Published as Lüttjohann A., van Luijtelaar G. (2013) Epilepsy Research DOI: http://dx.doi.org/10.1016/j.eplepsyres.2013.03.009
Abstract
Purpose: The site specific effects of two different types of electrical stimulation of the
thalamus on electroencephalic epileptic activity as generated in the cortico-thalamo- cortical systemwere investigated in genetic epileptic WAG/Rij rats, a well characterized and validated absence model.
Methods: First, 12 male rats received low frequency (double-pulse pairs of 2.5 Hz, 150
_A inten-sity and 30 s inter-pair-interval) open-loop stimulation to either the Ventral- Postero-Medial(VPM) or the Anterior Thalamic Nucleus (ATN) for 8 h. Second, rats received high frequency(130 Hz, pulse train of 1 s) closed-loop stimulation applied to either VPM or ATN whenever aspike-wave discharge (SWD) was automatically detected.
Results: Low frequency stimulation induced 8 Hz SWD-like afterdischarges (AD). AD
were frequently seen in VPM but rarely in ATN stimulated rats. AD, recorded in cortex and thalamus, showed a strong temporal coherence (visually assessed) and opposite spike polarities. Proper-ties of AD and spontaneous SWD were equally affected by the stimulation. Closed-loop high frequency stimulation disrupted spontaneous SWD with no difference between ATN and VPM stimulated rats. 89% of SWD could be disrupted leading to a decrease in average SWD duration from 9 to 1.5 s.
Conclusion: Low frequency stimulation induced AD, which strongly mimic SWD.
Moreover, the effects were site-specific. High frequency thalamic stimulation disrupts ongoing SWD probable by interfering with the slow firing pattern of cortico-thalamo- cortical neurons seen during SWD cycle. The absence of stimulation site specificity for high frequency stimulation might be dueto the fact that stimulation only started on average 1 s after SWD onset when SWD are alreadyfully expressed in the bidirectional cortico-thalamo-cortical resonance system.
Keywords Absence epilepsy; Spike-wavedischarges; Afterdischarges; Electricalstimulation; Anterior thalamus; Ventral-Postero-Medial thalamus; Cortico-thalamo-cortical system; WAG/Rij rats
Introduction
The generation of generalized spike and wave dis-charges (SWD), the electrophysiological hallmark of absence epilepsy, is known to rely on an intact cortico-thalamo-cortical network (Crunelli and Leresche, 2002; Danober et al., 1998; Onat et al., 2012; van Luijtelaar and Sitnikova,2006; van Luijtelaar et al., 2011b). Pathophysiological mechanisms of SWD generation are commonly studied intwo well described and validated genetic animal models, WAG/Rij and GAERS, which show spontaneous periods of reduced responsiveness, accompanied by SWD (Coenen and Van Luijtelaar, 2003; Depaulis and van Luijtelaar, 2006). In these models an epileptic-focus, located in the deep layers of the somatosensory-cortex, was identified as the site of SWD origin (Meeren et al., 2005, 2002; Polack et al., 2007,2009). Recently it was shown that low-frequency electrical stimulation of this epileptic- focus in WAG/Rij and GAERS rats could induce afterdischarges (AD), which strongly resem- bled SWD (Lüttjohann et al., 2011; Zheng et al., 2012).Like SWD, local-field-potential recordings of AD, measured in the deep somatosensory and on the motor-cortex, showed a
frequently induced during drowsiness and only seldom during deep sleep and active- wakefulness, indicating the same vigilance prefer-ence known for SWD occurrence (Drinkenburg et al., 1991; Lüttjohann et al., 2011; Smyk et al., 2011). Given this strong similarity between SWD and AD, the authors assumed that these AD, like SWD reflect a cortico-thalamo-cortical phenomenon, and that based on characteristics of AD, network properties relevant for the generation of SWD could be inferred. However, the recordings in our previous study were restricted to the cortex and it needs to be established whether AD are indeed mimicking SWD as far as the involvement of the thalamus is concerned. Therefore, it is investigated whether AD, similar to SWD, can be measured in both cortex and thalamus and whether thalamic stimulation is able to affect characteristics of SWD and AD similarly. Although the role of the cortex and thalamus in SWD initiation and spreading is widely recognized, the exact interactions between a cortical focus and different thalamic nuclei and their contributions in these processes still need to be elucidated (van Luijtelaar et al., 2011b). The VPM (ventral-postero-medial) thalamic nucleus is thought to be part of the key-network of SWD generation. It sends sensory information to and receives information from the somatosensory cortex via direct thalamo-cortical