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Polarity-dependent effect of low-frequency stimulation on amygdaloid kindling in rats
Brain Stimulation 6 (2013) 190e197
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Polarity-dependent effect of low-frequency stimulation on amygdaloid kindling in rats Zhenghao Xu a,1, Yi Wang a,1, Miaomiao Jin a, Jiaxing Yue a, Cenglin Xu a, Xiaoying Ying a, Dengchang Wu a, b, Shihong Zhang a, Zhong Chen a, * a
Department of Pharmacology, Key Laboratory of Medical Neurobiology of the Ministry of Health of China and Zhejiang Province Key Laboratory of Neurobiology, College of Pharmaceutical Sciences, School of Medicine, Zhejiang University, Hangzhou 310058, China b Department of Neurology, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 January 2012 Received in revised form 15 March 2012 Accepted 24 April 2012 Available online 1 June 2012
Background: Low-frequency stimulation (LFS, <5 Hz) has been proposed as an alternative option for the treatment of epilepsy. The stimulation pole, anode and cathode, may make different contributions to the anti-epileptic effect of LFS. Objective: To determine whether electrode polarity influences the anti-epileptic effect of LFS at the kindling focus in amygdaloid kindling rats. Methods: The effect of bipolar and monopolar (or unipolar) LFS at the amygdala in different polarity directions on amygdaloid kindling acquisition, kindled seizures and electroencephalogram (EEG) were tested. Results: Bipolar LFS in the same direction of polarity as the kindling stimulation but not in the reverse direction retarded kindling acquisition. Anodal rather than cathodal monopolar LFS attenuated kindling acquisition and kindled seizures. Bipolar LFS showed a stronger anti-epileptic effect than monopolar LFS. Furthermore, anodal LFS (both bipolar and monopolar) decreased, while cathodal LFS increased the power of the EEG from the amygdala; the main changes in power were in the delta (0.5e4 Hz) band, which was specifically increased during kindling acquisition. Conclusions: Our results provide the first evidence that the effect of LFS at the kindling focus on amygdaloid kindling in rats is polarity-dependent, and this may be due to the different effects of anodal and cathodal LFS on the activity in the amygdala, especially on the delta band activity. So, It is likely that the electrode polarity, especially that for anodal current, is a key factor affecting the clinical effects of LFS on epilepsy. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Low-frequency stimulation Polarity-dependent Bipolar and monopolar Electroencephalogram Kindling
Low-frequency stimulation (LFS, <5 Hz) targeting specific brain areas is emerging as an alternative option for the treatment of uncontrolled epilepsy, with the advantages of reversibility, adjustability and minimal invasion [1]. LFS using direct-current [2,3], square waves [4] or sine waves [5,6] suppresses seizures in rodents. Recently, our studies first demonstrated that LFS (monophasic square-wave pulses, 1 Hz) of the central piriform cortex and the cerebellar fastigial nucleus, regions other than the kindling focus, interferes with amygdaloid kindling seizures [7e9]. Therefore, LFS may be a promising approach for clinical anti-epileptic treatment. The authors report no conflicts of interest. * Corresponding author. Tel.: þ86 571 88208228; fax: þ86 571 88208422. E-mail address: [email protected] (Z. Chen). 1 These authors contributed equally to the paper. 1935-861X/$ e see front matter Ó 2013 Elsevier Inc. All rights reserved. doi:10.1016/j.brs.2012.04.010
The mechanism underlying LFS treatment for epilepsy is still unknown and the optimal strategy for stimulation remains unclear. The target and frequency of stimulation have been considered as crucial factors [1,10]. However, contradictory results of LFS at specific targets have frequently been found. For example: (1) It has been reported that focal LFS has an inhibitory effect (termed ‘quenching’ effect) on kindling seizures in rats [2]; however another study indicates that this is mediated by low-level direct-current leakage from the stimulator and not the LFS itself [3]. (2) D’Arcangelo et al. [11] reported that LFS at the entorhinal cortex (EC) depresses the ability of the EC to generate ictal activity in magnesium-free medium in mouse hippocampus-EC slices, while Solger et al. [12] found that LFS at the EC under similar conditions has no effect or even induces seizure-like events. (3) The effects of focal LFS treatment on patients with temporal lobe epilepsy are also complex [13,14]. Recently, we
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reported that there may be a “time-window” during the afterdischarge when LFS interferes with amygdaloid kindling; LFS delivered outside the window has no effect on the kindling and even aggravates kindling seizures [8,15,16]. The time-window phenomenon may partly explain some of the paradoxical features of LFS [11,12] and indicates that the time delay of closed-loop stimulation may be crucial for LFS treatment. Recently, Bekar et al. [17] reported that accumulation of adenosine at the cathode is crucial for the high-frequency stimulation-mediated attenuation of tremor. In contrast, brain-derived neurotrophic factor (BDNF) secretion at the anode may be a key mediator for the improved motor skill learning induced by transcranial direct-current stimulation (tDCS) of the motor cortex [18]. Both BDNF and adenosine may participate in epileptogenesis and modulate seizures [19e23]. Some other studies also showed that anodal and cathodal stimulation at the motor cortex may have different effects on neuronal activity [24e26]. Thus, it seems that anode and cathode may make different contributions to the antiepileptic effect of LFS. However, few reports have discussed the difference between anode and cathode in the LFS treatment of epilepsy. Therefore, the present study was designed to determine whether the electrode polarity affects the anti-epileptic effects of LFS at the kindling focus on amygdaloid kindling in rats. Materials and methods
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kindling stimulation (bipolar LFSþ), group 2 received reversed bipolar LFS (bipolar LFS), and group 3 received sham LFS (control). All rats were stimulated daily for 20 days. The LFS parameters were the same as in our previous study [15,16], that is, LFS (monophasic square-wave pulses, 100e150 mA, 1 Hz, 0.1 ms/pulse and 900 pulses) was delivered immediately after cessation of kindling stimulation. The criteria for using 100e150 mA for LFS were: if the ADT was less than 150 mA, the intensity of LFS was equal to the ADT; if the ADT was equal or greater than 150 mA, the intensity of LFS was equal to 150 mA. Effect of monopolar LFS on amygdaloid kindling acquisition To further define the polarity-specificity in the LFS treatment of epilepsy and exclude the influence of polarity of kindling stimulation, we implanted a tripolar electrode into the amygdala: two outer electrode tips were used for kindling stimulation and the middle electrode tip was used to deliver monopolar LFS to the kindling focus (Fig. 2A). The reference pole for monopolar LFS was attached to a neck muscle. Rats were divided into 4 groups matched for ADT. Group 1 received bipolar LFSþ; group 2 received anodal monopolar LFS (the anode in the kindling focus and the cathode in a neck muscle); group 3 received cathodal monopolar LFS (the cathode in the kindling focus and the anode in a neck muscle); and group 4 received sham LFS (control). All rats were stimulated daily for 20 days.
