos expression in SNr The effects of four stimulation frequencies: 80, 130, 260 and 390 Hz (n = 3 for each frequency) on ether-induced fos immunoreactivity were investigated. The stimulation was performed by rectangular, monopolar pulses, with pulse width of 60 μs and stimulation intensity between 150 and 300 μA (Digitimer Ltd, constant current isolated stimulator, England). In order to establish the current intensity, test procedures were conducted one at a time, as previously reported (Lado et al., 2003; Shehab et al., 2006). Initially, the current was set at 100 μA and then increased in 50 μA steps until consistent adaptable motor effects were induced. Noticed behavioral effects were mainly twitching of the contralateral forelimb and head turning and circling. After determining the current, the experiment was performed on the subsequent day to avoid any possible stimulation interference. The procedure involved unilateral stimulation applied on its own for 5 min followed by concomitant ether anesthesia and electric stimulation for 20 min. Thereafter and concurrently with animals' recovery from anesthesia (approximately 5 min), electric stimulation was turned-off. The rats were subsequently sacrificed and perfused as mentioned above (90 min after the turning off of electric stimulation) and brain sections were processed for c-fos immunocytochemistry.

Animals c-fos Immunocytochemistry Wistar male young adult rats weighing 160–180 g at the time of surgery were used. Rats were housed under standard conditions (12 h light-dark cycle), and were allowed free access to food and water. In all experiments, surgical anesthesia was induced by an intraperitoneal injection of a mixture of 0.93 ml/kg ketamine (100 mg/ml) and 0.93 ml/kg xylazine (20 mg/ml). Experiment 1 In this experiment the suppressive effects of four different HFS frequencies on ether-induced neuronal activity in SNr were examined by c-fos immunoreactivity. Electrode implantation

Brain sections were incubated with polyclonal rabbit anti-c-fos antibody (Gift from Dr Evans, 1:10000 or Santa Cruz 52, 1:1000) overnight. After rinsing in phosphate buffered saline PBS, the sections were incubated in biotinylated goat anti-rabbit IgG (Jackson, 1:500) for 1 h then in extravidin-peroxidase conjugate (Sigma, 1:1000) for another hour. To visualize any c-fos immunoreactivity the sections were incubated for 5–8 min in a solution of 25 mg diaminobenzidine (DAB) in 50 ml 0.1 M phosphate buffer (PB, pH 7.4) with 7.5 μl hydrogen peroxide (30%) and 1 ml nickel chloride (3.5%) added to intensify the reaction. Finally, the sections were rinsed in PB and mounted on gelatin-coated slides. After drying in air the sections were dehydrated in graded alcohol, cleared in xylene and mounted with DPX. All antibodies were diluted in PBS containing 0.3% Triton.

Under general anesthesia (as described above), concentric bipolar electrode (plastics one INC, MS 308-SPC electric concentric 26 GA-005in, 11 mm outer elect, 1 mm inner projection) was stereotaxically implanted in SNr. The diameter of the tip of the electrode is approximately 160 μm. The stereotaxic coordinates were 2.00 mm lateral to the midline, 1.5 mm anterior to the ear bars and 7.1 mm below the dura with the nose-bar set at +5 mm (above the ear bars). Electrodes were fixed to the skull using screws and dental cement. The animals were allowed to recover for a period of 3–4 days.

Counting of fos labeled nuclei

Effects of ether anesthesia on c-fos expression in SNr

This set of experiments assessed the ability of HFS to suppress ether-induced c-fos immunoreactivity. This was done by measuring the size of the area surrounding the tip of the electrode at which c-fos immunoreactivity was absent. This area was referred to as the “suppressive zone of DBS.” The size of the zone was measured in two

Normally, c-fos is not detected in SNr under control condition. In contrast, massive c-fos activation is induced in the neurons of SNr after ether anesthesia (Shehab and Julyan, 2002). In this experiment ether-

Counting of fos labeled nuclei was carried out on both sides of the brain (experimental v contralateral control SNr). Six sections were used in each animal; two from each of the three SNr levels (caudal, middle and rostral). One-way ANOVA and paired t-test were used for statistical analyses. Diameter of DBS suppression of fos expression (suppressive zone)

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sections at the middle level of SNr from each rat and expressed as a mean of the diameter of the suppressive zone.

frequencies (130 and 260 Hz) in suppressing tonic seizures using MES test.

