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Noninvasive transcranial direct current stimulation in a genetic absence model
Epilepsy & Behavior 26 (2013) 42–50
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Noninvasive transcranial direct current stimulation in a genetic absence model M. Zobeiri ⁎, G. van Luijtelaar Department of Biological Psychology, Donders Centre for Cognition, Radboud University Nijmegen, The Netherlands
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Article history: Received 2 August 2012 Revised 5 October 2012 Accepted 7 October 2012 Available online 30 November 2012 Keywords: Absence epilepsy WAG/Rij rats Repetitive cathodal stimulation Cathodal tDCS Spectral analyses Hyperpolarization SWDs EEG
a b s t r a c t The proposed area of onset for absence epilepsy characteristic of spontaneously occurring spike and slowwave discharges (SWDs) in the genetic absence rat model is the subgranular layer of the somatosensory cortex. Modulation of the hyperexcitable cortical foci by bilateral transcranial direct current stimulation (tDCS) might change the expression of SWDs. The effects of cathodal and anodal tDCS as well as cumulative effects of different intensities of repeated cathodal stimulation on EEG and behavior were examined. Cathodal tDCS reduced the number of SWDs during stimulation and affected the mean duration after stimulation both in an intensity-dependent manner. Behavior was changed after the highest stimulation intensity. Spectral analyses of the EEG during stimulation revealed an increase in sub-delta and delta frequency ranges, suggesting that cortical cells were hyperpolarized. Cathodal tDCS might be an effective non-invasive tool to decrease cortical excitability, presumably in focal zone in this genetic model. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Transcranial direct current stimulation (tDCS) is a non-invasive approach of brain stimulation which has been suggested as a helpful therapy in some psychological and neurological disorders [1–4]. The ability of weak DC stimulation in modulating the excitability of cerebral cortex has been previously demonstrated [5,6]. Although tDCS effects have been shown to be polarity-specific, anodal stimulation generally increases the excitability, while cathodal stimulation decreases the excitability of the stimulated zone, other factors such as intensity and duration of the applied current as well as orientation of the axons and dendrites in the induced electrical field are important parameters in the direction of cortical excitability changes, as well as prolongation of the tDCS effects [7–11]. Due to the modulatory effects on cortical excitability, tDCS might be considered as a therapeutic tool in suppression or reduction of epileptic seizures with focal origin; however, so far, only a few studies have been performed in this respect [12–17]. In vitro animal experiments on rat brain hippocampal slices demonstrated a potent role for DC stimulation in abolishing the epileptiform activity in a polarity-dependent manner [12–14]. In vivo studies in rats with induced epileptic seizures also revealed the ability of tDCS in suppression of convulsions or enhancement of seizure threshold as a result of cathodal tDCS [15,16]. The proposed anticonvulsant effects of tDCS depended on the duration and intensity of stimulation. Human clinical trials have also shown the efficacy of tDCS in suppression of ⁎ Corresponding author. Fax: +31 24 3616066. E-mail address: [email protected] (M. Zobeiri). 1525-5050/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yebeh.2012.10.018
epileptiform activity in adults with treatment-refractory epilepsy [17]. The results of these experiments suggest that cathodal tDCS is able to reduce the excitability of the cortex in the epileptogenic foci, and decrease the probability of the epileptiform activities, preferably outlasting the actual stimulation period. Moreover, a recent study on the efficacy of transcranial electrical stimulation has shown that closed-loop low frequency stimulation of the epileptic zone in a genetic animal model of absence epilepsy is able to reduce the mean duration of SWDs [18]. To investigate the efficacy of tDCS on absence epilepsy, we conducted the present study on the WAG/Rij strain of rats, a valid genetic model of absence epilepsy [19]. These rats show spontaneously occurring age-dependent spike and slow-wave discharges (SWDs) characteristic of absence epilepsy in both human and animals [20,21]. Spike and slow-wave discharges in WAG/Rij rats originate from a hyperexcitable focus, located bilaterally in the perioral region of somatosensory cortices [22,23]. It is expected that changing the excitability of cortical neurons in the epileptic foci by means of tDCS might influence SWDs. In the first experiment, the effects of cathodal and anodal stimulation on SWDs were explored and compared since it is not precisely known what the different forms of stimulation might do on SWDs typically for absence epilepsy. Next, acute (during stimulation), longer-lasting (1-hour 45-minute post-stimulation intervals), and long-term (24 h) effects of repetitive cathodal stimulation with two different intensities were investigated on SWD occurrence. Considering the negative relation between SWDs and behavior (motor activity) [24,25], and that changes in SWDs might be secondary to the changes in behavior induced by stimulation, the effects of tDCS on behavior were explored as well.
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2. Material and methods 2.1. Animals Twenty-six male WAG/Rij rats at the age of 6 months with mean body weight of 343±21 g were used. Animals were born in the laboratory of Biological Psychology, Donders Centre for Cognition, Radboud University Nijmegen, The Netherlands and housed in groups of 2 rats per cage before surgery and in separate cages immediately after operation, in 12:12 light/dark cycle (white lights] on at 20:30) with free access to food and water throughout the whole experiment. All experimental procedures were in accordance with the “Principles of laboratory animal care” (NIH publication No. 86-23, revised 1985) and local guidelines approved by the ethical committee of Radboud University Nijmegen (RU-DEC) and European Communities Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimize the number and degree of discomfort of animals used in this study. 2.2. EEG recording electrode For all rats, a tripolar EEG recording electrode set (Plastic One, Roanoke, VA, USA) was implanted stereotactically under deep inhalation isoflurane anesthesia (Pharmachemie BV, Haarlem, the Netherlands). The active EEG electrode was placed on the motor cortex of the right hemisphere (A/P: +2.0 mm, V/L: −2.0 mm) with two wires as ground and reference on top of the cerebellum; coordinates were determined according to the stereotactic atlas of Paxinos and Watson [26] (Fig. 1). 2.3. Transcranial DC stimulation electrodes Transcranial direct current stimulation was applied by using a constant current stimulator (Grass constant current units, Model CCU1A) through a bilateral epicranial electrode setup (Radboud University, Technical Support Group) consisting of three stainless steel electrodes with an inner diameter of 2.1 mm and a contact area of 3.5 mm 2 filled with electrode paste (Grass, EC33) for increasing the electrode contact area with the cranium, as previously used in an induced seizure model in rats [16]. The inner diameter of the electrode covers the cranium above the perioral region of the somatosensory cortex; stimulation electrodes were placed on both hemispheres considering that the foci are bilateral [27]. The two stimulation electrodes were fixed onto the cranium above the right and left somatosensory cortices (V/L −4.6 and V/L +4/6, respectively) and the reference electrode onto the cranium above the frontal cortex with no specific coordinate (Fig. 1b). Both stimulation and recording electrodes were fixed onto the skull via dental cement. All rats received a preoperative injection of 0.1 ml atropine (Pharmachemie BV, Haarlem, the Netherlands) and
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preoperative and postoperative (24 and 48 h after surgery) injection of 0.4 ml/kg Rimadyl (mix Rimadyl/NaCl 1/5, Pfizer Animal Health B.V., Capelle a/d Ijssel, The Netherlands) and were allowed to recover for 2 weeks. 2.4. EEG recording and transcranial direct current stimulation During the experiment, rats were placed in Plexiglas registration boxes (20 × 35 × 25 cm). They were connected with the recording and stimulation equipment via a swivel, which allows the rats to move freely. Each animal was connected to two isolated constant current units (CCU). The voltage source was a Dual Galvanic Isolated DC Power supply of 90 V (Radboud University Nijmegen, Technical Support Group (TSG)). The setup was built with one voltage source and four constant current units (CCU) which allowed stimulation of two freely moving rats at the same time by two separate reversible currents. The EEG signals were amplified by a physiological amplifier (TD 90087, Radboud University Nijmegen, TSG) filtered between 1 (High Pass) and 100 Hz (Low Pass) and digitized with a Dataq DI-720 acquisition USB device and Windaq software DATAQ (Instruments, Inc., Akron, OH, USA) onto a PC. The behavioral activity of the rats was registered by a Passive Infrared Registration system (PIR, RK2000DPC LuNAR PR Ceiling Mount, Rokonet RISCO Group S.A., Drogenbos, Belgium) placed on the top of registration box. To prevent electrical transients (stimulation break effects) and EEG signal loss (clipping), the current was automatically ramped up and down for 10 s at the onset and offset of stimulation [8]. The intensity of the applied current was kept in the safety limit range determined by previous studies during the three experiments [28]. The impedance of the stimulation electrodes was checked the day before the beginning of the experiment and ranged from 5 to 17.5 KΩ. 3. Study protocol 3.1. Experiment I Ten rats were randomly allocated in two groups. An eight-hour baseline EEG was recorded for each rat (9 AM–5 PM) the day prior to transcranial DC stimulation. During the stimulation, each rat received 4 series of 15-minute cathodal and anodal stimulation of 100 μA with an interval of 1 h and 45 min (105 min) in a counter-balanced order (Fig. 2) while EEG was recorded and behavior was monitored. 3.2. Experiments II and III In the second experiment, 4 sessions of 15-minute cathodal stimulation were applied (n = 8) at the same time of day as Exp I. In
Fig. 1. a: Hyperexcitable region of the somatosensory cortex, responsible in initiation of spike and wave discharges (SWDs) in WAG/Rij rats. Adapted from [22]. b: Placement of recording and stimulation electrodes on rat skull. : Tripolar EEG recording set with an active EEG wire on right motor cortex and reference and ground wires on cerebellum. ●: tDCS electrodes.