and cortico-thalamic connections. These cells project collaterals to the caudal reticular-thalamic-nucleus (RTN), which in turn sends GABAergic projections to the VPM (Descheneset al., 1998; Lu and Lin, 1993; Pinault and Deschenes, 1998).The anterior-thalamic-nucleus (ATN), by contrast, does not have any direct connections to the epileptic-focus. It is partof the limbic-loop and predominantly projects to the cingulate and the retrohippocampal cortex (Shibata, 1993a,b; Van Groen and Wyss, 1995). Furthermore, the ATN sends and receives projections to the rostral RTN (Gonzalo-Ruiz and Lieberman, 1995; Pinault and Deschenes, 1998). The sensitivity of stimulation to induce AD can be compared between VPM and ATN. If AD are indeed a type of SWD (Lüttjohann et al., 2011; Zheng et al., 2012), they should be recorded in the VPM and more readily induced via stimulation of the VPM than via ATN. While low-frequency thalamic stimulation is expected to induce epileptic activity in the form of AD, high-frequency thalamic stimulation (130 Hz) has antiepileptic effects and should abort ongoing SWD (Vercueil et al., 1998). The location specificity/sensitivity (VPM, ATN) of this type of stimulation will be investigated in the second part of the study. It is hypothesized that it is easier to disrupt SWD by VPM as compared to ATN stimulation. Whereas in the first part of the study low-frequency stimulation will be applied in an open-loop fashion, i.e. is continuously applied, high-frequency stimulation will be applied in a closed-loop fashion, i.e. only in reaction to SWD.
Method Subjects
12 male WAG/Rij rats, age 6 months, were used as experimental subjects. They were born and raised at the department of Biological Psychology, Donders Centre for Cognition, Radboud University Nijmegen, The Netherlands. Prior to surgery rats were housed in pairs (High Makrolon cages with Enviro-Dri bedding-material and cage-enrichment) with free access to food and water and were kept at a 12—12 h light—dark cycle (light off at 8.30 A.M.). After surgery rats were housed individually. The experiment was approved by the Ethical Committee on Animal Experimentation of Radboud University Nijmegen. Efforts were made to minimize the amount of discomfortand restrict the number of animals.
Surgery
Stereotactic surgery was performed under isoflurane anesthesia: WAG/Rij rats were divided in two groups, which differed with respect to the location of the stimulation electrodes (Group 1: local stimulation in the VPM; Group 2: local stimulation in the ATN). In both groups two tripolar electrode sets (Plastic-OneMS333/2a) were implanted. Each tripolar electrode set consisted of three stainless steel wires, isolated with polyimide, with a diameter of 0.2 mm. Only the tip of each electrode wire was un-isolated. In group 1 (n = 7) the three electrode wires of the first tripolar assembly were implanted in the VPM, right hemi-sphere (A/P: −3.3 mm, M/L: −2.5 mm, H: −7.2 mm). Wires were oriented in an anterior-posterior direction and had the following configuration: stimulation anode, stimulation cathode, active recording electrode. Tip distance between electrode tips was 0.3 mm to ensure localized stimulation and recording within the VPM. The first wire of the second electrode set was implanted epidurally on the motor cortex (A/P: +2 mm, M/L: −2 mm), right hemisphere and was used as active recording electrode. The second and third wire of this set were implanted on top of the cerebellum and functioned as ground and reference electrode respectively. In group 2 (n = 5) two electrode-wires of the first tripolar-assembly were implanted in the ATN right hemisphere (A/P:−1.4 mm, M/L: −1.5 mm, H: −6.4 mm) and functioned as stimulation anode and cathode. Again, wires were oriented in an anterior-posterior direction and had a tip distance of 0.3 mm to ensure localized stimulation. The third wire of this electrode set was epidurally implanted on the motor cortex (A/P: +2 mm, M/L: −2 mm), right hemisphere, and functioned as the active recording electrode. Of the second electrode set one wire was implanted in the VPM, right hemi-sphere (A/P: −3.3 mm, M/L: −2.5 mm, H: −7.2 mm) and functioned as the active recording electrode whereas the other two wires were implanted on top of the cerebellum as the ground and reference electrodes respectively. All coordinates were determined according to the stereo-tactic atlas of Paxinos and Watson (Paxinos and
Watson,1998). Electrode-assemblies were fixed to the skull with dental cement. Rats were preoperatively injected with Atropine and Rimadyl, and again with Rimadyl postoperatively (24 and 48 h after surgery). Rats were allowed to recover for 2 weeks.