Double and triple immunofluorescence staining and confocal microscopy

Bilateral electrode implantation in SNr

Double immunofluorescence labeling was carried out to verify whether the positive c-fos nuclei in SNr following ether anesthesia belong to neurons or glia. The sections were incubated overnight with a mixture of rabbit anti c-fos antibody (Santa Cruz 52, 1:100) and monoclonal mouse antibody (1:500) which was raised against nuclear protein (NeuN, Chemicon International Inc) known to be present in most of the neurons in CNS but not in glia (Mullen et al., 1992). After that the sections were rinsed with PBS three times and incubated with a mixture of donkey anti-mouse IgG conjugated with Alexa Fluor 488 (Molecular probes, 1:200) and donkey anti-rabbit IgG conjugated with lissamine rhodamine (LRSC, Jackson, 1:100) for 1 h. Sections were rinsed again in PBS, mounted on glass slides with glycerol based medium and stored in a fridge until examination. Double immunofluorescence labeling was also carried out to determine whether the positive c-fos nuclei belong to GABAergic neurons. The sections were incubated overnight with a mixture of mouse parvalbumin antibody (Sigma, 1:250), a marker of GABAergic neurons in SNr (Gerfen et al., 1985), and rabbit anti-fos antibody (1:100). Then sections were incubated with a mixture of donkey antimouse IgG conjugated with Alexa Fluor 488 (1:200) and donkey antirabbit IgG conjugated with LRSC for 1 h. After that the sections were rinsed with PBS and mounted on slides. Fluorescent and confocal (C1 Plus Nikon) microscopes were used for co-localization. Triple immunofluorescence labeling was carried out to determine whether the positive c-fos nuclei belong to neurons and not to astrocytes or to microglia. The sections were incubated overnight with a mixture of rabbit anti-Iba 1 (ionized calcium binding adapter molecule 1, Wako, 1:500, marker for microglia), mouse anti-GFAP (glial fibrillary acidic protein, marker for astrocytes, Vector, 1:200) and goat anti-cfos antibody (Santa Cruz 52, 1:100). After rinsing, sections were incubated with a mixture of donkey anti-rabbit IgG conjugated with cy5 (Jackson, 1:100), donkey anti-mouse IgG conjugated with Alexa Fluor 488 (1:200) and donkey anti-goat IgG conjugated with LRSC for 1 h. After that sections were rinsed in PBS, mounted on slides and stored in the fridge until examined by a confocal laser microscope for localization.

Under deep general anesthesia, two concentric bipolar electrodes were stereotaxically implanted in SNr at an angle of 15°. The stereotaxic co-ordinates were 4.00 mm lateral to the midline, 4.5– 5 mm posterior to bregma and 7.7–7.9 mm ventral to the skull with the nose-bar set at −3.5 mm (below the ear bars). Electrodes were chronically attached to the skull using screws and dental cement. The animals were allowed to recover for 3–4 days.

Controls Four sets of control experiments were carried out. In the first set of experiments, naïve rats (n = 3) were compared with another group of rats (n = 3) which were exposed to ether only and nigra sections were stained for c-fos. This was done to confirm our previous results (Shehab and Julyan, 2002), which showed that ether anesthesia induces c-fos in SNr in comparison with naïve untreated rats (n = 3). In the second set, 4 rats had electrode implanted in SNr only without stimulation. The aim of these experiments was to investigate whether the electrode implantation on its own causes activation of the c-fos immunoreactivity in SNr. In the third set of experiments, rats (n = 3) had electrodes implanted in SNr and were exposed to ether anesthesia. The aim of these experiments was to investigate if electrode implantation causes neuronal damage. In the fourth set, 12 rats had unilateral stimulation with 80, 130, 260 or 390 Hz (n = 3 for each group) as described above without exposure to ether anesthesia. The purpose of these experiments was to test the effect of deep brain stimulation on the basal level of c-fos. Experiment 2 In this experiment we investigated the effect of bilateral high frequency stimulation of SNr in suppressing tonic seizures. Based on the results of experiment 1 we compared the effects of two stimulation

