Experimental Neurology 211 (2008) 518–528

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Experimental Neurology

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Cortical and thalamic components of neocortical kindling-induced epileptogenesis inbehaving cats

Dragos A. Nita a, Youssouf Cissé a,1, Flavio Fröhlich b,c, Igor Timofeev a,⁎a Department of Anatomy and Physiology, Laval University, Centre de Recherche Université Laval Robert-Giffard, 2601 Ch. de la Canardière, Québec, Canada G1J 2G3b The Salk Institute for Biological Studies, Computational Neurobiology Laboratory, La Jolla, California 92037, USAc Division of Biological Sciences, Section of Neurobiology, University of California San Diego, La Jolla, California 92093, USA

a r t i c l e i n f o

⁎ Corresponding author. Fax: +1 418 663 8756.E-mail address: Igor.Timofeev@phs.ulaval.ca (I. Timo

1 Present address: The University of Tokushima, TheDivision of Enzyme Chemistry, 3-18-15 Kuramoto-cho, J

0014-4886/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.expneurol.2008.02.028

a b s t r a c t

Article history:Received 13 November 2007Revised 1 February 2008Accepted 25 February 2008Available online 14 March 2008

Kindling is an essential operating paradigm of the nervous system extensively used both as a model ofepileptogenesis and neuroplasticity. In a parallel study conducted on chronically implanted non-anesthetizedkindled cats, we report the occurrence of long-lasting slow oscillatory patterns (1.5–2 Hz) called outlastingactivities (OA) following the acute seizures (AS) induced by cortical stimulation. Here, we asked if OA observedin the neocortex of kindled animals are generated exclusively by the cortical networks or if they also rely onthe burst firing of thalamic neurons. We analyzed the electrophysiological patterns of synchronization ofcortical EEG (areas 4, 5, 7, 21, 17, 18, 22) and thalamic field (EThG) (ventral posterior lateral nucleus—VPL), andthe influence of modulatory systems originating in the pedunculo-pontine tegmentum (PPT) and locuscoeruleus (LC) on the discharge pattern of thalamic neurons during OA. Synchrony analysis of field recordingsshowed that during AS cortical paroxysmal activities preceded thalamic ones, while during OA this sequentialorder was reversed. During OA thalamic neurons regularly discharged bursts with the frequency of OA.Electrical stimulation of either PPT or LC during OA decreased both the probability of bursts in thalamocorticalneurons and the amplitude of OA. Yet, neither of them was able to block completely the expression of OA.Following PPT/LC stimulation the burst firing of thalamocortical neurons was replaced by tonic firing. Weconclude that the thalamus is involved in the mechanism of generation of OA but that it does not play anexclusive role.

© 2008 Elsevier Inc. All rights reserved.

Keywords:EpilepsyElectrophysiologyAfterdischargeOutlasting activitiesBurst firing

Introduction

Kindling is an essential operating paradigm of the nervous system,extensively used both as a model of epileptogenesis and neuroplas-ticity. In kindling, repeated administration of a weak stimulus thatinitially evokes no behavioral response produces gradually increasingparoxysmal EEG patterns and behavioral seizures over the course oftime, ultimately leading to generalized tonico-clonic seizures (God-dard, 1967). Once the effect of kindling is established, the inducedchange is persistent so that even the administration of a weakstimulus elicits generalized seizures (Goddard et al., 1969; McNamara,1984).

Among the wide range of mechanisms that may account forepileptogenesis during kindling synaptic potentiation and newcircuitry formation play a major role (reviewed in McNamara, 1994;Morimoto et al., 2004). However, by difference with previous studies,demonstrating kindling-induced morphological changes at the den-

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dritic and synaptic level in the hippocampus (Geinisman et al., 1988;Sutula, 1990; Hawrylak et al., 1993; Jiang et al., 1998) and amygdala(Nishizuka et al., 1991; Okada et al., 1993), there has been a lack ofevidence of long-lasting dendritic variations in the neocortex follow-ing kindling (Teskey et al., 1999). In the single documented attempt,Racine et al. found no changes in dendritic branching or spine densityin the anterior cortex of cortically kindled rats (Racine et al., 1975).Thus, there is no evidence at the structural level thatwould support thehypothesis of an extensive role of neocortex in epileptogenesis duringcortical kindling. Neocortical kindling is also characterized by arelatively high threshold of elicited acute seizure (AS), unstable seizuredevelopment, and difficulty in establishing a stable generalizedconvulsive seizure state (reviewed in Wake and Wada, 1976;Majkowski et al., 1981; Okamoto, 1982). However, in a parallel studywedemonstrated that neocortical kindling could be very efficient if thekindling procedure takes place during transition fromslow-wave sleepto waking state (Nita et al., 2008-this issue).