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Fig. 2. The stimulation protocol for the first experiment. The intensity of stimulation for both cathodal and anodal polarities was 100 μA with an interstimulation interval of 1 h and 45 min. All 10 rats (5 rats in each group) had an 8-h (from 9 AM to 5 PM) baseline EEG recording the day before stimulation. The EEG was also recorded for the same 8 h during the stimulation day. The behavioral activity of animals was monitored by using a Passive Infrared Registration system (PIR).
addition, the baseline, the current strength and after-stimulation intervals were similar to the first experiment (100 μA, 105 min). To determine if repetitive tDCS has any long-term effects, a second 8-hour post-stimulation EEG was recorded for each rat the day after cathodal DC stimulation. The same protocols (baseline, stimulation parameters, and their timing) were used for the third experiment except that the intensity of the cathodal tDCS was increased to 150 μA (n = 8). 3.3. Histological evaluation To evaluate the safety of repetitive tDCS at the given intensities, one week after the last stimulation session, 5 rats (two rats from the first experiment with mixed anodal and cathodal tDCS protocol, 1 rat from the second experiment and 2 rats from the last experiment with cathodal current stimulation intensity of 150 μA) underwent histological analysis of the stimulated regions by using light microscopy [28]. After transcardial perfusion of the rats, the brains were gently removed from the skulls, and paraffin sections from the stimulated regions (4 μm thickness; interval 40 μm) were prepared and further processed by hematoxylin and eosin (H&E) staining at the laboratory of Pathology (UMCN, Radboud University Nijmegen). Pathological alternations of the cortical cells such as edema, necrosis, karyopyknosis, karyolysis, and karyorrhexis were investigated. 4. Statistics 4.1. Experiment I The effects of stimulation on mean duration and number of SWDs as well as behavioral activity of animals during tDCS were tested in three separate repeated-measures mixed-design ANOVAs with the number and mean duration of SWDs or amplitude of the PIR as dependent variables. For all three analyses, the number of stimulation (first, second, third, or last stimulation) and day of experiment (baseline as first day and stimulation as second day) were used as within-subjects factors and groups (Fig. 2) of experiment as between-subjects factors. The effects of anodal vs. cathodal stimulation were tested with four independent t-tests.
The short-term aftereffects of tDCS were evaluated first during the seven 15-minute blocks after stopping the stimulation with orthogonal contrasts; in case of no dynamics, the data on the seven 15-minute blocks were pooled and analyzed in the same way as during tDCS.
4.2. Experiments II and III The effects of cathodal stimulation on number or mean duration of SWDs or behavioral activity during stimulation time points as well as at post-stimulation intervals were analyzed with separate repeated measures for each of the applied current intensities and each variable. The long-lasting aftereffect of repetitive cathodal stimulation in poststimulation day (8 h) was investigated by comparisons with the baseline day (day and time points as within-subjects factors). Two of the eight rats lost their EEG signal only during tDCS in the third experiment due to differences in impedance between the bilateral stimulation electrodes. These animals were not included in the statistical analyses for the acute effects of tDCS during stimulation; one rat lost its signal in two out of the 4 stimulation periods. The missing values for these two sessions were imputed by a multiple imputation method using the SPSS statistics version 19 with linear regression as a model for variables. The number of imputations was set at 5. Correlations between SWD parameters (number and mean duration of SWDs) and behavioral activity of the animals during stimulation and at seven 15-minute blocks after stimulation were calculated. Spectral content of 2 s segments (30 segments in 90% of the cases, for the other as much as possible) of passive wakefulness (low PIR accompanied by a desynchronized EEG with predominating beta) during stimulation and respective time points in the baseline were averaged with Brain Vision Analyzer (version 1.05, Brain Products GmbH, Gilching, Germany). Differences in absolute and relative power between the baseline and during stimulation were compared for each of six different frequency bands (sub-delta (b1 Hz), delta (1–4 Hz), theta (5–7 Hz), alpha (8–12 Hz), beta (13–30 Hz), and gamma (31– 100 Hz)) with an ANOVA, with day, time of day, and frequency bands as within-subjects factors. A p value b .05 was considered as significant for all statistical tests performed.
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5. Results 5.1. Exp I: effects of cathodal and anodal tDCS on the number and mean duration of SWDs 5.1.1. Effects of tDCS during stimulation Test of the acute tDCS effects on number of SWDs during stimulation revealed a significant group difference (F= 6.83; df= 1,8; p b 0.05), and a significant second-order interaction between the day of experiment (baseline vs. stimulation day), number of stimulation (first, second, third, and last stimulation), and groups of experiment (F= 3.62; df= 3,24; p b 0.05). Subsequent post-hoc tests unraveling this second-order interaction revealed a marginal difference between the second stimulation and third stimulation (F= 5.02; df= 1,8; p = 0.055), suggesting a change in the number of SWDs after the second consecutive stimulation with the same polarity (2 consecutive sessions of cathodal stimulation in group I and anodal stimulation in group II). Paired sample t-test between the 2nd and 3rd stimulation in both groups showed that there was an increase in SWDs for the animals that were anodal-stimulated for the second consecutive time. The results are displayed in Fig. 3. The result of independent-samples t-test revealed a marginal difference in the number of SWDs during the first 15-minute stimulation between the anodal and cathodal stimulated groups (n = 5 in both groups); the number was lower in the cathodal stimulated group. Tests on acute stimulation effects on mean duration of SWDs during stimulation showed a significant group effect (F = 7.63; df = 1,8; p b 0.05), a significant interaction between the number of stimulation
Fig. 3. Number of spike–wave discharges (SWDs) during four 15-minute periods of anodal and cathodal DC stimulation at a current intensity of 100 μA in (a) group I and (b) group II and their respective baseline time periods. Repeated-measures mixed-design revealed significant main group effects (F=6.83; df=1,8; p=0.031). Test of within-subjects contrast revealed a marginal significant interaction for day of experiment*, number of stimulation* and groups of experiment between levels 2 and 3. Data are displayed as mean± SEM. a: As shown during the first anodal stimulation, the number of SWDs was higher than the respective baseline time periods; however, this increase was not statistically significant (n=5). b: A slight decrease in the number of SWDs during the first 15-minute cathodal stimulation was not statistically different from baseline (n=5). However, the difference between anodal and cathodal stimulation on the stimulation day was marginally significant for the first stimulation period.
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and groups (F = 3.59; df = 3,24; p b 0.05), and a significant interaction between the day of experiment and number of stimulation for levels 2 and 3 of stimulation (F = 5.41; df = 1,8; p b 0.05). Subsequent post-hoc analysis showed a significant decrease in the mean duration of SWDs from the 3rd to 4th stimulation time points in group II. This indicates a significant decrease in the mean duration of SWDs during the last cathodal stimulation as compared with the previous anodal stimulation (t = 3.97; df = 4; p b 0.05). Further analysis of each stimulation condition within groups with their respective baseline values by paired sample t-test revealed an increase in the mean duration of SWDs during the second anodal stimulation in group II (t = 2.71; df = 4; p b 0.05). The mean duration of SWDs during other stimulation time points was not significantly different from the baseline values. Moreover, there were no differences between anodal and cathodal stimulation between groups. 5.1.2. Effects of tDCS at post-stimulation intervals Neither anodal nor cathodal stimulation had significant long-lasting aftereffects on the number or on the mean duration of SWDs in the 1-hour 45-minute post-stimulation intervals. 5.2. Effects of repetitive cathodal stimulation on the number and mean duration of SWDs (Exps II and III) 5.2.1. Exp II During the second experiment, four 15-minute periods of cathodal tDCS at the current strength of 100 μA were applied (n = 8). The result of repeated measures analyses revealed only a significant reduction of the number of SWDs on the stimulation day compared to baseline (F = 9.35; df = 1,7; p b 0.05; n = 8) (Fig. 4a). The mean duration of SWDs during stimulation time points did not change significantly from the baseline. No long-lasting aftereffects for cathodal tDCS on the number of SWDs were found in 1-hour 45-minute post-stimulation intervals; however, a significant increase in the mean duration of SWDs from the baseline values was observed (F = 10.89; df = 1,7; p b 0.05). The results are depicted in Fig. 5.
Fig. 4. Effects of repetitive cathodal transcranial direct current stimulation (tDCS) with intensity of (a) 100 and (b) 150 μA and post-stimulation intervals of 1 h and 45 min on number of spike–wave discharges (SWDs) as compared with the respective baseline time points. The data are displayed as mean ± SEM. The results of repeated measures revealed significant main effect of stimulation on the number of SWDs for both intensities (p b 0.05, n = 8 and n = 6 respectively).
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Fig. 5. Effects of cathodal transcranial direct current stimulation (tDCS) with the current strength of 100 μA on mean duration of spike–wave discharges (SWDs) (a) during four 15-minute stimulation periods and (b) in 1-hour 45-minute post-stimulation intervals as compared with the respective baseline time points. a: The mean duration of SWDs was not significantly different from baseline values during stimulation periods. b: A significant increase in the mean duration of SWDs in 1-hour 45-minute after-stimulation intervals was observed (pb 0.05). The data are displayed as mean±SEM. * Significant deviations of the mean duration of SWDs at indicated time points from respective baseline time points.