Effects of bilateral deep brain stimulation of SNr on tonic seizures The stimulation was performed by rectangular, monopolar pulses, with pulse width of 60 μs and stimulation intensity between 150 and 300 μA (Digitimer Ltd). The intensity of the current was determined on each side of SNr separately using same criteria as described above. Each animal was tested on three consecutive days; once with stimulation of 130 Hz, second day with 260 Hz and third day with control stimulation (in which the wires were connected to the electrodes but without stimulation). The order of stimulation was counterbalanced. The stimulation lasted for 10 min immediately followed by the removal of the stimulating electrodes and delivery of the electric shock (1 s of 40 mA, 50 Hz AC) through the ear-clip electrodes. Animals' behavior during the stimulation and MES was recorded by video camera; and duration of tonic hind limb extension (THE) was also recorded (Shehab et al., 1995a,b, 1996, 1997, 2006, 2007). For analyses, only THE lasting longer than 3 s (Shehab et al., 2007) were considered. Hematoxylin staining Animals were perfused following the last test. Serial sections (50 μm) were cut in cryostat. The sections were mounted on gelatin slides, left to dry in air then dehydrated in graded alcohol, washed with water, stained with hematoxylin for 8 min. The sections were then washed with water, cleared with acid alcohol and washed in running tap water for 3 min. Finally the sections were dehydrated again in graded alcohol, cleared in xylene and mounted with DPX. This method was used to reveal the sites of the tips of the electrodes. Results Effects of ether anesthesia on c-fos expression in SNr Brain sections at the midbrain from untreated (control) rats did not show any basal level of c-fos immunoreactivity in SNr. In contrast, numerous c-fos labeled nuclei were detected in SNr after exposing the rat to ether anesthesia for 20 min (Fig. 1). This confirms our previously reported result (Shehab and Julyan, 2002) that exposing rats to ether anesthesia induces c-fos immunoreactivity in the basal ganglia including SNr and the subthalamic nucleus (STN, Fig. 1). In the current study, the ether-induced c-fos expression in SNr was used as a model to investigate the effects of four stimulation frequencies (80, 130, 260 and 390 Hz). The stimulation was unilaterally introduced in the left SNr while the right SNr was used as control. To confirm that the c-fos labeled nuclei belong to neurons rather than glia, double immunofluorescence staining with antibody against NeuN was carried out. A clear co-localization between c-fos labeled nuclei and NeuN labeled cells was observed (Figs. 2a–c). In another set of sections, a clear co-localization between nuclei positive for parvalbumin, a known marker of GABAergic neurons in the SNr (Gerfen and Wilson, 1996; Gerfen et al., 1985), and fos labeled nuclei was found (Figs. 2d–f). Finally, triple immunofluorescence staining with c-fos, GFAP (marker of astrocytes) and Iba1 (marker of microglia) antibodies was carried out

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Fig. 1. c-fos immunoreactivity in coronal sections at the STN and different rostro-caudal levels of SNr of a naive control rat (a–d) compared with a rat exposed to ether anesthesia (e-h) and a rat with electrode implantation and exposed to ether anesthesia (i–l). No c-fos immunoreactivity can be detected in the STN (a) or in the rostral (b), middle (c), and caudal (d) levels of the SNr of the untreated rat, while a massive induction of c-fos immunoreactivity can be seen in all these nuclei in rat exposed to ether anesthesia (e–h). Electrode implantation and exposure to ether anesthesia showed no effect on c-fos expression and did not cause neuronal damage. Arrow in k indicates the site of the electrode tip at the middle level of SNr. STN, subthalamic nuclei; SNr, substantia nigra reticulata. Scale bar = 0.3 mm.

to find out whether the positive c-fos nuclei were astrocytes or microglia. None of c-fos labeled nuclei in SNr were GFAP or Iba1 positive (Fig. 3). Control experiments No c-fos could be detected in SNr of naïve rats, while massive induction of neuronal activation was found after ether exposure. Control experiments designed to examine whether the electrode caused c-fos activation showed only few c-fos positive nuclei around the tip of electrodes, which most likely belonged to glia. Furthermore, in rats, which were both exposed to ether anesthesia and had electrodes

implanted, but had no stimulation, similar c-fos immunoreactivity in SNr was observed to the one seen in rats exposed to ether alone. The presence of activation of c-fos immunoreactivity in the vicinity of the tip of the electrode strongly suggests that the electrode implantation did not cause neuronal death (Figs. 1i–l). This indicates that neurons in SNr are not damaged as they still have the capability of expressing c-fos. Electrical stimulation at either 80, 130, 260 or 390 Hz without exposure to ether, resulted in small c-fos labeled nuclei around the tip of the electrode and few scattered ones in the rest of nigra. Double immunofluorescence staining showed that some of the nuclei around the tip of the electrode were also GFAP positive. Being small in size and GFAP positive suggest that they are astrocytes (not shown). However,

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Fig. 2. Confocal images of a coronal section of the SNr of an ether anesthetized rat showing the relationship of c-fos labeled nuclei (red in a and d) to NeuN in (b) and parvalbumin in (e). A colocalization of c-fos labeled nuclei with NeuN positive nuclei is seen in (c) and with neurons containing parvalbumin seen in (f). Scale bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the presence of few scattered labeled neurons in SNr might suggest that although HFS stimulation of nigra has mainly inhibitory action, occasional excitatory effect cannot be ruled out. In order to find out whether HFS might affect c-fos expression in the contralateral SNr further analysis was carried out. The results showed that there was no significant difference between the number of c-fos labeled nuclei in the right (unstimulated) nigra in the experimental animals compared with nigra in animals which had only ether exposure (ANOVA one way test, F = 0.966, P = 0.467). Effects of unilateral high frequency stimulation on ether-induced c-fos in SNr The expression of c-fos immunoreactivity was observed in the rostro-caudal levels of the left SNr. The number of c-fos positive nuclei in the left stimulated and the right non-stimulated substantia nigra for