Data-acquisition, electrical stimulation and experimental procedure
Rats were placed individually in a 20 inch × 35 inch × 25 inchPlexiglas registration box and connected to the recording leads for LFP recording and electrical brain-stimulation. Leads were attached to a swivel-contact, which allowed registration and stimulation in the freely moving animals. The LFP signals were amplified with a physiological amplifier (TD90087, Radboud-University-Nijmegen, Electronic-Research-Group), filtered by a band-pass filter with cut-off points at 1(HP) and 100(LP) and a 50 Hz Notch filter, and digitalized with a constant sample-rate of 500 Hz by WINDAQ-recording-system (DATAQ-Instruments, Inc., Akron, OH, USA). Movements of the rat were registered by a Passive-Infrared-Registration system (PIR, RK2000DPCLuNAR PR-Ceiling-Mount, Rokonet RISCO-Group S.A., Dro- genbos, BE).
Experimental procedure part 1 (open-loop, low-frequency, thalamic stimulation): on experimental day1 (two weeks after surgery) a baseline recording was performed for 8 h during the dark phase using the above described recording settings (Baseline-LFS). The next day LFP recordings were recorded for 8 h and open-loop electrical stimulation was applied using the following stimulation settings (LFS-stim): double-pulse stimulation with a pulse- duration of 0.4 ms, an interpulse-interval of 400 ms (both previously used in our study on cortical stimulation (Lüttjohann et al., 2011)) and an intensity of 150 µA. Double-pulses were administered for a total of 8 h during the dark phase with a 30 s interval between each double-pulse pair; a total number of 960 double pulse pairswere administered to the rats. Experimental procedure part 2 (closed-loop, high-frequency, thalamic stimulation): two weeks after exper-iment one, rats underwent baseline recording during the first 3 h of the dark phase (Baseline-HFS). Next, rats were exposed to an intensity-threshold-test (ITT). In this test the LFP signal of the motor cortex-channel was analyzed by a previously developed and validated online SWD detection algorithm, which was shown to detect SWD with a sensitivity of 100% and a specificity of 96% (Ovchinnikov et al., 2010). It was implemented into a closed-loop DBS system: as soon as a SWD was detected (within 1 s) high-frequency stimulation(130 Hz, bi-phasic pulses, width 0.4 ms, 1 s pulse-train) was immediately administered to the rat. Stimulation started with a low intensity of 20 µA, and was increased in steps of 10 µA until 3 consecutive SWD were interrupted. The behavior of the rats during this intensity-test was closely observed. Next the rat was stimulated with the determined intensity during 1 h. If the rat had a sufficient amount of SWD (10 or more) and at least 90%
were disrupted, that specific intensity was considered the SWD less than 90% were disrupted, t
stimulated for another hour. The next day rats were subject to the final stimulation
(HFS-stim). In this stimulation session LFP signals were recorded during the first three hours of the dark phase (time-locked to the Baseline
the number of SWD) and closed
width 0.4 ms, 1 s pulse-train, and previously deter
was administered to the rat whenever a SWD was detected with the aid of the detection algorithm, functionally connected to
Figure 1 Scheme of experimental procedures along time axis. Note that
exposed to a reverse12/12 h light
LFS: low frequency stimulation, open loop; HFS:
threshold test or determination of stimulus intensity needed for the disruption HFS stimulation session (See text for more details).