Also, long-term effects of repetitive cathodal tDCS were investigated by comparing the effects of baseline with the post-stimulation day. There were no significant differences for the number and mean duration of SWDs between the baseline day and post-stimulation day; the number and mean duration of SWDs remained close to the pre-stimulation baseline EEG values during the whole 8 h of recording. 5.2.2. Exp III The number of SWDs during 15-minute 150 μA cathodal stimulation was significantly lower than the baseline (F=10.07; df=1,5; pb .05; n=6), for results please see Fig. 4b. The mean duration tended to be lower than baseline, although the difference was not statistically significant. The mean duration of SWDs in the post-stimulation intervals tended to be lower than that in the baseline. An orthogonal trend analyses of the changes across the seven 15-minute post-stimulation periods showed a significant linear and quadratic (Flin = 7.28; df= 1,7; p b .05; η2 = 0.51; Fquad = 21.26; df= 1,7; p b 0.01; η 2 = 0.75, n =8) orthogonal trend, demonstrating that the effect sizes as expressed by η 2 were large, and that the duration decreased over the 1-hour 45-minute interval, with an increase towards the end of the post-stimulation interval. Long-term effects on the number and mean duration were not found. Spectral analyses of the EEG during stimulation revealed that there was neither a main effect of day, time of day, nor an interaction with the time of day. However, the absolute power showed a day×frequency band interaction effect (F=12.65, df=5,20, pb .001); post-hoc tests showed that sub-delta and delta frequency bands were increased during stimulation compared to those in the baseline. The relative power also showed a day × frequency band interaction (F = 6.36, df = 5,20, p b .001); post-hoc tests revealed that sub-delta and delta frequency bands were increased and alpha and beta decreased (p's b .05) during stimulation compared to those in the baseline period. Fig. 6 illustrates the effects of 150 μA DC stimulation on the spectral content of the cortical EEG.
5.3.2. Exp II The repeated measures ANOVAs did not indicate a significant main effect of repetitive cathodal stimulation with an intensity of 100 μA on the behavioral activity of animals during stimulation as well as in the post-stimulation intervals. 5.3.3. Exp III When stimulated at 150 μA, a significant main effect of stimulation on the behavioral activity of the animals during stimulation was found (F = 41.31; df = 1,7; p = 0.00, n = 8). The results are presented in Fig. 7. The activity of the animals increased compared to that in the baseline. The ANOVA of the post-stimulation period data showed that the rats were less active compared with their respective baseline time points (F = 18.94; df = 1,7; p = 0.003). The repeated measures ANOVA on the PIR data in the post-stimulation intervals showed a significant time effect (F=2.75; df=1,7; pb .05). The orthogonal trend analyses of the changes across the seven 15-minute post-stimulation periods of the post-stimulation interval periods did not show a significant linear or quadratic trend. There were no clear systematic correlations between the PIR scores and SWD parameters (number and mean duration) on the baseline day. During stimulation, a high PIR score correlated generally negatively although non-significantly (n=6) with a low number of SWDs and short SWDs. However, systematic positive and often significant correlations between PIR and mean duration of SWDs were found in the last six post-stimulation intervals.
5.3. Effects of tDCS on behavioral activity of the animals 5.3.1. Exp I Although some of the rats tended to show more head scratching during the first two stimulation periods, there were no statistical differences in behavior as measured with the PIR during the four stimulation periods compared to baseline. The ANOVAs did not show main effects (day of experiment, stimulation number (day time points), or group of experiment) or interactions. There were also no differences between anodal and cathodal stimulation.
Fig. 6. Absolute power of the cortical EEG during 150 μA cathodal stimulation. Depicted is the average power of the various bands over the four stimulation periods compared with the mean of the respective baseline periods. Clear increase in EEG power in sub-delta and delta frequency ranges was found during stimulation,(p b 0.05, n = 5).
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Fig. 7. Effects of repeated cathodal transcranial direct current stimulation (tDCS) with an intensity of 150 μA on the behavioral activity of animals (a) during stimulation and (b) after stopping the stimulation, at post-stimulation intervals and compared with the respective baseline time points. a: The ANOVA revealed that rats were more active during the stimulation day compared to the same periods of the baseline day (p b 0.05, n = 8). b: The activity of animals during post-stimulation intervals was reduced compared to the same time points of the baseline periods (p b 0.05, n= 8). Data are displayed as mean amplitude of the PIR signal ± SEM.
Histological evaluation of the brains after H&E staining and by light microscopy did not show any abnormalities in the stimulated regions as a result of tDCS. 6. Discussion The main results of these series of experiments demonstrate that bilateral unipolar transcranial direct current stimulation of the somatosensory cortices in epileptic WAG/Rij rats is able to affect the occurrence of SWDs in a polarity- and intensity-dependent manner. 6.1. Comparison between the effects of anodal and cathodal tDCS on the number and mean duration of SWDs In our first experiment, the acute (during stimulation) and longlasting effects (in 1-hour 45-minute stimulation intervals) of both anodal and cathodal tDCS on the number and mean duration of SWDs and behavior were investigated. The lack of a clear stimulation effect and a significant second-order interaction and a group effect on the number of SWDs were indicative that the effects of stimulation were not very pronounced. Additionally, the post-hoc tests revealed that SWDs were marginally increased in group II when anodal stimulation occurred for the second consecutive time. Moreover, when the effects of the first time cathodal tDCS (group II) were compared to the effects of the first time anodal stimulation (group I), a marginal lower number of SWDs as a result of cathodal stimulation was observed. In other words, the differential effects of anodal versus cathodal stimulation on the number of SWDs were rather hidden, marginally visible during the first stimulation period; later, the effects in experiment I were even more obscure. The effects of stimulation on mean duration were also not clear. No clear main effect of stimulation and no significant interaction were found. A decrease in the mean duration of SWDs from the 3rd to 4th stimulation was found in group II, indicating a decrease in duration during the last cathodal stimulation as compared with the previous anodal stimulation. Further analysis of each stimulation condition with their baseline values showed an increase in the mean duration of SWDs during the second anodal stimulation for the animals of group II. There were no differences between anodal and cathodal stimulation between groups. The use of groups of rats with intermixed anodal and cathodal stimulation might have obscured a clear-cut view on what tDCS is able to do. The not well-pronounced effects of tDCS on SWDs in this mixed stimulation design might result from the reverse effects of the anodal and cathodal stimulation on excitability of the stimulated region as a result of membrane polarization [8,10]. As illustrated in previous animal and human studies, these changes might outlast the stimulation duration [8,5] and, therefore, they can
consequently influence the next stimulation. When two consecutive stimulation sessions are given, a somewhat larger effect can be noticed as illustrated by the increased potency of the second consecutive anodal stimulation on mean duration and number of SWDs. The enhanced excitability-changing ability of the repeated tDCS has been recently shown in a study in which repeated cathodal tDCS during the aftereffects of the first stimulation increased the efficacy of the previous stimulation [29]. No clear changes in the behavioral activity of the animals were found in the first experiment. 6.2. Low intensity repetitive cathodal tDCS has different effects on the number and mean duration of SWDs during stimulation 6.2.1. Effects during stimulation Repetitive cathodal stimulation (100 μA), as shown in experiment II, reduced the number of SWDs during stimulation. This finding is in line with the results of previous studies in which an antiepileptic effect for cathodal stimulation has been proposed [15–17] and can convincingly confirm the efficacy of cathodal tDCS which was, due to the design of our first experiment such as counterbalancing and number of animals per group, less clear. The excitability-diminishing properties of cathodal stimulation can result from membrane hyperpolarization [6,8]. The mean duration of SWDs did not change during stimulation. Differential effects on the number and mean duration of SWDs are rather common: they were described as a consequence of early maternal manipulations [21] and after the administration of the antiepileptic drug remacemide [30] and indicate that different mechanisms are involved in initiation and termination of SWDs. Studies on WAG/Rij rats has demonstrated that while the number of SWDs is determined by the excitability of the focal region, the duration of SWDs is determined by factors affecting cortico-thalamo-cortical network activity [23]. 6.2.2. Aftereffects of low-intensity repetitive cathodal tDCS on SWD parameters The number of SWDs during the post-stimulation periods did not differ from respective baseline time points. However, an increase in the mean duration of SWDs at these stimulation intervals was observed. The non-similar effects of cathodal stimulation during and after stimulation might be as a result of the separate mechanisms involved in induction of acute and post-stimulation (longer lasting) effects of tDCS. It has been demonstrated that acute effects of tDCS (during stimulation) are ion channel-dependent; however, the longer-lasting effects seem to be driven by synaptic modification [6,31]. For cathodal stimulation, an induction of NMDA receptor-mediated synaptic long-term depression (LTD) as a possible mechanism of action has been proposed [31]. However, the aftereffects of cathodal tDCS are not limited to synaptic