different frequencies of stimulation is shown in Figs. 4 and 5. The comparison of the effects of different stimulation frequencies on c-fos expression showed that all frequencies except 80 Hz were effective in suppressing c-fos activity (Fig. 5). A one-way repeated measures ANOVA showed a significant effect of frequency F(1,68) = 21.897, P = 0.0001. A post-hoc Tukey's HSD test showed significant difference between all stimulation frequencies and 80 Hz stimulation frequency (P = 0.0001 for all frequencies) indicating that 80 Hz was not effective in inhibiting c-fos expression. Further, there were no significant differences between 390 and 260 Hz stimulation (P = 0.511), 390 and 130 Hz stimulation (P = 0.501) or between 260 Hz and 130 stimulation frequency (P = 1.000). High frequency stimulation with 130, 260 and 390 Hz produced remarkable suppression of c-fos expression in the vicinity of the tip of the electrode (Figs. 4 and 5). Although there was clear c-fos suppression in all rostro-caudal levels of the left SNr after stimulation with these

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Fig. 3. Confocal images of a coronal section of the SNr of an ether anesthetized rat showing that c-fos positive nuclei (red, in a) are not colocalized with either Iba 1 (blue in b), a marker for microglia, or GFAP (green in c), a marker for astrocytes. In panel d, images a, b and c are merged. SNr substantia nigra reticulata. Scale bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

frequencies c-fos labeled nuclei were still observed in the periphery of SNr (for example see Fig. 4). The percentage of c-fos suppression compared with controls is shown in Table 1. In comparison with the right control side, statistical analysis showed that stimulation with 130 Hz produced significant suppression in all levels of left SNr (paired t-test, P = 0.005 for rostral, P = 0.001 for mid and P = 0.007 for caudal SNr) (Fig. 5, Table 1). Further statistical analysis showed that the stimulation with 260 Hz (paired t-test, P = 0.000 for rostral, P = 0.000 for mid and P = 0.004 for caudal) and 390 Hz (paired t-test, P = 0.011 for rostral, P = 0.000 for mid and P = 0.013 for caudal) also produced significant suppression in all levels of left SNr (Fig. 5, Table 1). Another one-way ANOVA test was carried out to find out whether different frequencies had different effects on c-fos suppression at different levels of nigra. Tukey's HSD results showed that there was no significant difference after stimulation with 130 Hz (rostral to caudal P = 1.000, rostral to middle P = 1.000, and middle to caudal P = 0.918), 260 Hz (rostral to caudal P = 0.097, rostral to middle P = 1.000, and middle to caudal P = 0.137) or 390 Hz (rostral to caudal P = 1.000, rostral to middle P = 0.689, and middle to caudal P = 0.475).

order to investigate whether the extent of the c-fos suppression in the stimulated SNr is influenced by the stimulation frequency. Although there was a general but not significant tendency of increase in the percentage of the c-fos suppression with increased stimulation frequency, the data showed that increasing the frequency from 130 Hz to 260 and 390 Hz did not significantly increase the extent of the inhibitory effect (ANOVA one way test, F = 0.524, P = 0.599) (Fig. 5, Table 1). In the second method, the spread of action of HFS was assessed by measuring the diameter of the suppressive zone at the area surrounding the tip of the electrode. We refer to this area as the “suppressive zone of DBS.” The diameter of the DBS suppressive zone was on average 1.3 mm in size. The average size of SNr, in the ventromedial–dorsolateral direction, was 2 mm. Thus, a 1.3 mm suppression zone would amount to inhibition of approximately 64% of initially activated neurons. There were no significant differences in the mean diameter of the suppressive zone (Table 1) between the three stimulation frequencies (ANOVA one way test, F = 1.314, P = 0.287).

Effects of the frequency range on ether-induced c-fos immunoreactivity in the SNr

These experiments investigated the potential suppressive effects of 130 and 260 Hz electrical stimulation on induced tonic seizures measured by the duration of THE. Histological examination confirmed that electrodes were successfully placed bilaterally, in both SNr, in 19 out of 28 rats. The implantations were in the middle of SNr (5.8 mm to 5.3 mm caudal to bregma according to Paxinos and Watson 1998) in 10 out of those 19 rats. In the remaining 9 rats the implantations were placed either in

Having established that HFS caused neuronal inhibition, it was important to investigate the extent of effects of the different frequencies and the spread of their action throughout the rostrocaudal/mediolateral levels of the SNr. Two methods were used. In the first method, formal counting of c-fos labeled nuclei was carried out in both sides of SNr in

Effects of bilateral high frequency stimulation on induced tonic seizures

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Fig. 4. Coronal sections at different rostro-caudal levels on SNr showing an example of the suppressive effect of HFS at 260 Hz on ether-induced c-fos immunoreactivity. Arrow in b indicates the site of the tip of the electrode at the middle level of SNr. Note the remarkable suppression of ether-induced c-fos immunoreactivity in all rostro-caudal levels of left SNr compared with contralateral right side. SNr, substantia nigra reticulata. Scale bar = 0.5 mm.