Animal surgery and amygdaloid kindling Effect of monopolar LFS on amygdaloid-kindled seizures Under pentobarbital sodium anesthesia (35 mg/kg), male SpragueeDawley rats (260e300 g, Grade II, Certificate No. SCXK2008-0033, Experimental Animal Center, Zhejiang Academy of Medical Science, Hangzhou, China) were mounted in a stereotaxic apparatus (512600, Stoelting, USA) and electrodes were implanted into the right basolateral amygdala (AP: 2.4 mm, L: 4.8 mm, V: 8.8 mm); the reference and ground screws were placed in the bone over the cerebellum (AP: 10.5 mm, L: 1.5 mm). All coordinates were measured from bregma according to the atlas of Paxinos and Watson [27]. Electrode location was histologically verified in all animals after the behavioral studies. The electrodes were made of twisted stainless-steel Teflon-coated wires (791500, diameter 0.125 mm, A.M. Systems, USA) insulated except for 0.5 mm at the tip and the maximal tip separation was 0.7e0.8 mm. After 7e10 days of recovery, the afterdischarge threshold (ADT) was determined (monophasic square-wave pulses, 60 Hz, 1 ms/pulse and 60 pulses) with a constant current stimulator (SEN-7203, SS-202J; Nihon Kohden, Tokyo, Japan). The stimulation intensity began at 40 mA, and was subsequently increased in 20 mA steps every 30 min until at least 5 s of afterdischarge (AD) was elicited. The intensity that first produced an AD (5 s or longer) was designated the ADT and used for daily kindling stimulation. The electroencephalogram (EEG) of the amygdala was recorded with a digital amplifier (Synamps RT, Neuroscan System, USA). Seizure severity was classified according to the Racine scale [28]: (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. Seizure stages 1e3 indicate focal seizures, while stages 4e5 are generalized seizures [29]. In addition to seizure stage, the AD duration (ADD) was recorded. When the animals exhibited three consecutive stage 5 seizures, they were regarded as fully kindled. Effect of bipolar LFS on amygdaloid kindling acquisition Rats were divided into 3 groups matched for ADT. Group 1 received bipolar LFS using the same electrodes and polarity as
When fully kindled, rats were divided into 3 groups matched by generalized seizure threshold (GST): Group 1 received anodal monopolar LFS; group 2 received cathodal monopolar LFS; and group 3 (control) received sham LFS. All rats were stimulated daily for 10 days. The GST was determined by increasing the current intensity in steps until a generalized seizure was elicited and used for daily kindling stimulation. The intensity of LFS was equal to 150 mA for all kindled rats. Amygdala EEG recording and analysis Raw EEG signals in a unipolar setting (about 5 min long) were recorded with band-pass filters spanning DC to 200 Hz and sampled at 1000 Hz. Recorded EEG signals were analyzed offline by Scan 4.5 (Neuroscan, Compumedics Ltd, Melbourne, Australia). The entire EEG time series was digitally band-pass filtered from 0.3 to 100 Hz, then divided into consecutive 4 s epochs (4096 points). EEG epochs with artifact were rejected by visual inspection and finally 50 artifact-free epochs were selected by random-sorting for spectral analysis. Spectral analysis using the fast Fourier transform (FFT) was run on the 4 s epoch with a Hanning window to avoid edge effects. The FFT output provided a total power for each 4 s epoch with a frequency resolution of 0.244 Hz between zero and 100 Hz. These 0.244 Hz frequency bins were subsequently averaged within 6 frequency bands: d (delta, 0.5e4 Hz); q (theta, 4e8 Hz); a (alpha, 8e12 Hz); b (beta, 12e30 Hz); g (gamma, 30e100 Hz); and total power (0e100 Hz). The EEG of the amygdala was recorded before and after LFS for about 5 min. To investigate the effect of bipolar LFS on amygdaloid EEG, rats were divided into two groups: group 1 received bipolar LFSþ; group 2 received sham LFS. To investigate the effect of monopolar LFS on amygdaloid EEG, rats were self-controlled for EEG recording. Rats were given sham LFS on the first day and divided into two groups on the second day: group 1 received anodal monopolar LFS; group 2 received cathodal monopolar LFS. In
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Figure 1. Effects of bipolar LFS on amygdaloid kindling acquisition. (A) Schematic of the polarities for kindling and bipolar LFS: “þ” and “” represent the anode and cathode of electrical stimulation. (B) Effects of LFS on the behavioral stage of seizures during amygdaloid kindling acquisition (n ¼ 9 for bipolar LFSþ, n ¼ 8 for bipolar LFS, n ¼ 9 for control). (C) Effect of LFS on the number of stimulations at each seizure stage and (D) required to reach each seizure stage. *P < 0.05, **P < 0.01 and ***P < 0.001 represent differences from the control group. ##P < 0.01 represents the difference between bipolar LFSþ and LFS.
addition, to investigate the effect of kindling stimulation on the activity of the amygdala, background EEG of the amygdala was also recorded before daily kindling stimulation during kindling acquisition.
Statistics Analysis of group differences in kindling acquisition was performed by two-way analysis of variance (ANOVA) for repeated
Figure 2. Effects of monopolar LFS on amygdaloid kindling acquisition. (A) Schematic of the polarities for kindling and LFS: “þ” and “” represent the anode and cathode of electrical stimulation. Cathodal monopolar LFS, the cathode in the kindling focus and the anode in a neck muscle; anodal monopolar LFS, the anode in the kindling focus and the cathode in a neck muscle. (B) Effects of LFS on the behavioral stage of seizures during amygdaloid kindling acquisition (n ¼ 10 for anodal monopolar LFS, n ¼ 10 for cathodal monopolar LFS, n ¼ 9 for bipolar LFSþ and n ¼ 10 for control). (C) Effects of LFS on afterdischarge duration (ADD) during amygdaloid kindling acquisition. (D) Effect of LFS on the number of stimulations at each seizure stage and (E) required to reach each seizure stage. (F) Number of rats in different seizure states at day 20. *P < 0.05, **P < 0.01 and ***P < 0.001 represent differences from the control; #P < 0.05, ##P < 0.01 and ###P < 0.001 represent differences from cathodal monopolar LFS; &&&P < 0.01 represents the difference between anodal monopolar LFS and bipolar LFSþ.