Corticothalamic (CT) neurons are reciprocally interconnected withthalamocortical (TC) neurons from different dorsal thalamic nuclei,and project to reticular (RE) neurons. This circuit generates both thephysiological EEG rhythms occurring during natural states of vigilance(Steriade et al., 1993), and the pathological developments of thenormal brain oscillations into seizures (reviewed in Steriade, 2003;

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Timofeev and Steriade, 2004). During cortically-generated spike-wave(SW) seizures in the intact thalamocortical circuit only aminority of TCneurons fires low-threshold spike-bursts during SW seizure, while thevast majority is steadily hyperpolarized and exhibits phasic inhibitorypost-synaptic potentials (IPSPs) induced by over-excited GABA-ergicreticular neurons (Steriade and Contreras, 1995; Pinault et al., 1998;Timofeev et al., 1998; Steriade and Timofeev, 2001; reviewed inCrunelli and Leresche, 2002). However, some recent studies in kindlingand pilocarpine models of epilepsy reported that the development ofspontaneous recurrent seizures in the corticothalamic system isassociatedwith increased T-type calciumcurrents in thalamic neurons,whichmay underlie the bursts of action potentials participating in SWactivity (Bertram et al., 2001; Su et al., 2002).

In the companionpaper (Nita et al., 2008-this issue)we reported thatacute seizures (AS) induced by kindling are followed by spontaneouslong-lasting (up to 2 h) slow activities (OA), persistent during sleep–wake cycle, during which animals are conscious, can walk and eat, andthat have a frequency in the EEG of 1.5–2 Hz. The same frequency is thefrequency of intrinsic delta activity of thalamocortical neurons (McCor-mick and Pape, 1990b). Thus, we hypothesized that the intrinsicproperties of TC neurons may contribute to the generation of OA.Using field potential andmultiunit recordings in kindled cats in vivo, wedemonstrate here that the thalamus indeed contributes to thegeneration of OA, but does not play an exclusive role.

Fig. 1. Development of burst firing in the thalamus during kindling. A) Experimental paradigmarea, the cortical stimulation electrode in green, periorbital EOG and the EMG electrodesstimulation are depicted in orange. B) Top panel contains four cortical EEG traces illustratincomes after the initial acute seizure (AS) induced by cortical electrical stimulation. Insets depiEEG recordings stand for: 1. left motor cortex (area 4), 2. left anterior associative cortex (C) Extracellular unit recordings in the thalamus and identification of spikes. Bursts of actioexample of burst recorded in the thalamus (indicated by a black triangle) depicted as fieldoccurring bursts in the thalamus during kindling procedure. Bursts were counted on 5 differlast day of experiment.

Materials and methods

Experiments were performed on 5 cats of both sexes (4 males and1 female). Since it is clear from both clinical observations (Scharfmanand MacLusky, 2006; Verrotti et al., 2007) and experimental research(Teskey et al., 1999) that gonadal hormones exert a profoundinfluence on excitability, seizures, and epilepsy (estrogens increasingneuronal excitability, while progesterone and androgens decreasingictal activity), all males were castrated and the female hysterecto-mized several months before the beginning of the experiments. Everyeffort was made to minimize the number of animals used and theirsuffering.

Surgical procedures were carried out in sterile condition underbarbiturate anesthesia (30mg/kg i.v.), following a pre-medicationwithacepromazine (0.3 mg/kg i.m.), butorphanol (0.3 mg/kg i.m.), atropine(0.05mg/kg i.m.) and ketamine (20mg/kg i.m.). The level of anesthesiawas continuously monitored by the aspect of electroencephalogram(EEG) and cardiac frequency (aiming 90–110 beats/min). Oxygensaturation of the arterial blood and end-tidal CO2were alsomonitored.General surgical procedures included cephalic vein canulation forsystemic liquid delivery (lactated Ringer's solution 5–10 ml/kg/h) andlidocaine (0.5%) infiltration of all pressure points or incision lines.Body temperature was maintained between 37 and 39 °C with aheating pad.