1250 1000

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0

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rostral (n = 4, 4.8 mm caudal to bregma) or caudal part (n = 5, 6.03 and 6.40 mm caudal to bregma) of SNr (Fig. 6). In all rats (n = 19) bilateral HFS at either 130 or 260 Hz failed to reduce the THE elicited by the electroshock. The mean THE scores following the electrical stimulation at 130 and 260 Hz were 7.02 ± 0.7 s and 7.3 ± 0.7 s, respectively. The mean THE score in the same animals when no electrical current was applied as a control, was 7.8 ± 0.3 s. Statistical analysis showed that there were no significant differences in means of the THE scores following bilateral electrical stimulation at either 130 Hz (Wilcoxon's signed rank test P = 0.777, Z = −0.283) or 260 Hz (Wilcoxon's signed rank test P = 0.573, Z = −0.563) compared with the control values. To test the potential effect of electrode position within SNr the effects of bilateral stimulation in which the electrodes were placed in the rostral, middle and caudal regions of nigra were re-analyzed. The mean THE scores following the electrical stimulation of the middle of nigra (n = 10) at 130 and 260 Hz were 6.2 ± 1 s and 6.8 ± 1 s, respectively. However, although the mean of these scores were shorter on average, there were no significant differences when compared with control values (7.5 ± 0.6 s) i.e. following the application of the electrode without the current, (Wilcoxon's signed rank test P = 0.515, Z = −0.652 for 130 Hz and P = 0.386, Z = −0.866 for 260 Hz). Similarly, there were no significant effects of HFS when it was applied either to rostral (n = 4, mean THE scores are 7.0 ± 1.2 s for control, 6.7 ± 1.3 s for 130 Hz and 7.4 ± 0.8 s for 260 Hz, Wilcoxon's signed rank test P = 0.715, Z = −0.365 for 130 Hz and P = 0.715, Z = −0.365 for 260 Hz) or caudal nigra (n = 5, mean THE scores are 8.6± 0.3 s for control, 8.9 ± 0.4 s for 130 Hz and 8 ± 0.9 s for 260 Hz, Wilcoxon's signed rank test P = 0.500, Z = −0.674 for 130 Hz and P = 0.686, Z = −0.405 for 260 Hz). Furthermore, no significant differences in the mean THE scores following application of electrodes without current were obtained on first, second or third day of stimulation (Kruskal–Wallis test P = 0.65). These results are consistent with previous studies (Shehab et al., 1995a, 1997) in which repeated exposure to electric shock over three consecutive days had no effects on the duration of THE under control conditions.

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Fig. 5. Histograms demonstrate the suppressive effects of four different frequency stimulations (80,130,260 and 390 Hz) on c-fos expression in three rostro-caudal levels of left SNr compared with right (control) side. Stimulation with 80 Hz produced no significant suppression of c-fos labeled nuclei in left SNr. In comparison, 130, 260 and 390 Hz stimulation produced significant suppressive effect.

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Table 1 Percentage of c-fos suppression in stimulated SNr (left side) compared to the control side (right side) and the diameter of the suppressive zone following the stimulation with 80, 130, 260 and 390 Hz. Frequency Hz

% of suppressed c-fos labeled nuclei in the stimulated SNr normalized to contra-lateral non-stimulated SNr ± SEM

Diameter of nigra mm ± SEM

Diameter of the suppressive zone mm ± SEM

% of the suppressed diameter

80 130 260 390

− 2 ± 3.9 59 ± 5.5 67 ± 5.2 64 ± 6.1

2.1 ± 0.03 2.1 ± 0.03 2.0 ± 0.05

1.4 ± 0.07 1.3 ± 0.04 1.2 ± 0.1

64.3 63.8 62.0

Discussion The main results of this study can be summarized as follows: (1) HFS of substantia nigra reticulata at130, 260 and 390 Hz produced remarkable and significant local neuronal inhibition while stimulation with a lower frequency, at 80 Hz, produced no significant inhibitory