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Figure 3. Effects of bipolar LFS at the amygdala on amygdaloid EEG. Power spectra before (A) and after LFS (B) (n ¼ 5 for each group). (C) Comparison of the power spectra of amygdaloid EEG among groups. (D), (E) and (F) Summary data from A, B and C. *P < 0.05 and **P < 0.01 represent differences from the control; #P < 0.05, ##P < 0.01 and ### P < 0.001 represent differences between anode and cathode.
measures followed by Fisher’s least significant difference (LSD) test. Comparisons of the number of stimulations for each seizure stage during kindling acquisition were made with one-way ANOVA followed by the LSD test. The paired-samples t-test was used to compare the power spectra of EEG between electrode tips (polarities), and the independent-samples t-test was applied to analyze the power spectra of the EEG between groups. In kindled seizures, one-way ANOVA followed by the LSD test was used to calculate the statistical significance. In the case of comparing the incidence of focal or generalized seizures, the c2 test was used. Data are presented as mean S.E.M. Statistical analysis was carried out using SPSS 16.0 for Windows. For all analyses, the tests were two-sided and a P < 0.05 was considered significant.
Results Polarity-dependent effect of bipolar LFS on kindling acquisition Bipolar LFSþ of the kindling focus retarded the progression of behavioral seizure stages during kindling acquisition (P < 0.001 and P < 0.01 compared with bipolar LFS and control groups, respectively; Fig. 1B). However, bipolar LFS had no effect on the progression of behavioral seizure stages during the 20 days of stimulation. Control animals were fully kindled after 17.3 0.8 stimulations. In contrast, only 1 out of 9 rats receiving bipolar LFSþ was fully kindled after 20 stimulations, and 5 rats remained at the stage of focal seizures. Four out of 8 rats receiving bipolar LFS were fully kindled, and all rats reached the stage of generalized seizures. We further calculated the number of stimulations needed to reach and remain at each seizure stage. Bipolar LFSþ increased the number of days for animals staying in stages 0 (P < 0.01; Fig. 1C) and 1 (P < 0.05; Fig. 1C), as well as increasing the number of days to each stage compared with control (P < 0.05; Fig. 1D). Bipolar LFS had no effect on the number of stimulations needed to reach and
remain at each seizure stage compared with control (P > 0.05; Fig. 1C and D).
Polarity-dependent effect of monopolar LFS on kindling acquisition Anodal monopolar LFS of the kindling focus slowed the progression of behavioral seizure stages (P < 0.01; Fig. 2B) and shortened the ADD (P < 0.05; Fig. 2C) during kindling acquisition compared with control, while cathodal monopolar LFS had no such effect (P > 0.05; Fig. 2B and C). Anodal monopolar LFS exerted a weaker effect on kindling progression than bipolar LFSþ (P < 0.01; Fig. 2B). The distribution of rats at the stage of focal seizures, generalized seizures and the fully kindled state in each group are shown in Fig. 2F. We further calculated the number of stimulations needed to reach and remain at each seizure stage. Anodal monopolar LFS increased the number of stimulations to stages 3e5 (P < 0.05 for each stage; Fig. 2D), while cathodal monopolar LFS had no effect. In the anodal monopolar LFS group, rats stayed longer in stages 1 and 2 (P < 0.05 and P < 0.01; Fig. 2C), whereas there was no effect in the cathodal monopolar LFS group (P > 0.05; Fig. 2C).
Polarity-dependent effect of bipolar LFS on amygdala EEG Before bipolar LFS, there was no significant difference in any frequency band of the amygdala EEG between the anode and cathode in both sham and bipolar LFS groups (P > 0.05; Fig. 3A and D). After 15 min of bipolar LFS, the power in d, q, b and total bands recorded by the cathodal electrode was much higher than that recorded by anodal electrode (P < 0.001, P < 0.01, P < 0.05 and P < 0.001; Fig. 3B and E). We further found that the change of power ratio (post-LFS power/pre-LFS power-1) in d and total bands at the anode in the bipolar LFS group was much lower, while that at the cathode was much higher than in the sham group (P < 0.001 and P < 0.001; Fig. 3C and F). In addition, the change of power ratio in
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Figure 4. Effects of monopolar LFS at the amygdala on amygdaloid EEG. (A) Summary data from power spectra of amygdaloid EEG before and after sham LFS (n ¼ 14) on day 1; (C) and (E) anodal monopolar LFS and cathodal monopolar LFS on day 2 (n ¼ 7 for each group). (B), (D) and (F) Summary data from comparing the power spectra of amygdaloid EEG before and after sham LFS, anodal monopolar LFS and cathodal monopolar LFS. *P < 0.05 and **P < 0.01 represent differences from the control electrode.
the b band at the cathode was slightly higher than that at the anode in the bipolar LFS group (P < 0.05; Fig. 3F). Polarity-dependent effect of monopolar LFS on amygdala EEG There was no significant difference in any frequency band of the amygdala EEG between the two electrodes before LFS or after sham LFS (P > 0.05; Fig. 4AeC and E). The power in the d band recorded by the stimulating electrode in anodal monopolar LFS (the anode) was much lower than that recorded by the non-stimulating control electrode (P < 0.05; Fig. 4C) after 15 min of anodal monopolar LFS. And the power in d, q and total bands recorded by the stimulating electrode of cathodal monopolar LFS (the cathode) was much higher than that recorded by the non-stimulating control electrode (P < 0.001, P < 0.01 and P < 0.01; Fig. 4E) after 15 min of cathodal monopolar LFS. Furthermore, comparison of the power ratio change (post-LFS power/pre-LFS power-1) in the different frequency bands of the amygdala EEG revealed that anodal monopolar LFS decreased the power in d and total bands (P < 0.001 and P < 0.05; Fig. 4D), while the cathodal monopolar LFS increased the power in d, b and total bands (P < 0.001, P < 0.01 and P < 0.01; Fig. 4D). Polarity-dependent effect of monopolar LFS on amygdaloid-kindled seizures There was no significant difference in any frequency band of the amygdaloid EEG between the kindling and sham groups on day 0.