Histology
At the end of both experimental subparts electrode positions were histologically verified. To this end a direct-current (9 V, 25
deeply anaesthetized rat. Rats were perfused with a potassiumferroc
phosphate solution. Brains were fixed in a 30% sucrose solution, 0.1 ml PBS, cut in 40 coronal slices with the aid of a microtome, and stained with Cresyl violet. Only animals with a confirmed electrode tip of the stimulation electro
statistical analyses (Fig. 2).
Data analysis
The number and duration of SWD and AD during baseline and
were quantified in the first experiment. Coherence, synchronicity between recor and general morphology of SWD and AD were assessed based on
Behavioral activity was quantified using the PIR values. SWD data were analyzed with a repeated-measures-ANOVA with either the number of SWD or SWD duration as dep
variable, group (ATN, VPM)as between
session) as within-subjects-factor 1 and time (hour 1
were disrupted, that specific intensity was considered the SWD-interruption
less than 90% were disrupted, the intensity was increased with 10 µA and the rat was stimulated for another hour. The next day rats were subject to the final stimulation
stim). In this stimulation session LFP signals were recorded during the first three hours locked to the Baseline-HFS session to exclude circadian effects on the number of SWD) and closed-loop high-frequency stimulation (130 Hz, bi
train, and previously deter-mined interruption-threshold as intensi to the rat whenever a SWD was detected with the aid of the
detection algorithm, functionally connected to the constant-current stimulator (Fig. 1).
Scheme of experimental procedures along time axis. Note that rats in this experiment were exposed to a reverse12/12 h light-dark-cycle with light off between 8.30 h and 20.30 h. Abbreviations: LFS: low frequency stimulation, open loop; HFS: high frequency stimulation, closed loop. ITT: intensity termination of stimulus intensity needed for the disruption of SWD to be used in the HFS stimulation session (See text for more details).
At the end of both experimental subparts electrode positions were histologically verified. To current (9 V, 25 µA, 10 s duration) was passed through each electrode in the deeply anaesthetized rat. Rats were perfused with a potassiumferrocyanide
phosphate solution. Brains were fixed in a 30% sucrose solution, 0.1 ml PBS, cut in 40 coronal slices with the aid of a microtome, and stained with Cresyl violet. Only animals with a confirmed electrode tip of the stimulation electrodes in the target regions were included in the
The number and duration of SWD and AD during baseline and open-loop LFS were quantified in the first experiment. Coherence, synchronicity between recor and general morphology of SWD and AD were assessed based on
Behavioral activity was quantified using the PIR values. SWD data were analyzed with a ANOVA with either the number of SWD or SWD duration as dep
variable, group (ATN, VPM)as between-subjects-factor, session (baseline, stimulation factor 1 and time (hour 1—hour8) as within-subjects
interruption-threshold. When A and the rat was stimulated for another hour. The next day rats were subject to the final stimulation-session stim). In this stimulation session LFP signals were recorded during the first three hours HFS session to exclude circadian effects on frequency stimulation (130 Hz, bi-phasic, pulse-
threshold as intensity) to the rat whenever a SWD was detected with the aid of the online-SWD
current stimulator (Fig. 1).