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mechanisms and possibly involve alterations in transmembrane protein and changes in PH as well [32]. Transcranial direct current stimulation can also induce changes in glial cells [33]. Glial cells have modulatory effects on neuronal excitability either by transport of glutamate from the extracellular space or by maintenance of extracellular ionic concentration and play an important role in epileptogenesis by regulating the extracellular concentrations of excitatory ions and neurotransmitters, as well as through other mechanisms [34]. Other factors besides levels of neurotransmitters and regional cerebral blood flow (rCBF) have been also found to change as a result of tDCS [35,36]; all these factors might influence the direction of tDCS aftereffects especially in an epileptic brain. In all, it is proposed that the decreased occurrence of SWDs during cathodal stimulation directly results from the excitability changes of the underlying tissue by a hyperpolarizing current and that the prolonged paroxysmal activity might be due to the gradual changes of the stimulated region over time. These changes, such as the increase in the mean duration of SWDs, do not occur suddenly; rather, they gradually appear. Moreover, the visibility of their effect on EEG parameters during stimulation might be masked by the direct effects of DC stimulation. Another explanation for the prolonged SWD duration after low intensity cathodal stimulation might be the effects of mild hyperpolarization of the cortex which has been demonstrated to increase the high-frequency bursts of action potentials. Studies on WAG/Rij rats have demonstrated that a light to moderate hyperpolarization of the intrinsically bursting cortical pyramidal cells and of the thalamocortical and reticular thalamic nuclei (RTN) can make these cells more prone to high-frequency bursts of action potentials [37]. Since pyramidal neurons of the somatosensory cortex in WAG/Rij rats are part of an extensive cortico-thalamo-cortical network, changes in the excitability of these cells might also affect neurotransmission within the network and prolong the duration of SWDs. It has been shown previously that cathodal DC stimulation changes the physiological network oscillations detectable on EEG [32]. 6.3. High-intensity repetitive cathodal tDCS suppresses number of SWDs and has more pronounced aftereffects on the mean duration of SWDs 6.3.1. Effects of repetitive cathodal stimulation during stimulation The suppressive (antiepileptic) effects of tDCS on the number of SWDs during stimulation increased by increasing the intensity of the cathodal tDCS from 100 to 150 μA. This finding is in accordance with the results of our first and second experiment and in line with the intensity-dependent efficacy of tDCS which has been demonstrated previously [16]. The reduction in the number of SWDs was accompanied by a non-significant reduction in the mean duration of SWDs. This can demonstrate that the animals had not only a lower number but also a shorter cumulative duration of SWDs. The behavioral activity of the animals during stimulation was significantly increased with this current intensity. The higher behavioral activity of the animals during the stimulation can be explained by a higher probability of the sensation of the current on the skull because of the increased intensity of stimulation or as a result of tDCS itself (cathodal at the somatosensory cortex and anodal at the frontal cortex). Considering the close relationship between SWDs and the state of vigilance, it is relevant to wonder whether the changes in SWDs' occurrence are due to changes in cortical excitability or are due to changes in behavior which might secondarily cause changes in SWDs. It is well demonstrated that SWDs preferentially occur during passive wakefulness, drowsiness, and light slow-wave sleep [25,38]. However, the suppressive effects of cathodal tDCS on the number of SWDs as found during stimulation in Exps II and III can be explained more likely by changes in membrane polarization than behavior. The EEG during 150 μA stimulation was clearly changed: its spectral analyses showed a large increase in the absolute and relative power of delta and sub-delta; others reported an increase of 3–7 Hz in the hour following cathodal stimulation in humans [32]. The non-REM sleep-like
EEG had sometimes an unusual rather stereotyped appearance of predominantly 1–2 Hz. First and interestingly, the behavior of the rat during stimulation was not sleep-like, but accompanied by higher motor activity than during the respective baseline periods. The increase of delta and the concomitant increase in motor activity point towards dissociation between EEG and behavior, a phenomenon well-known from pharmaco-EEG studies. The increase in slow frequencies can be considered as clear evidence for hyperpolarization of cortical neurons. Second, although the behavioral activity of the animals during the second experiment remained unchanged, a significant decrease in the number of SWDs during stimulation was observed. Third, the reduction in behavior in post-stimulation intervals in our third experiment was not accompanied by any changes in the number of SWDs. Therefore, it can be concluded that the changes in the number of SWDs as a consequence of cathodal DC stimulation are not likely due to changes in behavior or in changes to sleep, but might be a direct consequence of tDCS. 6.3.2. Post-stimulation The number of SWDs in post-stimulation intervals was not different from the baseline. Interestingly, and in contrast to100 μA cathodal stimulation, the mean duration of SWDs at the post-stimulation intervals showed strong changes over time, characterized by a negative linear and quadratic orthogonal trend confirming that a gradual decrease was accompanied by a return to baseline values towards the end. The clear decrease in the mean duration of the SWDs over the post-stimulation intervals was accompanied by a significant decrease in the behavioral activity of the rats in post-stimulation intervals. Although no orthogonal trends were found in the behavioral data in the post-stimulation intervals, a correlational analysis revealed a systematic negative correlation between the activity scores of the animals and mean duration of SWDs. This correlation did not exist in the baseline. In other words, tDCS might have induced a correlation between the PIR and mean duration of SWDs. Since effects of tDCS are not limited to the proposed stimulated region and behavioral changes can occur as a consequence of frontal stimulation, it is conceivable that tDCS indeed indirectly affects also behavioral parameters. Whether the changes in the mean duration of SWDs in post-stimulation intervals are directly due to the stimulation of the somatosensory cortex or secondarily as a result of activation of the other parts of the cortex is not immediately clear. 6.4. Aftereffects of cathodal stimulation The changes in the mean duration of SWDs only appeared at poststimulation intervals and the direction of these changes was intensitydependent. A current intensity of 150 μA reduced the mean duration of SWDs in post-stimulation intervals over time. Given the fact that different brain areas are responsible for the initiation and duration of SWDs, the delayed changes in the mean duration of SWDs compared to immediate (during stimulation) and non-lasting changes in the number of SWDs (at post-stimulation intervals) confirm the involvement of other regions besides the directly stimulated areas as a consequence of tDCS [35]. The significant negative linear and quadratic orthogonal trends show that the gradual decrease in mean duration was accompanied by a return to basal levels. This indicates that the excitability-changing aftereffects induced by tDCS are reversible and they decrease towards the end of 1-hour 45-minute post-stimulation intervals. Moreover, these reversible changes in excitability in addition to the results of the histological evaluation of the stimulated regions make is not likely that physiological damage of the brain occurred as a result of repetitive stimulation. The stimulation intensity during the study was kept in the safety limits demonstrated in a previous study in rats [28]. Higher-intensity cathodal stimulation induced clear post-stimulation effects in the form of orthogonal trends on the mean duration of SWDs;
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interestingly, this trend was not observed with lower intensity cathodal stimulation. Low-intensity prolonged SWDs at post-stimulation intervals. Obviously, the effects of cathodal tDCS on mean duration are also intensity-specific. Weaker currents might preferentially influence the activity of the most superficial inhibitory cortical interneurons; the net effect of their hyperpolarization might be excitation of the pyramidal neurons. Higher-intensity stimulation might target the deeper cortical layers with their different neuronal composition, afferent and efferent thalamic projections, thereby either layer IV with its inputs from the thalamic relay cells or layers V and VI with their cortical outputs reentering to the thalamic circuitry. It is well-known that the direction of excitability changes induced by scalp DC stimulation can be intensity-dependent. Stronger and weaker currents might therefore differ in their physiological sites of action in the cerebral cortex and in their consequences on behavior [9]. To investigate the long-term effects of cathodal tDCS on parameters of SWDs, an eight-hour post-stimulation baseline EEG was recorded the day after the last stimulation session. Earlier studies have demonstrated that long-term effects are quite possible for cognitive and neuropsychiatric symptoms [39,40]. The effects of repetitive cathodal stimulation on SWDs did not persist the next day. It has been already shown that intensity and duration of the stimulation are important factors in prolongation of tDCS' aftereffects [41]. A previous study on optimal intervals for long-term effects of repeated cathodal stimulation demonstrated an extra role for repetition (interstimulation) intervals [29]. This can explain our lack of long-term aftereffects since the aftereffects of cathodal stimulation during post-stimulation periods reduced towards the end of the stimulation intervals, and the next stimulation might have taken place after the termination of the aftereffects of the previous stimulation. However, the search for optimal and safe stimulation parameters including the interstimulation intervals can now begin. Its outcomes might however be specific for the type of disturbances in this genetic model. 7. Conclusion The results of this study demonstrate that bilateral cathodal tDCS, targeting the bilateral foci in a genetic absence model, has 1) short lasting antiepileptic effects on the number of SWDs and 2) longerlasting intensity-dependent effects on the mean duration of SWDs. The aftereffects of cathodal tDCS on the mean duration of SWDs appeared to elevate by time and might be associated with modulatory effects of cathodal stimulation on cortical excitability induced by hyperpolarization. Although the mechanisms remain somewhat enigmatic and methodological improvements might be necessary, to the best of our knowledge, this is the first experiment which evaluates the antiepileptic effects of tDCS on a genetic animal model of human absence epilepsy. Acknowledgments The authors would like to thank Annika Lüttjohann for her role in the first phase of this study, Gerard van Oijen, Saskia Hermeling, Hans Krijnen, Norbert Hermesdorf for their technical assistance and support and Dr. Benno Kuesters (UMCN) for his skillful histological evaluation of the brain slices. This work was granted by the Brain Gain Smart Mix program of The Netherlands Ministry of the Economic Affairs and The Netherlands Ministry of the Education, Culture and Science. References [1] Fregni F, Boggio PS, Nitsche MA, Marcolin MA, Rigonatti SP, Pascual-Leone A. Treatment of major depression with transcranial direct current stimulation. Bipolar Disord 2006;8:203-4.