When the rats were fully kindled, the power in the d band of the kindled group was much higher than that of the sham group (P < 0.05; Fig. 5A). The summary data of the power in all bands are shown in Fig. 5A. In addition, anodal monopolar LFS decreased the incidence of generalized seizures (P < 0.001; Fig. 5B) and reduced the average seizure stage (P < 0.001; Fig. 5C). However, cathodal monopolar LFS had no effect on kindled seizures compared with control (P > 0.05; Fig. 5B and C). Discussion In the present study, we were interested to find that both bipolar and monopolar LFS of the kindling focus retarded the progression of kindling acquisition, in which bipolar LFS had a stronger effect than monopolar LFS. More interestingly, bipolar LFSþ but not LFS at the kindling focus retarded amygdaloid kindling acquisition; and anodal rather than cathodal monopolar LFS at the kindling focus attenuated amygdaloid kindling and amygdaloid-kindled seizures. So the results of the present study provide the first direct evidence of a polarity-specific phenomenon for LFS at the kindling focus that interferes with amygdaloid kindling seizures in rats. It is likely that the electrode polarity may be a key factor affecting the clinical effects of LFS on epilepsy. Recently, it was reported that the blocking effect of directcurrent fields on seizure-like discharges in low-calcium CA1 slices is influenced by the direction of the current fields [30,31]. We were interested to find that the effect of bipolar LFSþ was dramatically
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Figure 5. Effects of amygdala kindling stimulation on amygdaloid EEG and effects of monopolar LFS at the amygdala on kindled seizures. (A) Summary data from power spectra of amygdaloid EEG on days 0, 7 and 17 during kindling acquisition (n ¼ 7 for kindling; n ¼ 5 for sham kindling; EEG recorded by the middle tip of the tripolar electrode). (B) Mean incidence of generalized seizures (GS) and (C) seizure stages during 10 days of LFS treatment (n ¼ 5 for anodal monopolar LFS; n ¼ 6 for cathodal monopolar LFS; n ¼ 5 for control). *P < 0.05 and ***P < 0.001 represent differences from the control; ##P < 0.01 represent differences between anodal and cathodal monopolar LFS.
different from that of bipolar LFS on kindling acquisition. The effect of bipolar LFSþ is in accord with our previous findings [15,32] that LFS markedly retards the kindling acquisition process. Rats receiving bipolar LFSþ remained at the stage of focal seizures after 20 stimulations; that is, bipolar LFSþ may be effective in avoiding generalized but not partial seizures. This effect is also induced by other types of electrical stimulation such as vagal nerve stimulation [33]. In contrast, bipolar LFS did not prolonged the number of days to each stage during kindling acquisition; that is, bipolar LFS had no effect on kindling acquisition. It has also been reported that the effect of tDCS over the sensorimotor cortex on the threshold of localized seizure activity depends on the polarity in a rat cortical ramp model of epilepsy [36]. Different electrical fields may have different direct effects on neuronal circuit-building [34,35]. Even though the mechanism remains unclear, these findings at least suggest that a polarity-specific phenomenon exists in the LFS treatment of epilepsy, and the electrode polarity might be crucial for this treatment. This feature may partly explain the inconsistent results in some clinical studies and animal experiments [2,3,11e13], in which LFS may be delivered to the same target but with a different direction of current. To further define the polarity-specific phenomenon in the LFS treatment of epilepsy, we used a tripolar electrode and monopolar LFS. Monopolar electrical stimulation is mostly applied clinically for deep brain stimulation [10]. Typically, the cathode is in the brain target because it activates neurons more easily than the anode, and the anode is often used as a reference and placed in a muscle or on the skin [10,37]. However, so far, there is no definite consensus with respect to the selection of bipolar or monopolar stimulation modes. In the present study, we were surprised to find that anodal but not cathodal monopolar LFS at the kindling focus had a remarkable anti-epileptogenesis effect in kindling rats and an anticonvulsive effect in kindled rats. These results provide direct evidence that a polarity-dependent phenomenon exists in LFS at the kindling focus to interfere with amygdaloid kindling in rats and suggest that
anodal current might be important for the LFS treatment of epilepsy. In addition, we found that the anti-epileptic effect of monopolar LFS was weaker than bipolar LFS at the kindling focus. It is likely that bipolar stimulation is more suitable than monopolar stimulation for the focal LFS treatment of epilepsy. Furthermore, we recorded and analyzed amygdaloid EEG to determine whether LFS directly affects the activity of the amygdala in a polarity-dependent manner. We were interested to find that anodal LFS (both bipolar and monopolar) mainly decreased the power of d activity in the amygdala, while cathodal LFS increased it, and slightly increased that of b activity. Moreover, the power of d activity was increased during kindling acquisition. So far, a close relationship between d activity and epilepsy has been proposed but is still unclear: d activity has been considered as an EEG marker of the epileptic focus [38e45]; the slow oscillation in the d band facilitates hippocampal afterdischarges in urethane-anesthetized rats [46]; and many anti-epileptic drugs, such as carbamazepine [47e49], valproate [49], levetiracetam [49,50] and diazepam [51], also influence the background d activity of the EEG. Thus, although we have no further direct evidence, our results additionally and strongly suggest that d activity may be important for epileptogenesis, and interfering with the d activity of the kindling focus may contribute to the anti-epileptic effect of LFS, especially monopolar LFS. The mechanism underlying LFS treatment for epilepsy is still unknown. There are several possibilities for the mechanism of the polarity-dependent effect of LFS on epileptogenesis and d activity: (1) Enhanced benzodiazepine binding may be involved in the antiepileptic effects of repetitive LFS (1 Hz) [4] and benzodiazepines such as diazepam suppress d activity [51]. So, the effect of anodal LFS in suppressing d band activity and retarding kindling acquisition might be associated with the enhanced benzodiazepine binding detected after repetitive LFS. (2) Bekar et al. [17] reported that the release of adenosine at the cathode is crucial for deep brain stimulation-mediated attenuation of tremor. Adenosine activation
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of the A1 receptor enhances d EEG activity [52]. Thus, it is possible that the cathodal polarity LFS enhanced d EEG activity by inducing the releasing of adenosine [53], and this may have no effect on epileptogenesis because high concentrations of extracellular adenosine, induced by kindling stimulation and epileptiform activity, may aggravate epileptogenesis by activating the A2 receptor [54]. In addition, Mohammad-Zadeh et al. found that LFS prevents the kindling-induced elevation of cyclic AMP (cAMP) levels in kindled animals [20]. It has also been reported that anodal direct-current may modulate the adenosine-sensitive generation of cAMP in rat cerebral cortex [55]. So, the adenosine-cAMP pathway might also participate in the anti-epileptogenesis and d activity modulation effects of anodal LFS. (3) Although no significant changes in background g band were detected as a consequence of LFS or kindling stimulation in the present study, g oscillations are important in the brain with epilepsy [56]. Recently, Lévesque et al. [57] reported that synchronized g oscillations (30e50 Hz) in the amygdalo-hippocampal network are associated with seizure propagation and severity. LFS might reduce the ADDs and seizure severity of amygdaloid kindling by directly influencing the synchronization of neural oscillations during ADs. In conclusion, our results provide the first and direct evidence that the anti-epileptic effect of LFS at the kindling focus on amygdaloid kindling in rats is polarity-dependent, and this may be due to anodal and cathodal LFS having different effects on activity in the kindling focus, especially d activity. Bipolar stimulation may be more effective than monopolar stimulation for the focal LFS treatment of epilepsy. So, It is likely that the electrode polarity, especially that for anodal current, is a key factor affecting the clinical effects of LFS on epilepsy.
Acknowledgments This work was funded by the National Basic Research of China 973 Program (2011CB504403) and by the National Natural Science Foundation of China (81030061, 30725047 and 81173042). We are grateful to Dr. IC Bruce for reading the manuscript.