rats in this experiment were cycle with light off between 8.30 h and 20.30 h. Abbreviations: high frequency stimulation, closed loop. ITT: intensity of SWD to be used in the
At the end of both experimental subparts electrode positions were histologically verified. To A, 10 s duration) was passed through each electrode in the yanide-formaldehyde- phosphate solution. Brains were fixed in a 30% sucrose solution, 0.1 ml PBS, cut in 40 µm coronal slices with the aid of a microtome, and stained with Cresyl violet. Only animals with a des in the target regions were included in the
loop LFS-stimulation were quantified in the first experiment. Coherence, synchronicity between recording channels and general morphology of SWD and AD were assessed based on visual inspection. Behavioral activity was quantified using the PIR values. SWD data were analyzed with a ANOVA with either the number of SWD or SWD duration as dependent factor, session (baseline, stimulation-
analyzed via a repeated-measures ANOVA with the number of AD as dependent variable, group (VPM, ATN) as between
factor. Since not all rats showed AD during all hours of stimulation analyzed via a 2-way ANOVA with group (VPM, ATN) as between
(hour1 - hour8) as between-subjects
SWD (intensity-threshold) was compared via an unpaired
grouping-factor. The percentage of disrupted SWD was analyzed via a repeated analysis with session (intensity
factor and group (VPM, ATN) as between duration of SWD as recorded during the HFS were analyzed via a repeated
subjects-factor and session (baseline, HFS analyses were performed using SPSS19
Figure 2 Histological verification of electrode position (pictures adapted from Paxinos and Watson
(1998)). Blue dots indicate the position
rats with a verified electrode tip in the targeted region were
measures ANOVA with the number of AD as dependent variable, group (VPM, ATN) as between-subject-factor and time (hour 1 - hour 8) as within
factor. Since not all rats showed AD during all hours of stimulation, AD way ANOVA with group (VPM, ATN) as between-subjects
subjects-factor 2. For the second experiment, intensity to disrupt threshold) was compared via an unpaired t-test with group (VPM, ATN) as factor. The percentage of disrupted SWD was analyzed via a repeated
analysis with session (intensity-threshold-test, HFS-stimulation-session) as within factor and group (VPM, ATN) as between-subject-factor. Number, mean duration
of SWD as recorded during the HFS-baseline-session and HFS-
were analyzed via a repeated-measures ANOVA with group (VPM,ATN) as between factor and session (baseline, HFS-stim) as within-subjects-factor. All statistical analyses were performed using SPSS19-software (SPSSinc., Chicago, USA).
Histological verification of electrode position (pictures adapted from Paxinos and Watson position of the electrode tips within the various thalamic nuclei, only rats with a verified electrode tip in the targeted region were included in the statistical analyses.
measures ANOVA with the number of AD as dependent variable, hour 8) as within-subjects-
, AD-duration was subjects-factor 1 and time factor 2. For the second experiment, intensity to disrupt test with group (VPM, ATN) as factor. The percentage of disrupted SWD was analyzed via a repeated-measures session) as within-subject- actor. Number, mean duration and total
-stimulation session measures ANOVA with group (VPM,ATN) as between-
factor. All statistical software (SPSSinc., Chicago, USA).
Histological verification of electrode position (pictures adapted from Paxinos and Watson of the electrode tips within the various thalamic nuclei, only
Results
Low-frequent, open-loop, thalamic stimulation induces afterdischarges (AD)
Low-frequency (2.5 Hz) thalamic stimulation induced AD: they were characterized by a rhythmic spike and wave pattern of 8 Hz
expressed following the stimulation induced evoked potentials both the VPM and the motor-
both channels showed a strong temporal coherence. As is known for SWD (Meeren et al., 2002; Sitnikova and van Luijtelaar, 2007), spike
opposite (Fig. 3A). Rats were always motionless during AD except for rhythmic twitching movements of the whiskers.
Figure 3 Afterdischarges (AD) induced by low frequency thalamic stimulation. (A) Exemplary
recording of an AD induced via stimulation of the VPM. (B) Average number of stimulation induced AD via stimulation of either VPM or ATN. (C) Average duration of ATN
induced AD across stimulation-time.
The ANOVA on AD number revealed a
loop, thalamic stimulation induces afterdischarges (AD)
quency (2.5 Hz) thalamic stimulation induced AD: they were characterized by a rhythmic spike and wave pattern of 8 Hz which was immediately (without obvious latency expressed following the stimulation induced evoked potentials. AD were always recorded in
-cortex recording-channel. Upon visual inspection
both channels showed a strong temporal coherence. As is known for SWD (Meeren et al., Sitnikova and van Luijtelaar, 2007), spike-polarity in cortex and thalamus were opposite (Fig. 3A). Rats were always motionless during AD except for rhythmic twitching
Afterdischarges (AD) induced by low frequency thalamic stimulation. (A) Exemplary an AD induced via stimulation of the VPM. (B) Average number of stimulation induced AD via stimulation of either VPM or ATN. (C) Average duration of ATN-stimulation or VPM
time.
The ANOVA on AD number revealed a significant main effect of group (F
loop, thalamic stimulation induces afterdischarges (AD)
quency (2.5 Hz) thalamic stimulation induced AD: they were characterized by a without obvious latency) . AD were always recorded in Upon visual inspection recordings in