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Noninvasive transcranial direct current stimulation in a genetic absence model M. Zobeiri ⁎, G. van Luijtelaar Department of Biological Psychology, Donders Centre for Cognition, Radboud University Nijmegen, The Netherlands
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Article history: Received 2 August 2012 Revised 5 October 2012 Accepted 7 October 2012 Available online 30 November 2012 Keywords: Absence epilepsy WAG/Rij rats Repetitive cathodal stimulation Cathodal tDCS Spectral analyses Hyperpolarization SWDs EEG
a b s t r a c t The proposed area of onset for absence epilepsy characteristic of spontaneously occurring spike and slowwave discharges (SWDs) in the genetic absence rat model is the subgranular layer of the somatosensory cortex. Modulation of the hyperexcitable cortical foci by bilateral transcranial direct current stimulation (tDCS) might change the expression of SWDs. The effects of cathodal and anodal tDCS as well as cumulative effects of different intensities of repeated cathodal stimulation on EEG and behavior were examined. Cathodal tDCS reduced the number of SWDs during stimulation and affected the mean duration after stimulation both in an intensity-dependent manner. Behavior was changed after the highest stimulation intensity. Spectral analyses of the EEG during stimulation revealed an increase in sub-delta and delta frequency ranges, suggesting that cortical cells were hyperpolarized. Cathodal tDCS might be an effective non-invasive tool to decrease cortical excitability, presumably in focal zone in this genetic model. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Transcranial direct current stimulation (tDCS) is a non-invasive approach of brain stimulation which has been suggested as a helpful therapy in some psychological and neurological disorders [1–4]. The ability of weak DC stimulation in modulating the excitability of cerebral cortex has been previously demonstrated [5,6]. Although tDCS effects have been shown to be polarity-specific, anodal stimulation generally increases the excitability, while cathodal stimulation decreases the excitability of the stimulated zone, other factors such as intensity and duration of the applied current as well as orientation of the axons and dendrites in the induced electrical field are important parameters in the direction of cortical excitability changes, as well as prolongation of the tDCS effects [7–11]. Due to the modulatory effects on cortical excitability, tDCS might be considered as a therapeutic tool in suppression or reduction of epileptic seizures with focal origin; however, so far, only a few studies have been performed in this respect [12–17]. In vitro animal experiments on rat brain hippocampal slices demonstrated a potent role for DC stimulation in abolishing the epileptiform activity in a polarity-dependent manner [12–14]. In vivo studies in rats with induced epileptic seizures also revealed the ability of tDCS in suppression of convulsions or enhancement of seizure threshold as a result of cathodal tDCS [15,16]. The proposed anticonvulsant effects of tDCS depended on the duration and intensity of stimulation. Human clinical trials have also shown the efficacy of tDCS in suppression of ⁎ Corresponding author. Fax: +31 24 3616066. E-mail address: [email protected] (M. Zobeiri). 1525-5050/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yebeh.2012.10.018
epileptiform activity in adults with treatment-refractory epilepsy [17]. The results of these experiments suggest that cathodal tDCS is able to reduce the excitability of the cortex in the epileptogenic foci, and decrease the probability of the epileptiform activities, preferably outlasting the actual stimulation period. Moreover, a recent study on the efficacy of transcranial electrical stimulation has shown that closed-loop low frequency stimulation of the epileptic zone in a genetic animal model of absence epilepsy is able to reduce the mean duration of SWDs [18]. To investigate the efficacy of tDCS on absence epilepsy, we conducted the present study on the WAG/Rij strain of rats, a valid genetic model of absence epilepsy [19]. These rats show spontaneously occurring age-dependent spike and slow-wave discharges (SWDs) characteristic of absence epilepsy in both human and animals [20,21]. Spike and slow-wave discharges in WAG/Rij rats originate from a hyperexcitable focus, located bilaterally in the perioral region of somatosensory cortices [22,23]. It is expected that changing the excitability of cortical neurons in the epileptic foci by means of tDCS might influence SWDs. In the first experiment, the effects of cathodal and anodal stimulation on SWDs were explored and compared since it is not precisely known what the different forms of stimulation might do on SWDs typically for absence epilepsy. Next, acute (during stimulation), longer-lasting (1-hour 45-minute post-stimulation intervals), and long-term (24 h) effects of repetitive cathodal stimulation with two different intensities were investigated on SWD occurrence. Considering the negative relation between SWDs and behavior (motor activity) [24,25], and that changes in SWDs might be secondary to the changes in behavior induced by stimulation, the effects of tDCS on behavior were explored as well.
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2. Material and methods 2.1. Animals Twenty-six male WAG/Rij rats at the age of 6 months with mean body weight of 343±21 g were used. Animals were born in the laboratory of Biological Psychology, Donders Centre for Cognition, Radboud University Nijmegen, The Netherlands and housed in groups of 2 rats per cage before surgery and in separate cages immediately after operation, in 12:12 light/dark cycle (white lights] on at 20:30) with free access to food and water throughout the whole experiment. All experimental procedures were in accordance with the “Principles of laboratory animal care” (NIH publication No. 86-23, revised 1985) and local guidelines approved by the ethical committee of Radboud University Nijmegen (RU-DEC) and European Communities Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimize the number and degree of discomfort of animals used in this study. 2.2. EEG recording electrode For all rats, a tripolar EEG recording electrode set (Plastic One, Roanoke, VA, USA) was implanted stereotactically under deep inhalation isoflurane anesthesia (Pharmachemie BV, Haarlem, the Netherlands). The active EEG electrode was placed on the motor cortex of the right hemisphere (A/P: +2.0 mm, V/L: −2.0 mm) with two wires as ground and reference on top of the cerebellum; coordinates were determined according to the stereotactic atlas of Paxinos and Watson [26] (Fig. 1). 2.3. Transcranial DC stimulation electrodes Transcranial direct current stimulation was applied by using a constant current stimulator (Grass constant current units, Model CCU1A) through a bilateral epicranial electrode setup (Radboud University, Technical Support Group) consisting of three stainless steel electrodes with an inner diameter of 2.1 mm and a contact area of 3.5 mm 2 filled with electrode paste (Grass, EC33) for increasing the electrode contact area with the cranium, as previously used in an induced seizure model in rats [16]. The inner diameter of the electrode covers the cranium above the perioral region of the somatosensory cortex; stimulation electrodes were placed on both hemispheres considering that the foci are bilateral [27]. The two stimulation electrodes were fixed onto the cranium above the right and left somatosensory cortices (V/L −4.6 and V/L +4/6, respectively) and the reference electrode onto the cranium above the frontal cortex with no specific coordinate (Fig. 1b). Both stimulation and recording electrodes were fixed onto the skull via dental cement. All rats received a preoperative injection of 0.1 ml atropine (Pharmachemie BV, Haarlem, the Netherlands) and
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preoperative and postoperative (24 and 48 h after surgery) injection of 0.4 ml/kg Rimadyl (mix Rimadyl/NaCl 1/5, Pfizer Animal Health B.V., Capelle a/d Ijssel, The Netherlands) and were allowed to recover for 2 weeks. 2.4. EEG recording and transcranial direct current stimulation During the experiment, rats were placed in Plexiglas registration boxes (20 × 35 × 25 cm). They were connected with the recording and stimulation equipment via a swivel, which allows the rats to move freely. Each animal was connected to two isolated constant current units (CCU). The voltage source was a Dual Galvanic Isolated DC Power supply of 90 V (Radboud University Nijmegen, Technical Support Group (TSG)). The setup was built with one voltage source and four constant current units (CCU) which allowed stimulation of two freely moving rats at the same time by two separate reversible currents. The EEG signals were amplified by a physiological amplifier (TD 90087, Radboud University Nijmegen, TSG) filtered between 1 (High Pass) and 100 Hz (Low Pass) and digitized with a Dataq DI-720 acquisition USB device and Windaq software DATAQ (Instruments, Inc., Akron, OH, USA) onto a PC. The behavioral activity of the rats was registered by a Passive Infrared Registration system (PIR, RK2000DPC LuNAR PR Ceiling Mount, Rokonet RISCO Group S.A., Drogenbos, Belgium) placed on the top of registration box. To prevent electrical transients (stimulation break effects) and EEG signal loss (clipping), the current was automatically ramped up and down for 10 s at the onset and offset of stimulation [8]. The intensity of the applied current was kept in the safety limit range determined by previous studies during the three experiments [28]. The impedance of the stimulation electrodes was checked the day before the beginning of the experiment and ranged from 5 to 17.5 KΩ. 3. Study protocol 3.1. Experiment I Ten rats were randomly allocated in two groups. An eight-hour baseline EEG was recorded for each rat (9 AM–5 PM) the day prior to transcranial DC stimulation. During the stimulation, each rat received 4 series of 15-minute cathodal and anodal stimulation of 100 μA with an interval of 1 h and 45 min (105 min) in a counter-balanced order (Fig. 2) while EEG was recorded and behavior was monitored. 3.2. Experiments II and III In the second experiment, 4 sessions of 15-minute cathodal stimulation were applied (n = 8) at the same time of day as Exp I. In
Fig. 1. a: Hyperexcitable region of the somatosensory cortex, responsible in initiation of spike and wave discharges (SWDs) in WAG/Rij rats. Adapted from [22]. b: Placement of recording and stimulation electrodes on rat skull. : Tripolar EEG recording set with an active EEG wire on right motor cortex and reference and ground wires on cerebellum. ●: tDCS electrodes.