. EEG electrodes (circles) are depicted in blue together with the corresponding corticalplaced in neck muscles are indicated in white, while the electrodes used for PPT/LCg the occurrence of outlasting activities (OA) following the postictal depression whichct the expansions from the underlined periods in upper panel. Numbers on the left of thearea 5), 3. left posterior associative cortex (area 7), and 4. left visual cortex (area 21).n potentials (inter-spike interval b5 ms) are indicated by black triangles. D) ExpandedEEG, filtered unit recording and pseudospikes. E) Average frequency of spontaneouslyent epochs of 5 min of wake and are presented as normalized values with respect to the

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Coaxial bipolar EEG electrodes (FHC Inc., USA) (with the inner polein the cortical depth at about 0.8–1 mm and the outer pole placed atthe cortical surface) were bilaterally placed in the motor cortex,anterior and posterior associative cortex, auditory cortex, primary andsecondary visual cortex (cortical areas 3, 4, 5, 7, 17, 18, 21, and 22). Abundle of tungsten microelectrodes with tip impedances between 9.3and 11.4 MΩ (FHC Inc., USA) used to record the neuronal firing patternwas stereotaxically lowered in the thalamic ventral posterior lateral(VPL) nucleus. Additional pairs of recording electrodes were placedaround the orbit and neck muscles in order to monitor the states of

Fig. 2. Cortical and thalamic components during slow-wave sleep (SWS), acute seizures (AS) aVPL thalamic nucleus during SWS, AS, and OA. B) Expansions of periods shaded in grey in pan1-second windows. D) Same cross-correlograms as in panel C plotted as a function of time. EDuring SWS cortical and thalamic activities appear quasi-simultaneous, during AS the corticaand thalamus leads the cortex.

vigilance by recording the electro-oculogram (EOG) and electromyo-gram (EMG). In all animals, three Ag/AgCl references were implanted:two of them along the skull in the region of the external auditorycanals on both sides, and one over the nasium in the frontal bone (seeschema in Fig. 1A).

In all cats the electrode used for stimulation was placed in the leftassociative cortex (area 5). The kindling stimuli consisted in 40–60 s ofa 50 Hz rectangular pulse-trainwith stimulation intensity in the rangeof 0.5–1.5 mA. Our stimulating parameters lie within the range ofvalues used in the majority of kindling studies. Kindling procedure

nd outlasting activities (OA). A) Simultaneous field recording of cortical area 5 right andel A. C) Sequential cross-correlograms between cortical and thalamic field computed onach vertical bar corresponds to a color-coded version of a cross-correlogram in panel C.l activities precede thalamic activities while during OA this sequential order is reversed

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consisted in stimulating the cortical site 5 times per day up tooccurrence of AS followed by OA (after 5–7 days of kindling). After thismoment, we applied 1–2 electrical stimulation per day up to the endof experiments.

Bipolar concentric stimulating electrodes (FHC Inc., USA) wereplaced into the locus coeruleus (LC) nucleus (stereotaxic coordinates:1 mm posterior, 3 mm lateral and −1 mm depth) and pedunculo-pontine tegmental (PPT) area (stereotaxic coordinates: 1 mm anterior,3 mm lateral and −2.5 mm depth) (Reinoso-Suarez, 1961). The correctplacement of electrodes was tested by passing stimulating currentpulses through the electrodes (0.1 ms pulse duration, 100 Hz at 0.1–1.0 mA for 2 s) seeking an activating effect in the EEG, and wasconfirmed by electrolytic lesions on Nissl (thionine) stained sections.

The skull was reconstituted using acrylic dental cement and a fewbolts were placed in the cement to allow non-painful fixation of thecat's head in a stereotaxic frame. Animals were kept under observa-tion up to the full recovery and they received analgesic medication(anafen 2 mg/kg s.c.) for the next 48–72 h. After the recovery period(2–3 days), cats were trained to stay with the head restrained in astereotaxic frame and the body suspended in a rubber bag, whichallowed free body movements for 2–4 h/day, and usually in less than aweek they were fully conditioned displaying clearly identifiable statesof waking, SWS, and REM sleep, and being able to stay in the frame forseveral hours.

Behavioral responses evoked by the stimulation were monitoredusing a night-shoot video surveillance camera. Generalized behavio-rally manifest seizures appearing outside the stimulating sessionwereconsidered an “end limit point” of these experiments, as consentedwith the local ethic committee.