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Polarity-dependent effect of low-frequency stimulation on amygdaloid kindling in rats Zhenghao Xu a,1, Yi Wang a,1, Miaomiao Jin a, Jiaxing Yue a, Cenglin Xu a, Xiaoying Ying a, Dengchang Wu a, b, Shihong Zhang a, Zhong Chen a, * a
Department of Pharmacology, Key Laboratory of Medical Neurobiology of the Ministry of Health of China and Zhejiang Province Key Laboratory of Neurobiology, College of Pharmaceutical Sciences, School of Medicine, Zhejiang University, Hangzhou 310058, China b Department of Neurology, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 January 2012 Received in revised form 15 March 2012 Accepted 24 April 2012 Available online 1 June 2012
Background: Low-frequency stimulation (LFS, <5 Hz) has been proposed as an alternative option for the treatment of epilepsy. The stimulation pole, anode and cathode, may make different contributions to the anti-epileptic effect of LFS. Objective: To determine whether electrode polarity influences the anti-epileptic effect of LFS at the kindling focus in amygdaloid kindling rats. Methods: The effect of bipolar and monopolar (or unipolar) LFS at the amygdala in different polarity directions on amygdaloid kindling acquisition, kindled seizures and electroencephalogram (EEG) were tested. Results: Bipolar LFS in the same direction of polarity as the kindling stimulation but not in the reverse direction retarded kindling acquisition. Anodal rather than cathodal monopolar LFS attenuated kindling acquisition and kindled seizures. Bipolar LFS showed a stronger anti-epileptic effect than monopolar LFS. Furthermore, anodal LFS (both bipolar and monopolar) decreased, while cathodal LFS increased the power of the EEG from the amygdala; the main changes in power were in the delta (0.5e4 Hz) band, which was specifically increased during kindling acquisition. Conclusions: Our results provide the first evidence that the effect of LFS at the kindling focus on amygdaloid kindling in rats is polarity-dependent, and this may be due to the different effects of anodal and cathodal LFS on the activity in the amygdala, especially on the delta band activity. So, It is likely that the electrode polarity, especially that for anodal current, is a key factor affecting the clinical effects of LFS on epilepsy. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Low-frequency stimulation Polarity-dependent Bipolar and monopolar Electroencephalogram Kindling
Low-frequency stimulation (LFS, <5 Hz) targeting specific brain areas is emerging as an alternative option for the treatment of uncontrolled epilepsy, with the advantages of reversibility, adjustability and minimal invasion [1]. LFS using direct-current [2,3], square waves [4] or sine waves [5,6] suppresses seizures in rodents. Recently, our studies first demonstrated that LFS (monophasic square-wave pulses, 1 Hz) of the central piriform cortex and the cerebellar fastigial nucleus, regions other than the kindling focus, interferes with amygdaloid kindling seizures [7e9]. Therefore, LFS may be a promising approach for clinical anti-epileptic treatment. The authors report no conflicts of interest. * Corresponding author. Tel.: þ86 571 88208228; fax: þ86 571 88208422. E-mail address: [email protected] (Z. Chen). 1 These authors contributed equally to the paper. 1935-861X/$ e see front matter Ó 2013 Elsevier Inc. All rights reserved. doi:10.1016/j.brs.2012.04.010
The mechanism underlying LFS treatment for epilepsy is still unknown and the optimal strategy for stimulation remains unclear. The target and frequency of stimulation have been considered as crucial factors [1,10]. However, contradictory results of LFS at specific targets have frequently been found. For example: (1) It has been reported that focal LFS has an inhibitory effect (termed ‘quenching’ effect) on kindling seizures in rats [2]; however another study indicates that this is mediated by low-level direct-current leakage from the stimulator and not the LFS itself [3]. (2) D’Arcangelo et al. [11] reported that LFS at the entorhinal cortex (EC) depresses the ability of the EC to generate ictal activity in magnesium-free medium in mouse hippocampus-EC slices, while Solger et al. [12] found that LFS at the EC under similar conditions has no effect or even induces seizure-like events. (3) The effects of focal LFS treatment on patients with temporal lobe epilepsy are also complex [13,14]. Recently, we
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reported that there may be a “time-window” during the afterdischarge when LFS interferes with amygdaloid kindling; LFS delivered outside the window has no effect on the kindling and even aggravates kindling seizures [8,15,16]. The time-window phenomenon may partly explain some of the paradoxical features of LFS [11,12] and indicates that the time delay of closed-loop stimulation may be crucial for LFS treatment. Recently, Bekar et al. [17] reported that accumulation of adenosine at the cathode is crucial for the high-frequency stimulation-mediated attenuation of tremor. In contrast, brain-derived neurotrophic factor (BDNF) secretion at the anode may be a key mediator for the improved motor skill learning induced by transcranial direct-current stimulation (tDCS) of the motor cortex [18]. Both BDNF and adenosine may participate in epileptogenesis and modulate seizures [19e23]. Some other studies also showed that anodal and cathodal stimulation at the motor cortex may have different effects on neuronal activity [24e26]. Thus, it seems that anode and cathode may make different contributions to the antiepileptic effect of LFS. However, few reports have discussed the difference between anode and cathode in the LFS treatment of epilepsy. Therefore, the present study was designed to determine whether the electrode polarity affects the anti-epileptic effects of LFS at the kindling focus on amygdaloid kindling in rats. Materials and methods
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kindling stimulation (bipolar LFSþ), group 2 received reversed bipolar LFS (bipolar LFS), and group 3 received sham LFS (control). All rats were stimulated daily for 20 days. The LFS parameters were the same as in our previous study [15,16], that is, LFS (monophasic square-wave pulses, 100e150 mA, 1 Hz, 0.1 ms/pulse and 900 pulses) was delivered immediately after cessation of kindling stimulation. The criteria for using 100e150 mA for LFS were: if the ADT was less than 150 mA, the intensity of LFS was equal to the ADT; if the ADT was equal or greater than 150 mA, the intensity of LFS was equal to 150 mA. Effect of monopolar LFS on amygdaloid kindling acquisition To further define the polarity-specificity in the LFS treatment of epilepsy and exclude the influence of polarity of kindling stimulation, we implanted a tripolar electrode into the amygdala: two outer electrode tips were used for kindling stimulation and the middle electrode tip was used to deliver monopolar LFS to the kindling focus (Fig. 2A). The reference pole for monopolar LFS was attached to a neck muscle. Rats were divided into 4 groups matched for ADT. Group 1 received bipolar LFSþ; group 2 received anodal monopolar LFS (the anode in the kindling focus and the cathode in a neck muscle); group 3 received cathodal monopolar LFS (the cathode in the kindling focus and the anode in a neck muscle); and group 4 received sham LFS (control). All rats were stimulated daily for 20 days.