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Fig. 2. The stimulation protocol for the first experiment. The intensity of stimulation for both cathodal and anodal polarities was 100 μA with an interstimulation interval of 1 h and 45 min. All 10 rats (5 rats in each group) had an 8-h (from 9 AM to 5 PM) baseline EEG recording the day before stimulation. The EEG was also recorded for the same 8 h during the stimulation day. The behavioral activity of animals was monitored by using a Passive Infrared Registration system (PIR).
addition, the baseline, the current strength and after-stimulation intervals were similar to the first experiment (100 μA, 105 min). To determine if repetitive tDCS has any long-term effects, a second 8-hour post-stimulation EEG was recorded for each rat the day after cathodal DC stimulation. The same protocols (baseline, stimulation parameters, and their timing) were used for the third experiment except that the intensity of the cathodal tDCS was increased to 150 μA (n = 8). 3.3. Histological evaluation To evaluate the safety of repetitive tDCS at the given intensities, one week after the last stimulation session, 5 rats (two rats from the first experiment with mixed anodal and cathodal tDCS protocol, 1 rat from the second experiment and 2 rats from the last experiment with cathodal current stimulation intensity of 150 μA) underwent histological analysis of the stimulated regions by using light microscopy [28]. After transcardial perfusion of the rats, the brains were gently removed from the skulls, and paraffin sections from the stimulated regions (4 μm thickness; interval 40 μm) were prepared and further processed by hematoxylin and eosin (H&E) staining at the laboratory of Pathology (UMCN, Radboud University Nijmegen). Pathological alternations of the cortical cells such as edema, necrosis, karyopyknosis, karyolysis, and karyorrhexis were investigated. 4. Statistics 4.1. Experiment I The effects of stimulation on mean duration and number of SWDs as well as behavioral activity of animals during tDCS were tested in three separate repeated-measures mixed-design ANOVAs with the number and mean duration of SWDs or amplitude of the PIR as dependent variables. For all three analyses, the number of stimulation (first, second, third, or last stimulation) and day of experiment (baseline as first day and stimulation as second day) were used as within-subjects factors and groups (Fig. 2) of experiment as between-subjects factors. The effects of anodal vs. cathodal stimulation were tested with four independent t-tests.
The short-term aftereffects of tDCS were evaluated first during the seven 15-minute blocks after stopping the stimulation with orthogonal contrasts; in case of no dynamics, the data on the seven 15-minute blocks were pooled and analyzed in the same way as during tDCS.
4.2. Experiments II and III The effects of cathodal stimulation on number or mean duration of SWDs or behavioral activity during stimulation time points as well as at post-stimulation intervals were analyzed with separate repeated measures for each of the applied current intensities and each variable. The long-lasting aftereffect of repetitive cathodal stimulation in poststimulation day (8 h) was investigated by comparisons with the baseline day (day and time points as within-subjects factors). Two of the eight rats lost their EEG signal only during tDCS in the third experiment due to differences in impedance between the bilateral stimulation electrodes. These animals were not included in the statistical analyses for the acute effects of tDCS during stimulation; one rat lost its signal in two out of the 4 stimulation periods. The missing values for these two sessions were imputed by a multiple imputation method using the SPSS statistics version 19 with linear regression as a model for variables. The number of imputations was set at 5. Correlations between SWD parameters (number and mean duration of SWDs) and behavioral activity of the animals during stimulation and at seven 15-minute blocks after stimulation were calculated. Spectral content of 2 s segments (30 segments in 90% of the cases, for the other as much as possible) of passive wakefulness (low PIR accompanied by a desynchronized EEG with predominating beta) during stimulation and respective time points in the baseline were averaged with Brain Vision Analyzer (version 1.05, Brain Products GmbH, Gilching, Germany). Differences in absolute and relative power between the baseline and during stimulation were compared for each of six different frequency bands (sub-delta (b1 Hz), delta (1–4 Hz), theta (5–7 Hz), alpha (8–12 Hz), beta (13–30 Hz), and gamma (31– 100 Hz)) with an ANOVA, with day, time of day, and frequency bands as within-subjects factors. A p value b .05 was considered as significant for all statistical tests performed.
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5. Results 5.1. Exp I: effects of cathodal and anodal tDCS on the number and mean duration of SWDs 5.1.1. Effects of tDCS during stimulation Test of the acute tDCS effects on number of SWDs during stimulation revealed a significant group difference (F= 6.83; df= 1,8; p b 0.05), and a significant second-order interaction between the day of experiment (baseline vs. stimulation day), number of stimulation (first, second, third, and last stimulation), and groups of experiment (F= 3.62; df= 3,24; p b 0.05). Subsequent post-hoc tests unraveling this second-order interaction revealed a marginal difference between the second stimulation and third stimulation (F= 5.02; df= 1,8; p = 0.055), suggesting a change in the number of SWDs after the second consecutive stimulation with the same polarity (2 consecutive sessions of cathodal stimulation in group I and anodal stimulation in group II). Paired sample t-test between the 2nd and 3rd stimulation in both groups showed that there was an increase in SWDs for the animals that were anodal-stimulated for the second consecutive time. The results are displayed in Fig. 3. The result of independent-samples t-test revealed a marginal difference in the number of SWDs during the first 15-minute stimulation between the anodal and cathodal stimulated groups (n = 5 in both groups); the number was lower in the cathodal stimulated group. Tests on acute stimulation effects on mean duration of SWDs during stimulation showed a significant group effect (F = 7.63; df = 1,8; p b 0.05), a significant interaction between the number of stimulation
Fig. 3. Number of spike–wave discharges (SWDs) during four 15-minute periods of anodal and cathodal DC stimulation at a current intensity of 100 μA in (a) group I and (b) group II and their respective baseline time periods. Repeated-measures mixed-design revealed significant main group effects (F=6.83; df=1,8; p=0.031). Test of within-subjects contrast revealed a marginal significant interaction for day of experiment*, number of stimulation* and groups of experiment between levels 2 and 3. Data are displayed as mean± SEM. a: As shown during the first anodal stimulation, the number of SWDs was higher than the respective baseline time periods; however, this increase was not statistically significant (n=5). b: A slight decrease in the number of SWDs during the first 15-minute cathodal stimulation was not statistically different from baseline (n=5). However, the difference between anodal and cathodal stimulation on the stimulation day was marginally significant for the first stimulation period.
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and groups (F = 3.59; df = 3,24; p b 0.05), and a significant interaction between the day of experiment and number of stimulation for levels 2 and 3 of stimulation (F = 5.41; df = 1,8; p b 0.05). Subsequent post-hoc analysis showed a significant decrease in the mean duration of SWDs from the 3rd to 4th stimulation time points in group II. This indicates a significant decrease in the mean duration of SWDs during the last cathodal stimulation as compared with the previous anodal stimulation (t = 3.97; df = 4; p b 0.05). Further analysis of each stimulation condition within groups with their respective baseline values by paired sample t-test revealed an increase in the mean duration of SWDs during the second anodal stimulation in group II (t = 2.71; df = 4; p b 0.05). The mean duration of SWDs during other stimulation time points was not significantly different from the baseline values. Moreover, there were no differences between anodal and cathodal stimulation between groups. 5.1.2. Effects of tDCS at post-stimulation intervals Neither anodal nor cathodal stimulation had significant long-lasting aftereffects on the number or on the mean duration of SWDs in the 1-hour 45-minute post-stimulation intervals. 5.2. Effects of repetitive cathodal stimulation on the number and mean duration of SWDs (Exps II and III) 5.2.1. Exp II During the second experiment, four 15-minute periods of cathodal tDCS at the current strength of 100 μA were applied (n = 8). The result of repeated measures analyses revealed only a significant reduction of the number of SWDs on the stimulation day compared to baseline (F = 9.35; df = 1,7; p b 0.05; n = 8) (Fig. 4a). The mean duration of SWDs during stimulation time points did not change significantly from the baseline. No long-lasting aftereffects for cathodal tDCS on the number of SWDs were found in 1-hour 45-minute post-stimulation intervals; however, a significant increase in the mean duration of SWDs from the baseline values was observed (F = 10.89; df = 1,7; p b 0.05). The results are depicted in Fig. 5.
Fig. 4. Effects of repetitive cathodal transcranial direct current stimulation (tDCS) with intensity of (a) 100 and (b) 150 μA and post-stimulation intervals of 1 h and 45 min on number of spike–wave discharges (SWDs) as compared with the respective baseline time points. The data are displayed as mean ± SEM. The results of repeated measures revealed significant main effect of stimulation on the number of SWDs for both intensities (p b 0.05, n = 8 and n = 6 respectively).
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Fig. 5. Effects of cathodal transcranial direct current stimulation (tDCS) with the current strength of 100 μA on mean duration of spike–wave discharges (SWDs) (a) during four 15-minute stimulation periods and (b) in 1-hour 45-minute post-stimulation intervals as compared with the respective baseline time points. a: The mean duration of SWDs was not significantly different from baseline values during stimulation periods. b: A significant increase in the mean duration of SWDs in 1-hour 45-minute after-stimulation intervals was observed (pb 0.05). The data are displayed as mean±SEM. * Significant deviations of the mean duration of SWDs at indicated time points from respective baseline time points.