Fig. 3. Effect of pedunculo-pontine tegmentum (PPT) activation on sleep oscillations and outcat and activates the EEG. Negative deflections in the EEG during wake are associated witexpression of OA, but diminishes the amplitude of thalamic EThG. C) Fluctuations in power spindicated by arrows. D) Quantification of area under the FFT graph (on 0–4 Hz domain) beforamplitude of EEG and EThG before and after the PPT stimulation during natural sleep and O

At the end of experiments, or at the first sign of clinically manifestseizures outside the experimental protocol, animals received a lethaldose of barbiturate. All experimental procedures were performed inaccordance with the guidelines of the Canadian Council on AnimalCare and the U.S. National Institutes of Health Guide for the Care andUse of Laboratory Animals and were approved by the Committee forAnimal Care of Laval University.

Data analysis

Extracellular EThG consisted in both single- and multiunit record-ings. Analysis was performed only on data originating from individualunits. In the case of a multiunit recording the extraction of putativesingle units fromextracellular recording traceswas performed in threesteps: i) signal preprocessing, ii) event extraction, and iii) spike sorting.All analysis was performed in Matlab (The MathWorks, Natick, MA)with a customdesigned algorithm. For this process, each electrodewastreated separately since no time-locked events on several recordingsites were observed. i) The extracellular traces (sampling rate 20 kHz)were digitally filtered (Butterworth filter, 10th order, cut-off frequen-cies [400 Hz, 5000 Hz], Matlab) to remove the slow frequency signalcomponent not due to action potential firing. ii) The filtered signal wassubjected to amanually chosen threshold (negative threshold ofminusfive standard deviations of the extracellular trace) to extract presumedspikes. Each time the threshold was crossed, a corresponding 2 mswaveform snipped was extracted and stored as a vector (length: 40samples). Events that were clearly artifacts based on their amplitudewere manually excluded. iii) Putative single units were found with aspike-sorting algorithm (adapted from Fee et al., 1996).

lasting activity (OA). A) Electrical stimulation of PPT during slow-wave sleep awakes theh eye movements. B) Electrical stimulation during OA does not completely block theectra (FFT) related to the conditions depicted in panels A and B. The correspondences aree and after the PPT stimulation during natural sleep and OA. E) Variation of the averageA. (⁎ indicates pb0.05)

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The spike sorting was a two-step process. First, the waveformvectors were grouped into an overly large number of subclusters withthe K-means clustering algorithm. The number of subclusters wasmanually set to a value around ten times as high as the number ofexpected single units to be found. Subclusters that consisted of lessthan twenty waveform vectors were excluded from the subsequentanalysis. Second, subclusters were iteratively merged to form clustersthat represent putative single units. Linkage analysis of the subclustercentroids was used as a guide for manual merging of subclusters.Finally, spike sorting results were assessed by testing for refractoryperiod violations and by visual inspection of resulting waveformclusters in a reduced two-dimensional space determined by principalcomponent analysis. Waveform stability was evaluated by plotting thefirst principal component of all spikes as a function of time.

Inter-spike intervals b5 ms were considered bursts and werecounted on 5 epochs of 5 min each during waking state. Averagedvalues from all experimental animals for spontaneous burst incidencewere normalized to the incidence observed in the 30th day of thekindling protocol. Cross-correlograms between cortical and thalamicelectrodes were performed on 1-second windows with 50% overlap,color-coded, and successively displayed in dynamic cross-correlogramgraphs (see Fig. 2D). FFT quantifications (see Figs. 3C and 4C) wereperformed by averaging the area under the FFT graph on the 0–4 Hzwindow in all 5 experimental animals from 5 different epochs. Theaverage EEG amplitude was computed as a mean of the amplitudes ofvoltage deflection between successive positive and negative EEG peakson a 1-minute window. Autocorrelograms (Figs. 5B and 6B) weregenerated on successive windows of 5 s length. Threshold for statisticalsignificance was pb0.05 (paired Student t-test). The probability of

Fig. 4. Effect of locus coeruleus (LC) activation on sleep oscillations and outlasting activity (OAEEG. B) Electrical stimulation during OA does not completely block the expression of OA inC) Fluctuations in power spectra (FFT) corresponding to the conditions depicted in panels A a0–4 Hz domain) before and after the PPT stimulation during natural sleep and OA. E) Variaduring natural sleep and OA. (⁎ indicates pb0.05)

discharging during the negative or positive phase of the OA wascalculated for each presumed single unit on a 10-secondwindow beforethe stimulation of PPT or LC and on a 10-second window that followedthe stimulation.