Animal surgery and amygdaloid kindling Effect of monopolar LFS on amygdaloid-kindled seizures Under pentobarbital sodium anesthesia (35 mg/kg), male SpragueeDawley rats (260e300 g, Grade II, Certificate No. SCXK2008-0033, Experimental Animal Center, Zhejiang Academy of Medical Science, Hangzhou, China) were mounted in a stereotaxic apparatus (512600, Stoelting, USA) and electrodes were implanted into the right basolateral amygdala (AP: 2.4 mm, L: 4.8 mm, V: 8.8 mm); the reference and ground screws were placed in the bone over the cerebellum (AP: 10.5 mm, L: 1.5 mm). All coordinates were measured from bregma according to the atlas of Paxinos and Watson [27]. Electrode location was histologically verified in all animals after the behavioral studies. The electrodes were made of twisted stainless-steel Teflon-coated wires (791500, diameter 0.125 mm, A.M. Systems, USA) insulated except for 0.5 mm at the tip and the maximal tip separation was 0.7e0.8 mm. After 7e10 days of recovery, the afterdischarge threshold (ADT) was determined (monophasic square-wave pulses, 60 Hz, 1 ms/pulse and 60 pulses) with a constant current stimulator (SEN-7203, SS-202J; Nihon Kohden, Tokyo, Japan). The stimulation intensity began at 40 mA, and was subsequently increased in 20 mA steps every 30 min until at least 5 s of afterdischarge (AD) was elicited. The intensity that first produced an AD (5 s or longer) was designated the ADT and used for daily kindling stimulation. The electroencephalogram (EEG) of the amygdala was recorded with a digital amplifier (Synamps RT, Neuroscan System, USA). Seizure severity was classified according to the Racine scale [28]: (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. Seizure stages 1e3 indicate focal seizures, while stages 4e5 are generalized seizures [29]. In addition to seizure stage, the AD duration (ADD) was recorded. When the animals exhibited three consecutive stage 5 seizures, they were regarded as fully kindled. Effect of bipolar LFS on amygdaloid kindling acquisition Rats were divided into 3 groups matched for ADT. Group 1 received bipolar LFS using the same electrodes and polarity as
When fully kindled, rats were divided into 3 groups matched by generalized seizure threshold (GST): Group 1 received anodal monopolar LFS; group 2 received cathodal monopolar LFS; and group 3 (control) received sham LFS. All rats were stimulated daily for 10 days. The GST was determined by increasing the current intensity in steps until a generalized seizure was elicited and used for daily kindling stimulation. The intensity of LFS was equal to 150 mA for all kindled rats. Amygdala EEG recording and analysis Raw EEG signals in a unipolar setting (about 5 min long) were recorded with band-pass filters spanning DC to 200 Hz and sampled at 1000 Hz. Recorded EEG signals were analyzed offline by Scan 4.5 (Neuroscan, Compumedics Ltd, Melbourne, Australia). The entire EEG time series was digitally band-pass filtered from 0.3 to 100 Hz, then divided into consecutive 4 s epochs (4096 points). EEG epochs with artifact were rejected by visual inspection and finally 50 artifact-free epochs were selected by random-sorting for spectral analysis. Spectral analysis using the fast Fourier transform (FFT) was run on the 4 s epoch with a Hanning window to avoid edge effects. The FFT output provided a total power for each 4 s epoch with a frequency resolution of 0.244 Hz between zero and 100 Hz. These 0.244 Hz frequency bins were subsequently averaged within 6 frequency bands: d (delta, 0.5e4 Hz); q (theta, 4e8 Hz); a (alpha, 8e12 Hz); b (beta, 12e30 Hz); g (gamma, 30e100 Hz); and total power (0e100 Hz). The EEG of the amygdala was recorded before and after LFS for about 5 min. To investigate the effect of bipolar LFS on amygdaloid EEG, rats were divided into two groups: group 1 received bipolar LFSþ; group 2 received sham LFS. To investigate the effect of monopolar LFS on amygdaloid EEG, rats were self-controlled for EEG recording. Rats were given sham LFS on the first day and divided into two groups on the second day: group 1 received anodal monopolar LFS; group 2 received cathodal monopolar LFS. In
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Figure 1. Effects of bipolar LFS on amygdaloid kindling acquisition. (A) Schematic of the polarities for kindling and bipolar LFS: “þ” and “” represent the anode and cathode of electrical stimulation. (B) Effects of LFS on the behavioral stage of seizures during amygdaloid kindling acquisition (n ¼ 9 for bipolar LFSþ, n ¼ 8 for bipolar LFS, n ¼ 9 for control). (C) Effect of LFS on the number of stimulations at each seizure stage and (D) required to reach each seizure stage. *P < 0.05, **P < 0.01 and ***P < 0.001 represent differences from the control group. ##P < 0.01 represents the difference between bipolar LFSþ and LFS.
addition, to investigate the effect of kindling stimulation on the activity of the amygdala, background EEG of the amygdala was also recorded before daily kindling stimulation during kindling acquisition.
Statistics Analysis of group differences in kindling acquisition was performed by two-way analysis of variance (ANOVA) for repeated
Figure 2. Effects of monopolar LFS on amygdaloid kindling acquisition. (A) Schematic of the polarities for kindling and LFS: “þ” and “” represent the anode and cathode of electrical stimulation. Cathodal monopolar LFS, the cathode in the kindling focus and the anode in a neck muscle; anodal monopolar LFS, the anode in the kindling focus and the cathode in a neck muscle. (B) Effects of LFS on the behavioral stage of seizures during amygdaloid kindling acquisition (n ¼ 10 for anodal monopolar LFS, n ¼ 10 for cathodal monopolar LFS, n ¼ 9 for bipolar LFSþ and n ¼ 10 for control). (C) Effects of LFS on afterdischarge duration (ADD) during amygdaloid kindling acquisition. (D) Effect of LFS on the number of stimulations at each seizure stage and (E) required to reach each seizure stage. (F) Number of rats in different seizure states at day 20. *P < 0.05, **P < 0.01 and ***P < 0.001 represent differences from the control; #P < 0.05, ##P < 0.01 and ###P < 0.001 represent differences from cathodal monopolar LFS; &&&P < 0.01 represents the difference between anodal monopolar LFS and bipolar LFSþ.