effect; (2) increasing the frequency of stimulation from 130 to 390 Hz did not have any additional inhibitory effect; (3) the average diameter of the inhibitory zone with 130, 260 and 390 Hz stimulations was approximately 1.3 mm suggesting that on average 63% of SNr was inhibited with the electrodes and stimulation frequencies used in this study and (4) despite the considerable inhibitory effect of HFS of SNr neurons at 130 and 260 Hz, stimulation with these frequencies failed to produce an anticonvulsant effect, as no significant reduction in the duration of tonic seizures was obtained. Inhibitory effects of HFS The results of the first part of experiment 1 showed that there was no basal level of c-fos immunoreactivity in the SNr (Applegate et al., 1995; Shehab and Julyan, 2002; Shehab et al., 1992; Wirtshafter and Asin, 1995) or STN (Ruskin and Marshall, 1995; Shehab et al., 2006; Shehab and Julyan, 2002) and confirmed the results of our previous study (Shehab and Julyan, 2002) by demonstrating that exposure to etherinduced massive c-fos immunoreactivity in the SNr and STN. c-fos is one of the immediate genes that can be induced in most of CNS cells by various stimuli (Herrera and Robertson, 1996; Morgan et al., 1987; Morgan and Curran, 1991). It is, therefore, considered as a marker for neuronal activation (Bullitt, 1990; Dragunow and Faull, 1989; Herrera and Robertson, 1996). The source of c-fos induction in the basal ganglia following ether anesthesia is not known. However, it has been shown that the induction of c-fos immunoreactivity in the hypothalamus requires receptors activation rather than spike activity (Luckman et al., 1994). Recently we reported that one of the possibilities of the etherinduced c-fos in the basal ganglia is related to excitatory stimulation which probably involves the activation of the STN (Shehab and Julyan, 2002). This was based on histological evidence which showed that muscimol (GABA-agonist) injection into the STN (Shehab et al., 2006) or SNr (Shehab and Julyan, 2002) suppressed c-fos expression in STN and SNr respectively. Therefore, this study used ether-induced c-fos expression in the SNr as a model to investigate whether high-frequency stimulation applied to the SNr could have the same inhibitory effect as muscimol. Electrical stimulation with four different frequencies (80, 130, 260 and 390 Hz) was used. The results indicate that HFS at 130, 260 and 390 Hz suppress the c-fos immunoreactivity while lower frequency stimulation at 80 Hz did not suppress the c-fos immunoreactivity in the SNr. Similar c-fos suppressive effects have been previously shown when muscimol was injected into SNr (Shehab and Julyan, 2002) or STN (Shehab et al., 2006) indicating that HFS has, in these kind of experiments, an inhibitory neuronal effect. Effect of bilateral stimulation of SNr on tonic seizures

Fig. 6. Schematic diagram of coronal sections of rat midbrain at rostro-caudal levels of substantia SNr. Filled circles represent the sites of the tips of the implanted electrodes bilaterally inside SNr. Empty symbols represent sites of the tips of the electrodes implanted outside SNr either unilaterally or on both sides. The number beside each section represents its distance in millimeters caudal to bregma according to Paxinos and Watson (1998). cp, cerebral peduncle; SNc, substantia nigra compacta; SNr, substantia nigra reticulata.

The role of SNr in epileptic seizure suppression has been well documented (Depaulis et al., 1994; Gale, 1988; Garant and Gale, 1987; Iadarola and Gale, 1982; Moshé et al., 1994; Shehab et al., 1996). Experimental studies showed that pharmacological inhibition of SNr in different epilepsy models produces anticonvulsant effects and suppresses seizures including those which are likely to originate from the forebrain, as well as tonic motor seizures that are likely to originate from the brainstem (Browning and Nelson, 1986; Gale, 1988; Gale and Browning, 1988). Further studies have shown that DBS of nigra is