Results

Acute elicited seizures consisting in spike-wave (SW) complexes at1–2 Hz (Fig. 1B) were evoked after 4–5 days of suprathresholdelectrical stimulation of the neocortex. As we previously reported thisinitial AS is followed by a postictal depression and, afterwards, by aspontaneous pattern of long-lasting paroxysmal EEG oscillationwith afrequency of ~2 Hz, called “outlasting activities” (Nita et al., 2008-thisissue). During OA cats were conscious, were able to walk and eat, butdisplayed localized body jerks. OA proved to be dramaticallyinfluenced by the natural occurring states of vigilance in cortical andthalamic networks; being obvious during waking state and slow-wavesleep (SWS), and completely abolished during rapid eye movements(REM) sleep (Nita et al., 2008-this issue).

Since CTand TC neurons are tightly interconnected in a circuitry thatgenerates the main EEG rhythms and mediates the pathologicaldevelopments of normal brain oscillations into seizures (Steriade et al.,1993), we aimed at understanding the involvement of TC neurons in theexpression of OA. Therefore, we envisaged that TC neurons couldcontribute to the generation of OA. Extracellular multiunit recordingsperformed during waking state in VPL nucleus revealed the occurrenceof bursts of action potentials in the firing pattern of TC neurons (Fig. 1Cand D) with an inter-spike interval of 4–5 ms. The gradual increase inincidence of spontaneously occurring bursts over the course of the

). A) Electrical stimulation of LC during slow-wave sleep awakes the cat and activates thethe cortical EEG, but, similar to PPT stimulation, it diminishes the amplitude of EThG.nd B. Similar arrangement as in Fig. 3. D) Quantification of area under the FFT graph (ontion of the average amplitude of the EEG and EThG before and after the LC stimulation

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kindling procedure is presented in Fig. 1D as incidence frequencynormalized to the frequency of bursts observed on the 30th day after theoccurrence of the first AS.

To further study the role of the thalamus in the generation of OAwecorrelated the EThG from VPL nucleus with the EEG from the targetcortical areas during the SWS, during both the initial and final periods oftheAS, andduringOA (Fig. 2A andB), starting fromday 20of the kindlingprotocolwhenOAwere generalizedon thewhole cortical surface. DuringSWS, the EEG of the anterior medial part of cortical area 5 and VPLnucleus showed a high level of correlation (average peak level of 0.86±0.07) and a minimal time shift of few milliseconds. At the onset of thestimulation-evoked cortical AS the EThG recording from theVPL nucleusdid not display highly synchronous activities with the cortical EEG. Theaverage correlation coefficient was decreased to 0.43±0.17. As the ASdeveloped, the thalamus became gradually involved and, sometimes,EThG recordings displayed sharp spike-waves at the end of the AS. Inthese cases the average coefficient of correlation increased to 0.72±0.12and the peak in the cross-correlogram was shifted to 71.34±9.21 ms,showing a correspondingly delay of thalamic activities. When OAexpressed after the postictal depression the average correlation levelwas 0.67±0.16 but the peak in the correlogram was now shifted in theopposite direction by an average of −94.85±14.32 ms. These observa-tions demonstrate that during OA the thalamus is activated prior to thecortex on each cycle of the paroxysmal oscillation.

Fig. 5. Influence of pedunculo-pontine tegmentum (PPT) activation on the cellular thalamicthalamic field EEG and unit activity during OA. Stimulation of PPT is indicated as a black rpresented in the bottom panel. Note the loss of stable shift in the peak of the correlation folland histograms of inter-spike interval probability before and after stimulation. PPT activatioreduces the probability of burst discharges.

Since the thalamus seemed to play amajor role in the generation ofthe OA by both showing an increased incidence of bursting neurons,which further can recruit cortical neurons in the oscillatory pattern ofOA (Fig. 1), and by preceding the target cortical areas in the expressionof OA (Fig. 2), we attempted to block the generation of OA by electricalactivation of two modulatory brainstem systems: the cholinergicpedunculo-pontine tegmental area (PPT) and noradrenergic locuscoeruleus (LC), which both depolarize TC neurons and thus abolishintrinsic bursting (McCormick and Pape, 1988; Pape and McCormick,1989; Curro Dossi et al., 1991; McCormick, 1992a,b).