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Figure 3. Effects of bipolar LFS at the amygdala on amygdaloid EEG. Power spectra before (A) and after LFS (B) (n ¼ 5 for each group). (C) Comparison of the power spectra of amygdaloid EEG among groups. (D), (E) and (F) Summary data from A, B and C. *P < 0.05 and **P < 0.01 represent differences from the control; #P < 0.05, ##P < 0.01 and ### P < 0.001 represent differences between anode and cathode.
measures followed by Fisher’s least significant difference (LSD) test. Comparisons of the number of stimulations for each seizure stage during kindling acquisition were made with one-way ANOVA followed by the LSD test. The paired-samples t-test was used to compare the power spectra of EEG between electrode tips (polarities), and the independent-samples t-test was applied to analyze the power spectra of the EEG between groups. In kindled seizures, one-way ANOVA followed by the LSD test was used to calculate the statistical significance. In the case of comparing the incidence of focal or generalized seizures, the c2 test was used. Data are presented as mean S.E.M. Statistical analysis was carried out using SPSS 16.0 for Windows. For all analyses, the tests were two-sided and a P < 0.05 was considered significant.
Results Polarity-dependent effect of bipolar LFS on kindling acquisition Bipolar LFSþ of the kindling focus retarded the progression of behavioral seizure stages during kindling acquisition (P < 0.001 and P < 0.01 compared with bipolar LFS and control groups, respectively; Fig. 1B). However, bipolar LFS had no effect on the progression of behavioral seizure stages during the 20 days of stimulation. Control animals were fully kindled after 17.3 0.8 stimulations. In contrast, only 1 out of 9 rats receiving bipolar LFSþ was fully kindled after 20 stimulations, and 5 rats remained at the stage of focal seizures. Four out of 8 rats receiving bipolar LFS were fully kindled, and all rats reached the stage of generalized seizures. We further calculated the number of stimulations needed to reach and remain at each seizure stage. Bipolar LFSþ increased the number of days for animals staying in stages 0 (P < 0.01; Fig. 1C) and 1 (P < 0.05; Fig. 1C), as well as increasing the number of days to each stage compared with control (P < 0.05; Fig. 1D). Bipolar LFS had no effect on the number of stimulations needed to reach and
remain at each seizure stage compared with control (P > 0.05; Fig. 1C and D).
Polarity-dependent effect of monopolar LFS on kindling acquisition Anodal monopolar LFS of the kindling focus slowed the progression of behavioral seizure stages (P < 0.01; Fig. 2B) and shortened the ADD (P < 0.05; Fig. 2C) during kindling acquisition compared with control, while cathodal monopolar LFS had no such effect (P > 0.05; Fig. 2B and C). Anodal monopolar LFS exerted a weaker effect on kindling progression than bipolar LFSþ (P < 0.01; Fig. 2B). The distribution of rats at the stage of focal seizures, generalized seizures and the fully kindled state in each group are shown in Fig. 2F. We further calculated the number of stimulations needed to reach and remain at each seizure stage. Anodal monopolar LFS increased the number of stimulations to stages 3e5 (P < 0.05 for each stage; Fig. 2D), while cathodal monopolar LFS had no effect. In the anodal monopolar LFS group, rats stayed longer in stages 1 and 2 (P < 0.05 and P < 0.01; Fig. 2C), whereas there was no effect in the cathodal monopolar LFS group (P > 0.05; Fig. 2C).
Polarity-dependent effect of bipolar LFS on amygdala EEG Before bipolar LFS, there was no significant difference in any frequency band of the amygdala EEG between the anode and cathode in both sham and bipolar LFS groups (P > 0.05; Fig. 3A and D). After 15 min of bipolar LFS, the power in d, q, b and total bands recorded by the cathodal electrode was much higher than that recorded by anodal electrode (P < 0.001, P < 0.01, P < 0.05 and P < 0.001; Fig. 3B and E). We further found that the change of power ratio (post-LFS power/pre-LFS power-1) in d and total bands at the anode in the bipolar LFS group was much lower, while that at the cathode was much higher than in the sham group (P < 0.001 and P < 0.001; Fig. 3C and F). In addition, the change of power ratio in
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Figure 4. Effects of monopolar LFS at the amygdala on amygdaloid EEG. (A) Summary data from power spectra of amygdaloid EEG before and after sham LFS (n ¼ 14) on day 1; (C) and (E) anodal monopolar LFS and cathodal monopolar LFS on day 2 (n ¼ 7 for each group). (B), (D) and (F) Summary data from comparing the power spectra of amygdaloid EEG before and after sham LFS, anodal monopolar LFS and cathodal monopolar LFS. *P < 0.05 and **P < 0.01 represent differences from the control electrode.
the b band at the cathode was slightly higher than that at the anode in the bipolar LFS group (P < 0.05; Fig. 3F). Polarity-dependent effect of monopolar LFS on amygdala EEG There was no significant difference in any frequency band of the amygdala EEG between the two electrodes before LFS or after sham LFS (P > 0.05; Fig. 4AeC and E). The power in the d band recorded by the stimulating electrode in anodal monopolar LFS (the anode) was much lower than that recorded by the non-stimulating control electrode (P < 0.05; Fig. 4C) after 15 min of anodal monopolar LFS. And the power in d, q and total bands recorded by the stimulating electrode of cathodal monopolar LFS (the cathode) was much higher than that recorded by the non-stimulating control electrode (P < 0.001, P < 0.01 and P < 0.01; Fig. 4E) after 15 min of cathodal monopolar LFS. Furthermore, comparison of the power ratio change (post-LFS power/pre-LFS power-1) in the different frequency bands of the amygdala EEG revealed that anodal monopolar LFS decreased the power in d and total bands (P < 0.001 and P < 0.05; Fig. 4D), while the cathodal monopolar LFS increased the power in d, b and total bands (P < 0.001, P < 0.01 and P < 0.01; Fig. 4D). Polarity-dependent effect of monopolar LFS on amygdaloid-kindled seizures There was no significant difference in any frequency band of the amygdaloid EEG between the kindling and sham groups on day 0.
When the rats were fully kindled, the power in the d band of the kindled group was much higher than that of the sham group (P < 0.05; Fig. 5A). The summary data of the power in all bands are shown in Fig. 5A. In addition, anodal monopolar LFS decreased the incidence of generalized seizures (P < 0.001; Fig. 5B) and reduced the average seizure stage (P < 0.001; Fig. 5C). However, cathodal monopolar LFS had no effect on kindled seizures compared with control (P > 0.05; Fig. 5B and C). Discussion In the present study, we were interested to find that both bipolar and monopolar LFS of the kindling focus retarded the progression of kindling acquisition, in which bipolar LFS had a stronger effect than monopolar LFS. More interestingly, bipolar LFSþ but not LFS at the kindling focus retarded amygdaloid kindling acquisition; and anodal rather than cathodal monopolar LFS at the kindling focus attenuated amygdaloid kindling and amygdaloid-kindled seizures. So the results of the present study provide the first direct evidence of a polarity-specific phenomenon for LFS at the kindling focus that interferes with amygdaloid kindling seizures in rats. It is likely that the electrode polarity may be a key factor affecting the clinical effects of LFS on epilepsy. Recently, it was reported that the blocking effect of directcurrent fields on seizure-like discharges in low-calcium CA1 slices is influenced by the direction of the current fields [30,31]. We were interested to find that the effect of bipolar LFSþ was dramatically
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Figure 5. Effects of amygdala kindling stimulation on amygdaloid EEG and effects of monopolar LFS at the amygdala on kindled seizures. (A) Summary data from power spectra of amygdaloid EEG on days 0, 7 and 17 during kindling acquisition (n ¼ 7 for kindling; n ¼ 5 for sham kindling; EEG recorded by the middle tip of the tripolar electrode). (B) Mean incidence of generalized seizures (GS) and (C) seizure stages during 10 days of LFS treatment (n ¼ 5 for anodal monopolar LFS; n ¼ 6 for cathodal monopolar LFS; n ¼ 5 for control). *P < 0.05 and ***P < 0.001 represent differences from the control; ##P < 0.01 represent differences between anodal and cathodal monopolar LFS.