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anticonvulsant against forebrain seizures (Feddersen et al., 2007; Shi et al., 2006; Velisek et al., 2002). However, the results of this study demonstrate that DBS did not produce a significant inhibition of brainstem tonic seizures in the MES model, in spite of its suppressive effects on ether-induced SNr activation. In support of this effect, Usui et al. (2005) reported that although HFS of STN suppressed secondary generalized limbic seizures in rats, the same treatment of nigra had no effect. A similar phenomenon was shown when the STN was manipulated as an anticonvulsant region. It has been shown that pharmacological suppression of the STN has anticonvulsant effects in a range of animal models of epilepsy, including forebrain limbic motor seizures (Dybdal and Gale, 2000), flurothyl-induced seizures (Velísková et al., 1996), amygdala kindling (Deransart et al., 1998) and generalized nonconvulsive seizures (Deransart et al., 1996). In addition, HFS of the STN can also suppress absence seizures (Vercueil et al., 1998) and flurothyl-induced convulsions (Lado et al., 2003). However, muscimolinduced inhibition, neurotoxic lesion and HFS of STN all failed to induce any reliable protective effect against the generalized motor seizures induced by electroshock, although all of these manipulations are likely to suppress activity in the STN (Shehab et al., 2006). Interestingly, these findings are consistent with previous reports which showed that both muscimol injections (Velísková et al., 1996) and HFS of the STN at 130 Hz (Lado et al., 2003) increase the threshold for inducing clonic seizures, but has no effect on tonic seizure activity in the flurothyl model of epilepsy. In support of the notion that the chemical inhibition of the SNr does not suppress tonic seizures in all epilepsy models, Deransart et al. (2001) found that bilateral injection of muscimol into SNr suppressed the clonic component but had no effect on tonic seizures in audiogenic rats. Taken together, the data of this study and of our previous work (Shehab et al., 2006) demonstrate that high frequency stimulation of SNr and STN at 130 and 260 Hz fails to produce significant anticonvulsant effects in the MES model of tonic seizures. HFS of SNr fails to suppress tonic seizures in the MES model of epilepsy In such a case of negative findings, there are several issues that need to be addressed to demonstrate that the lack of positive effects is not due to any failures in procedure. Location of electrodes Previous studies indicate that the anticonvulsant effects of SN manipulation can be region specific. Thus it has been shown that injections of muscimol or zolpidem (an agonist of benzodiapezine receptor) (Moshé et al., 1994; Velísková et al., 1998) or HFS (Velisek et al., 2002) were anticonvulsant if they were applied in rostral nigra but ineffective in caudal nigra against seizures induced by fluorothyl. However, in our previous experiments we reported that for the present electroshock model, muscimol-induced suppression of activity in caudal nigra was the most effective in suppressing tonic seizures (Shehab et al., 1996). Therefore, in the current study we compared the effects of HFS at the rostral, middle and caudal levels of nigra. The results showed that at none of the locations did 130 or 260 Hz produce any significant reduction of the mean duration of the tonic hind limb extension. It is therefore concluded that, under conditions of the present experiments, there were no regions of substantia nigra where the application of DBS had anticonvulsant effects in the electroshock model. Potential effect of the size of inhibition We reported previously that a dose of 60 ng of muscimol, which was sufficient to suppress tonic hind limb extension in the MES test (Shehab et al., 1996), caused approximately a suppressive zone with a diameter of 2.4 mm (Shehab and Julyan, 2002). In the present experiments the

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diameter of the SNr (ventromedially–dorsolaterally) was approximately 2 mm on average. Assuming that the effects of HFS extend uniformly in the rostrocaudal and mediolateral directions, our results show, for the first time, that the diameter of the suppressive zone was approximately 1.3 mm for all three stimulation frequencies (Table 1), which means that around 62–64% out of the initially activated SNr neurons are suppressed by DBS. Thus, the difference between the size of the suppressive zone caused by the chemical injection of muscimol and electrical stimulation with HFS might be one of the reasons that may explain the failure of HFS to significantly reduce the THE in the MES test. This prompts a consequent question: is it essential to suppress all neurons of SNr in order to effectively induce an anticonvulsant effect on tonic seizures? Furthermore, muscimol is a chemical compound and only has a local inhibitory action on neurons in the vicinity of the tip of the injecting needle. In case of the HFS, the field of the electrical stimulation is not a fixed panel surrounding the electrode, implying that the three dimensional effect of the induced electrical stimulation may also affect the neighboring structures including the cerebral peduncle, afferent terminals terminating in SNr and efferent fibers originating from SNr. Stimulation of these structures might also interfere with the local neuronal inhibitory outcome which might in turn antagonize the anticonvulsant effects of nigral inhibition. These two issues may account for differences in effectiveness between the two treatments. Duration of HFS Another technical issue potentially accounting for the lack of HFS anticonvulsive effect is related to the duration of the bilateral stimulation of SNr. However, there is no reference related to the duration of the HFS optimal to produce anticonvulsant effect. In previous studies, HFS was applied for a period of time ranging from 5 s to 1 h (Feddersen et al., 2007; Shehab et al., 2006; Shi et al., 2006; Usui et al., 2005; Velisek et al., 2002; Vercueil et al., 1998). In the present study the duration of 10 min was used, and this did not produce anticonvulsant effects at either 130 or 260 Hz. Although, it is very likely that HFS for 10 min duration is enough to silence the neuronal activity in SNr, it is worth investigating whether a shorter or a longer duration of stimulation could be effective to produce anticonvulsant effects. Parameters of the stimulation: current intensity and frequency The present study tested stimulation frequencies at 130 and 260 Hz. Those frequencies were chosen based on previous clinical trials and animals experiments (Benazzouz et al., 2000; Boex et al., 2007; Chabardes et al., 2002; Handforth et al., 2006; Lado et al., 2003; Velisek et al., 2002; Vercueil et al., 1998; Wille et al., 2011). Electrical stimulation at 130 Hz has anticonvulsant effects in different animal models of epilepsy (Lado et al., 2003; Velisek et al., 2002; Vercueil et al., 1998) and in epileptic patients (Boex et al., 2007; Chabardes et al., 2002; Handforth et al., 2006). However, although the results of this study are in accordance with earlier results, confirming that stimulation with130 and 260 Hz causes a significant neuronal inhibition, the same stimulation frequencies were not anticonvulsant against tonic seizures. At this point of the investigation, it cannot be ruled out that frequencies higher than 260 would be required to suppress tonic seizures induced by electroshock. However, we believe it is unlikely that increasing the frequency of stimulation would have produced positive effects in suppressing tonic seizures for the following reasons. First, our results show that increasing the stimulation from 130 to 260 and 390 Hz did not increase the suppressive effects on c-fos expression. This makes the possibility of having an anticonvulsant effect following an increase in stimulation frequency doubtful. Secondly, using a genetic model of absence epilepsy, Feddersen et al. (2007) found that although stimulation of nigra at 60 Hz produced anticonvulsant effects, an increase of the stimulation frequency up to 500 Hz did not produce any further changes of convulsions threshold. Thirdly, Lado et al. (2003)