Also, long-term effects of repetitive cathodal tDCS were investigated by comparing the effects of baseline with the post-stimulation day. There were no significant differences for the number and mean duration of SWDs between the baseline day and post-stimulation day; the number and mean duration of SWDs remained close to the pre-stimulation baseline EEG values during the whole 8 h of recording. 5.2.2. Exp III The number of SWDs during 15-minute 150 μA cathodal stimulation was significantly lower than the baseline (F=10.07; df=1,5; pb .05; n=6), for results please see Fig. 4b. The mean duration tended to be lower than baseline, although the difference was not statistically significant. The mean duration of SWDs in the post-stimulation intervals tended to be lower than that in the baseline. An orthogonal trend analyses of the changes across the seven 15-minute post-stimulation periods showed a significant linear and quadratic (Flin = 7.28; df= 1,7; p b .05; η2 = 0.51; Fquad = 21.26; df= 1,7; p b 0.01; η 2 = 0.75, n =8) orthogonal trend, demonstrating that the effect sizes as expressed by η 2 were large, and that the duration decreased over the 1-hour 45-minute interval, with an increase towards the end of the post-stimulation interval. Long-term effects on the number and mean duration were not found. Spectral analyses of the EEG during stimulation revealed that there was neither a main effect of day, time of day, nor an interaction with the time of day. However, the absolute power showed a day×frequency band interaction effect (F=12.65, df=5,20, pb .001); post-hoc tests showed that sub-delta and delta frequency bands were increased during stimulation compared to those in the baseline. The relative power also showed a day × frequency band interaction (F = 6.36, df = 5,20, p b .001); post-hoc tests revealed that sub-delta and delta frequency bands were increased and alpha and beta decreased (p's b .05) during stimulation compared to those in the baseline period. Fig. 6 illustrates the effects of 150 μA DC stimulation on the spectral content of the cortical EEG.
5.3.2. Exp II The repeated measures ANOVAs did not indicate a significant main effect of repetitive cathodal stimulation with an intensity of 100 μA on the behavioral activity of animals during stimulation as well as in the post-stimulation intervals. 5.3.3. Exp III When stimulated at 150 μA, a significant main effect of stimulation on the behavioral activity of the animals during stimulation was found (F = 41.31; df = 1,7; p = 0.00, n = 8). The results are presented in Fig. 7. The activity of the animals increased compared to that in the baseline. The ANOVA of the post-stimulation period data showed that the rats were less active compared with their respective baseline time points (F = 18.94; df = 1,7; p = 0.003). The repeated measures ANOVA on the PIR data in the post-stimulation intervals showed a significant time effect (F=2.75; df=1,7; pb .05). The orthogonal trend analyses of the changes across the seven 15-minute post-stimulation periods of the post-stimulation interval periods did not show a significant linear or quadratic trend. There were no clear systematic correlations between the PIR scores and SWD parameters (number and mean duration) on the baseline day. During stimulation, a high PIR score correlated generally negatively although non-significantly (n=6) with a low number of SWDs and short SWDs. However, systematic positive and often significant correlations between PIR and mean duration of SWDs were found in the last six post-stimulation intervals.
5.3. Effects of tDCS on behavioral activity of the animals 5.3.1. Exp I Although some of the rats tended to show more head scratching during the first two stimulation periods, there were no statistical differences in behavior as measured with the PIR during the four stimulation periods compared to baseline. The ANOVAs did not show main effects (day of experiment, stimulation number (day time points), or group of experiment) or interactions. There were also no differences between anodal and cathodal stimulation.
Fig. 6. Absolute power of the cortical EEG during 150 μA cathodal stimulation. Depicted is the average power of the various bands over the four stimulation periods compared with the mean of the respective baseline periods. Clear increase in EEG power in sub-delta and delta frequency ranges was found during stimulation,(p b 0.05, n = 5).
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Fig. 7. Effects of repeated cathodal transcranial direct current stimulation (tDCS) with an intensity of 150 μA on the behavioral activity of animals (a) during stimulation and (b) after stopping the stimulation, at post-stimulation intervals and compared with the respective baseline time points. a: The ANOVA revealed that rats were more active during the stimulation day compared to the same periods of the baseline day (p b 0.05, n = 8). b: The activity of animals during post-stimulation intervals was reduced compared to the same time points of the baseline periods (p b 0.05, n= 8). Data are displayed as mean amplitude of the PIR signal ± SEM.
Histological evaluation of the brains after H&E staining and by light microscopy did not show any abnormalities in the stimulated regions as a result of tDCS. 6. Discussion The main results of these series of experiments demonstrate that bilateral unipolar transcranial direct current stimulation of the somatosensory cortices in epileptic WAG/Rij rats is able to affect the occurrence of SWDs in a polarity- and intensity-dependent manner. 6.1. Comparison between the effects of anodal and cathodal tDCS on the number and mean duration of SWDs In our first experiment, the acute (during stimulation) and longlasting effects (in 1-hour 45-minute stimulation intervals) of both anodal and cathodal tDCS on the number and mean duration of SWDs and behavior were investigated. The lack of a clear stimulation effect and a significant second-order interaction and a group effect on the number of SWDs were indicative that the effects of stimulation were not very pronounced. Additionally, the post-hoc tests revealed that SWDs were marginally increased in group II when anodal stimulation occurred for the second consecutive time. Moreover, when the effects of the first time cathodal tDCS (group II) were compared to the effects of the first time anodal stimulation (group I), a marginal lower number of SWDs as a result of cathodal stimulation was observed. In other words, the differential effects of anodal versus cathodal stimulation on the number of SWDs were rather hidden, marginally visible during the first stimulation period; later, the effects in experiment I were even more obscure. The effects of stimulation on mean duration were also not clear. No clear main effect of stimulation and no significant interaction were found. A decrease in the mean duration of SWDs from the 3rd to 4th stimulation was found in group II, indicating a decrease in duration during the last cathodal stimulation as compared with the previous anodal stimulation. Further analysis of each stimulation condition with their baseline values showed an increase in the mean duration of SWDs during the second anodal stimulation for the animals of group II. There were no differences between anodal and cathodal stimulation between groups. The use of groups of rats with intermixed anodal and cathodal stimulation might have obscured a clear-cut view on what tDCS is able to do. The not well-pronounced effects of tDCS on SWDs in this mixed stimulation design might result from the reverse effects of the anodal and cathodal stimulation on excitability of the stimulated region as a result of membrane polarization [8,10]. As illustrated in previous animal and human studies, these changes might outlast the stimulation duration [8,5] and, therefore, they can
consequently influence the next stimulation. When two consecutive stimulation sessions are given, a somewhat larger effect can be noticed as illustrated by the increased potency of the second consecutive anodal stimulation on mean duration and number of SWDs. The enhanced excitability-changing ability of the repeated tDCS has been recently shown in a study in which repeated cathodal tDCS during the aftereffects of the first stimulation increased the efficacy of the previous stimulation [29]. No clear changes in the behavioral activity of the animals were found in the first experiment. 6.2. Low intensity repetitive cathodal tDCS has different effects on the number and mean duration of SWDs during stimulation 6.2.1. Effects during stimulation Repetitive cathodal stimulation (100 μA), as shown in experiment II, reduced the number of SWDs during stimulation. This finding is in line with the results of previous studies in which an antiepileptic effect for cathodal stimulation has been proposed [15–17] and can convincingly confirm the efficacy of cathodal tDCS which was, due to the design of our first experiment such as counterbalancing and number of animals per group, less clear. The excitability-diminishing properties of cathodal stimulation can result from membrane hyperpolarization [6,8]. The mean duration of SWDs did not change during stimulation. Differential effects on the number and mean duration of SWDs are rather common: they were described as a consequence of early maternal manipulations [21] and after the administration of the antiepileptic drug remacemide [30] and indicate that different mechanisms are involved in initiation and termination of SWDs. Studies on WAG/Rij rats has demonstrated that while the number of SWDs is determined by the excitability of the focal region, the duration of SWDs is determined by factors affecting cortico-thalamo-cortical network activity [23]. 6.2.2. Aftereffects of low-intensity repetitive cathodal tDCS on SWD parameters The number of SWDs during the post-stimulation periods did not differ from respective baseline time points. However, an increase in the mean duration of SWDs at these stimulation intervals was observed. The non-similar effects of cathodal stimulation during and after stimulation might be as a result of the separate mechanisms involved in induction of acute and post-stimulation (longer lasting) effects of tDCS. It has been demonstrated that acute effects of tDCS (during stimulation) are ion channel-dependent; however, the longer-lasting effects seem to be driven by synaptic modification [6,31]. For cathodal stimulation, an induction of NMDA receptor-mediated synaptic long-term depression (LTD) as a possible mechanism of action has been proposed [31]. However, the aftereffects of cathodal tDCS are not limited to synaptic