different from that of bipolar LFS on kindling acquisition. The effect of bipolar LFSþ is in accord with our previous findings [15,32] that LFS markedly retards the kindling acquisition process. Rats receiving bipolar LFSþ remained at the stage of focal seizures after 20 stimulations; that is, bipolar LFSþ may be effective in avoiding generalized but not partial seizures. This effect is also induced by other types of electrical stimulation such as vagal nerve stimulation [33]. In contrast, bipolar LFS did not prolonged the number of days to each stage during kindling acquisition; that is, bipolar LFS had no effect on kindling acquisition. It has also been reported that the effect of tDCS over the sensorimotor cortex on the threshold of localized seizure activity depends on the polarity in a rat cortical ramp model of epilepsy [36]. Different electrical fields may have different direct effects on neuronal circuit-building [34,35]. Even though the mechanism remains unclear, these findings at least suggest that a polarity-specific phenomenon exists in the LFS treatment of epilepsy, and the electrode polarity might be crucial for this treatment. This feature may partly explain the inconsistent results in some clinical studies and animal experiments [2,3,11e13], in which LFS may be delivered to the same target but with a different direction of current. To further define the polarity-specific phenomenon in the LFS treatment of epilepsy, we used a tripolar electrode and monopolar LFS. Monopolar electrical stimulation is mostly applied clinically for deep brain stimulation [10]. Typically, the cathode is in the brain target because it activates neurons more easily than the anode, and the anode is often used as a reference and placed in a muscle or on the skin [10,37]. However, so far, there is no definite consensus with respect to the selection of bipolar or monopolar stimulation modes. In the present study, we were surprised to find that anodal but not cathodal monopolar LFS at the kindling focus had a remarkable anti-epileptogenesis effect in kindling rats and an anticonvulsive effect in kindled rats. These results provide direct evidence that a polarity-dependent phenomenon exists in LFS at the kindling focus to interfere with amygdaloid kindling in rats and suggest that
anodal current might be important for the LFS treatment of epilepsy. In addition, we found that the anti-epileptic effect of monopolar LFS was weaker than bipolar LFS at the kindling focus. It is likely that bipolar stimulation is more suitable than monopolar stimulation for the focal LFS treatment of epilepsy. Furthermore, we recorded and analyzed amygdaloid EEG to determine whether LFS directly affects the activity of the amygdala in a polarity-dependent manner. We were interested to find that anodal LFS (both bipolar and monopolar) mainly decreased the power of d activity in the amygdala, while cathodal LFS increased it, and slightly increased that of b activity. Moreover, the power of d activity was increased during kindling acquisition. So far, a close relationship between d activity and epilepsy has been proposed but is still unclear: d activity has been considered as an EEG marker of the epileptic focus [38e45]; the slow oscillation in the d band facilitates hippocampal afterdischarges in urethane-anesthetized rats [46]; and many anti-epileptic drugs, such as carbamazepine [47e49], valproate [49], levetiracetam [49,50] and diazepam [51], also influence the background d activity of the EEG. Thus, although we have no further direct evidence, our results additionally and strongly suggest that d activity may be important for epileptogenesis, and interfering with the d activity of the kindling focus may contribute to the anti-epileptic effect of LFS, especially monopolar LFS. The mechanism underlying LFS treatment for epilepsy is still unknown. There are several possibilities for the mechanism of the polarity-dependent effect of LFS on epileptogenesis and d activity: (1) Enhanced benzodiazepine binding may be involved in the antiepileptic effects of repetitive LFS (1 Hz) [4] and benzodiazepines such as diazepam suppress d activity [51]. So, the effect of anodal LFS in suppressing d band activity and retarding kindling acquisition might be associated with the enhanced benzodiazepine binding detected after repetitive LFS. (2) Bekar et al. [17] reported that the release of adenosine at the cathode is crucial for deep brain stimulation-mediated attenuation of tremor. Adenosine activation
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of the A1 receptor enhances d EEG activity [52]. Thus, it is possible that the cathodal polarity LFS enhanced d EEG activity by inducing the releasing of adenosine [53], and this may have no effect on epileptogenesis because high concentrations of extracellular adenosine, induced by kindling stimulation and epileptiform activity, may aggravate epileptogenesis by activating the A2 receptor [54]. In addition, Mohammad-Zadeh et al. found that LFS prevents the kindling-induced elevation of cyclic AMP (cAMP) levels in kindled animals [20]. It has also been reported that anodal direct-current may modulate the adenosine-sensitive generation of cAMP in rat cerebral cortex [55]. So, the adenosine-cAMP pathway might also participate in the anti-epileptogenesis and d activity modulation effects of anodal LFS. (3) Although no significant changes in background g band were detected as a consequence of LFS or kindling stimulation in the present study, g oscillations are important in the brain with epilepsy [56]. Recently, Lévesque et al. [57] reported that synchronized g oscillations (30e50 Hz) in the amygdalo-hippocampal network are associated with seizure propagation and severity. LFS might reduce the ADDs and seizure severity of amygdaloid kindling by directly influencing the synchronization of neural oscillations during ADs. In conclusion, our results provide the first and direct evidence that the anti-epileptic effect of LFS at the kindling focus on amygdaloid kindling in rats is polarity-dependent, and this may be due to anodal and cathodal LFS having different effects on activity in the kindling focus, especially d activity. Bipolar stimulation may be more effective than monopolar stimulation for the focal LFS treatment of epilepsy. So, It is likely that the electrode polarity, especially that for anodal current, is a key factor affecting the clinical effects of LFS on epilepsy.
Acknowledgments This work was funded by the National Basic Research of China 973 Program (2011CB504403) and by the National Natural Science Foundation of China (81030061, 30725047 and 81173042). We are grateful to Dr. IC Bruce for reading the manuscript.
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