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used flurothyl model of epilepsy and showed that stimulation of the subthalamic nucleus at 130 Hz produced an anticonvulsant effect, whereas increasing the stimulation frequency to 260 Hz did not have an additional effect on seizures. However, it should be noted that increasing stimulation frequency to 800 Hz resulted in a decrease of seizures threshold, eventually becoming pro-convulsant (Lado et al., 2003). It should also be pointed out that the intensity of the current used in the present study was set just below the intensity required to elicit motor effect. Therefore, the use of larger currents, which could be needed in order to produce a reliable anticonvulsant effect and concurrently eliciting motor side effects, would clearly be unacceptable in clinical use. Timing of HFS Finally, the electrical stimulation was switched off before rats received electric shock. This was deemed important in order to prevent the exposure of animals to two different routes of electrical stimulations (one through HFS and one through the MES test) at the same time. Such simultaneous application of two electrical currents would possibly interfere with the effects of each. Moreover, the short period of time between switching off the brain stimulation and the introduction of the electrical shock (approximately 1–2 s) could hardly be the reason for the negative results. This is further supported by data showing that HFS of the STN, with 130 Hz, induces a decrease in the neuronal activity with and after-effect lasting from 50 to120 s (Benazzouz et al., 1995). Thus, taken altogether there is sufficient evidence suggesting that the negative anticonvulsant effect of the HFS in the MES test was not due to methodological limitations. Conclusions In conclusion, the results of our study confirm the potent inhibitory effects of HFS at 130, 260 and 390 Hz on SNr neurons. The average diameter of the inhibitory effects of HFS at these frequencies was approximately 1.3 mm. However, in spite of its inhibitory effect, HFS of SNr, with current frequencies (130 and 260 Hz) and intensities of stimulation, is ineffective in suppressing tonic seizures originating from hindbrain structures. This suggests that it may not be adequate for treatment of epileptic patients who suffer from generalized tonic seizures. Further studies using different kinds of electrodes, which would allow near-complete inhibition of nigra, or using different periods of stimulation, like on-off current application, might shed more light on the use of HFS in tonic epileptic seizures. Acknowledgments This work was supported by research grants from the Sheikh Hamdan Award for Medical SciencesMRG-24/2005–2006 and NRF/UAE University (31M013). We thank Muhammed Madathil for histochemical support and Dr. R. Bernsen and Professor N. Nagelkerke for their help in statistical analyses. We also thank Dr Rima Aboudan for her editing of the paper. References Applegate, C.D., Pretel, S., Piekut, D.T., 1995. The substantia nigra pars reticulata, seizures and Fos expression. Epilepsy Res. 20, 31–39. Benabid, A.L., Wallace, B., Mitrofanis, J., Xia, R., Piallat, B., Chabardes, S., Berger, F., 2005. A putative generalized model of the effects and mechanism of action of high frequency electrical stimulation of the central nervous system. Acta Neurol. Belg. 105, 149–157. Benazzouz, A., Piallat, B., Pollak, P., Benabid, A.L., 1995. Responses of substantia nigra pars reticulata and globus pallidus complex to high frequency stimulation of the subthalamic nucleus in rats: electrophysiological data. Neurosci. Lett. 189, 77–80. Benazzouz, A., Gao, D.M., Ni, Z.G., Piallat, B., Bouali-Benazzouz, R., Benabid, A.L., 2000. Effect of high-frequency stimulation of the subthalamic nucleus on the neuronal activities of the substantia nigra pars reticulata and ventrolateral nucleus of the thalamus in the rat. Neuroscience 99, 289–295.

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