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mechanisms and possibly involve alterations in transmembrane protein and changes in PH as well [32]. Transcranial direct current stimulation can also induce changes in glial cells [33]. Glial cells have modulatory effects on neuronal excitability either by transport of glutamate from the extracellular space or by maintenance of extracellular ionic concentration and play an important role in epileptogenesis by regulating the extracellular concentrations of excitatory ions and neurotransmitters, as well as through other mechanisms [34]. Other factors besides levels of neurotransmitters and regional cerebral blood flow (rCBF) have been also found to change as a result of tDCS [35,36]; all these factors might influence the direction of tDCS aftereffects especially in an epileptic brain. In all, it is proposed that the decreased occurrence of SWDs during cathodal stimulation directly results from the excitability changes of the underlying tissue by a hyperpolarizing current and that the prolonged paroxysmal activity might be due to the gradual changes of the stimulated region over time. These changes, such as the increase in the mean duration of SWDs, do not occur suddenly; rather, they gradually appear. Moreover, the visibility of their effect on EEG parameters during stimulation might be masked by the direct effects of DC stimulation. Another explanation for the prolonged SWD duration after low intensity cathodal stimulation might be the effects of mild hyperpolarization of the cortex which has been demonstrated to increase the high-frequency bursts of action potentials. Studies on WAG/Rij rats have demonstrated that a light to moderate hyperpolarization of the intrinsically bursting cortical pyramidal cells and of the thalamocortical and reticular thalamic nuclei (RTN) can make these cells more prone to high-frequency bursts of action potentials [37]. Since pyramidal neurons of the somatosensory cortex in WAG/Rij rats are part of an extensive cortico-thalamo-cortical network, changes in the excitability of these cells might also affect neurotransmission within the network and prolong the duration of SWDs. It has been shown previously that cathodal DC stimulation changes the physiological network oscillations detectable on EEG [32]. 6.3. High-intensity repetitive cathodal tDCS suppresses number of SWDs and has more pronounced aftereffects on the mean duration of SWDs 6.3.1. Effects of repetitive cathodal stimulation during stimulation The suppressive (antiepileptic) effects of tDCS on the number of SWDs during stimulation increased by increasing the intensity of the cathodal tDCS from 100 to 150 μA. This finding is in accordance with the results of our first and second experiment and in line with the intensity-dependent efficacy of tDCS which has been demonstrated previously [16]. The reduction in the number of SWDs was accompanied by a non-significant reduction in the mean duration of SWDs. This can demonstrate that the animals had not only a lower number but also a shorter cumulative duration of SWDs. The behavioral activity of the animals during stimulation was significantly increased with this current intensity. The higher behavioral activity of the animals during the stimulation can be explained by a higher probability of the sensation of the current on the skull because of the increased intensity of stimulation or as a result of tDCS itself (cathodal at the somatosensory cortex and anodal at the frontal cortex). Considering the close relationship between SWDs and the state of vigilance, it is relevant to wonder whether the changes in SWDs' occurrence are due to changes in cortical excitability or are due to changes in behavior which might secondarily cause changes in SWDs. It is well demonstrated that SWDs preferentially occur during passive wakefulness, drowsiness, and light slow-wave sleep [25,38]. However, the suppressive effects of cathodal tDCS on the number of SWDs as found during stimulation in Exps II and III can be explained more likely by changes in membrane polarization than behavior. The EEG during 150 μA stimulation was clearly changed: its spectral analyses showed a large increase in the absolute and relative power of delta and sub-delta; others reported an increase of 3–7 Hz in the hour following cathodal stimulation in humans [32]. The non-REM sleep-like
EEG had sometimes an unusual rather stereotyped appearance of predominantly 1–2 Hz. First and interestingly, the behavior of the rat during stimulation was not sleep-like, but accompanied by higher motor activity than during the respective baseline periods. The increase of delta and the concomitant increase in motor activity point towards dissociation between EEG and behavior, a phenomenon well-known from pharmaco-EEG studies. The increase in slow frequencies can be considered as clear evidence for hyperpolarization of cortical neurons. Second, although the behavioral activity of the animals during the second experiment remained unchanged, a significant decrease in the number of SWDs during stimulation was observed. Third, the reduction in behavior in post-stimulation intervals in our third experiment was not accompanied by any changes in the number of SWDs. Therefore, it can be concluded that the changes in the number of SWDs as a consequence of cathodal DC stimulation are not likely due to changes in behavior or in changes to sleep, but might be a direct consequence of tDCS. 6.3.2. Post-stimulation The number of SWDs in post-stimulation intervals was not different from the baseline. Interestingly, and in contrast to100 μA cathodal stimulation, the mean duration of SWDs at the post-stimulation intervals showed strong changes over time, characterized by a negative linear and quadratic orthogonal trend confirming that a gradual decrease was accompanied by a return to baseline values towards the end. The clear decrease in the mean duration of the SWDs over the post-stimulation intervals was accompanied by a significant decrease in the behavioral activity of the rats in post-stimulation intervals. Although no orthogonal trends were found in the behavioral data in the post-stimulation intervals, a correlational analysis revealed a systematic negative correlation between the activity scores of the animals and mean duration of SWDs. This correlation did not exist in the baseline. In other words, tDCS might have induced a correlation between the PIR and mean duration of SWDs. Since effects of tDCS are not limited to the proposed stimulated region and behavioral changes can occur as a consequence of frontal stimulation, it is conceivable that tDCS indeed indirectly affects also behavioral parameters. Whether the changes in the mean duration of SWDs in post-stimulation intervals are directly due to the stimulation of the somatosensory cortex or secondarily as a result of activation of the other parts of the cortex is not immediately clear. 6.4. Aftereffects of cathodal stimulation The changes in the mean duration of SWDs only appeared at poststimulation intervals and the direction of these changes was intensitydependent. A current intensity of 150 μA reduced the mean duration of SWDs in post-stimulation intervals over time. Given the fact that different brain areas are responsible for the initiation and duration of SWDs, the delayed changes in the mean duration of SWDs compared to immediate (during stimulation) and non-lasting changes in the number of SWDs (at post-stimulation intervals) confirm the involvement of other regions besides the directly stimulated areas as a consequence of tDCS [35]. The significant negative linear and quadratic orthogonal trends show that the gradual decrease in mean duration was accompanied by a return to basal levels. This indicates that the excitability-changing aftereffects induced by tDCS are reversible and they decrease towards the end of 1-hour 45-minute post-stimulation intervals. Moreover, these reversible changes in excitability in addition to the results of the histological evaluation of the stimulated regions make is not likely that physiological damage of the brain occurred as a result of repetitive stimulation. The stimulation intensity during the study was kept in the safety limits demonstrated in a previous study in rats [28]. Higher-intensity cathodal stimulation induced clear post-stimulation effects in the form of orthogonal trends on the mean duration of SWDs;
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interestingly, this trend was not observed with lower intensity cathodal stimulation. Low-intensity prolonged SWDs at post-stimulation intervals. Obviously, the effects of cathodal tDCS on mean duration are also intensity-specific. Weaker currents might preferentially influence the activity of the most superficial inhibitory cortical interneurons; the net effect of their hyperpolarization might be excitation of the pyramidal neurons. Higher-intensity stimulation might target the deeper cortical layers with their different neuronal composition, afferent and efferent thalamic projections, thereby either layer IV with its inputs from the thalamic relay cells or layers V and VI with their cortical outputs reentering to the thalamic circuitry. It is well-known that the direction of excitability changes induced by scalp DC stimulation can be intensity-dependent. Stronger and weaker currents might therefore differ in their physiological sites of action in the cerebral cortex and in their consequences on behavior [9]. To investigate the long-term effects of cathodal tDCS on parameters of SWDs, an eight-hour post-stimulation baseline EEG was recorded the day after the last stimulation session. Earlier studies have demonstrated that long-term effects are quite possible for cognitive and neuropsychiatric symptoms [39,40]. The effects of repetitive cathodal stimulation on SWDs did not persist the next day. It has been already shown that intensity and duration of the stimulation are important factors in prolongation of tDCS' aftereffects [41]. A previous study on optimal intervals for long-term effects of repeated cathodal stimulation demonstrated an extra role for repetition (interstimulation) intervals [29]. This can explain our lack of long-term aftereffects since the aftereffects of cathodal stimulation during post-stimulation periods reduced towards the end of the stimulation intervals, and the next stimulation might have taken place after the termination of the aftereffects of the previous stimulation. However, the search for optimal and safe stimulation parameters including the interstimulation intervals can now begin. Its outcomes might however be specific for the type of disturbances in this genetic model. 7. Conclusion The results of this study demonstrate that bilateral cathodal tDCS, targeting the bilateral foci in a genetic absence model, has 1) short lasting antiepileptic effects on the number of SWDs and 2) longerlasting intensity-dependent effects on the mean duration of SWDs. The aftereffects of cathodal tDCS on the mean duration of SWDs appeared to elevate by time and might be associated with modulatory effects of cathodal stimulation on cortical excitability induced by hyperpolarization. Although the mechanisms remain somewhat enigmatic and methodological improvements might be necessary, to the best of our knowledge, this is the first experiment which evaluates the antiepileptic effects of tDCS on a genetic animal model of human absence epilepsy. Acknowledgments The authors would like to thank Annika Lüttjohann for her role in the first phase of this study, Gerard van Oijen, Saskia Hermeling, Hans Krijnen, Norbert Hermesdorf for their technical assistance and support and Dr. Benno Kuesters (UMCN) for his skillful histological evaluation of the brain slices. This work was granted by the Brain Gain Smart Mix program of The Netherlands Ministry of the Economic Affairs and The Netherlands Ministry of the Education, Culture and Science. References [1] Fregni F, Boggio PS, Nitsche MA, Marcolin MA, Rigonatti SP, Pascual-Leone A. Treatment of major depression with transcranial direct current stimulation. Bipolar Disord 2006;8:203-4.
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