The stimulationof PPTduring SWS induced anEEGactivation (Fig. 3A)revealed by a decrease in the amplitude of cortical EEG from 1.27±0.16 mV to 0.14±0.15 mV and of EThG from 1.32±0.07 mV to 0.18±0.09 mV (pb0.05). This activation was accompanied by an amplitudediminution of oscillations in the 1–4 Hz range (Fig. 3E). The power in the0–4 Hz band of the power spectrum significantly decreased (pb0.05)from 2.11±0.32 mV2×Hz to 1.21±0.34 mV2×Hz for cortical electrodes,and from 1.81±0.17 mV2×Hz to 1.21±0.16 mV2×Hz for thalamicelectrodes (Fig. 3D). Both variations were statistical significant(pb0.05). However, when PPT electrical stimulation was applied duringOA (Fig. 3B) the average amplitude of cortical components of OA onlyslightly decreased from 1.61±0.21 mV to 1.53±0.19 mV (not statisticallysignificant), while the amplitude of the OA recorded in the thalamusdecreased from 2.71±0.29 mV to 1.93±0.3 mV (pb0.05) (Fig. 3E). The

activities during OA. A) Top panel depicts a cortical field EEG recording together withectangle. The sequential cross-correlogram between cortical and thalamic field EEG isowing PPT stimulation. B) Spike-triggered auto-correlograms of thalamic unit recordingn switches the shape of the auto-correlogram from oscillatory to tonic discharge, and

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diminution of the amplitude of the 2Hz FFT peak of thalamic componentof OAwas reflected in a decrease in the area of the0–4Hz frequency bandfrom 3.11±0.2 mV2×Hz to 2.65±0.18 mV2×Hz (pb0.05), while thecorresponding frequency band for cortical electrodes did not significantlychange (from 1.96±0.25 mV2×Hz to 1.99±0.27 mV2×Hz — Fig. 3C, D).

A similar behavior of the EEG amplitude and of the components ofthe power spectra for cortical and thalamic EEG electrodes wasobserved following the electrical stimulation of LC (Fig. 4A–C). Incontrol condition, during SWS, LC stimulation decreased the ampli-tude of the cortical EEG from 1.25±0.14 mV to 0.18±0.16 mV (pb0.05)and of the thalamic EThG from 1.31±0.08 mV to 0.23±0.11 mV(pb0.05) (Fig. 4E). The frequency band measuring slow rhythmsdecreased in power from 1.66±0.31 mV2×Hz to 1.03±0.28 mV2×Hz incortical electrodes (pb0.05), and from 1.31±0.15 mV2×Hz to 0.23±0.12 mV2×Hz in thalamic electrodes (pb0.05, Fig. 4D). During OA theamplitude of the cortical EEG slightly changed from 1.5±0.24 mV to1.52±0.23 mV (not statistically significant), and the thalamic EThGamplitude decreased from 2.5±0.28 mV to 1.7±0.25 mV (pb0.05)(Fig. 4E). The area under the 2 Hz FFT peak of OAwas not significantlychanged in the power spectrum of cortical EEG (1.44±0.21 mV2×Hzvs. 1.39±0.26 mV2×Hz) but it was significantly diminished in thepower spectrum of thalamic electrodes (2.91±0.3 mV2×Hz vs. 2.17±0.29 mV2×Hz, pb0.05) (Fig. 4D). Following both PPT and LCstimulation during SWS, the activated period lasted for less than1 min, and thereafter the animal returned to SWS state.

Fig. 6. Influence of locus coeruleus (LC) activation on the cellular thalamic activities during OAunit activity during OA. Stimulation of LC is indicated as a black rectangle. The sequential crosNote the loss of stable shift in the peak of the correlation following LC stimulation. B) Spikeinterval probability before and after stimulation. LC activation switches the shape of the audischarges.

The main frequency peak in the power spectra during OA was notshifted by either PPT or LC electrical stimulation, and remained stableat a frequency of ~2 Hz (Figs. 3C and 4C).

The dynamic cross-correlation between cortical and thalamic fieldEEG before and after the electrical stimulation of cholinergic (Fig. 5A)and noradrenergic (Fig. 6A) modulatory systems showed a desyn-chronization in the phase shift of the correlogram peak amplitudetriggered by the stimulation. Before the stimulation of either PPT(Fig. 5A) or LC (Fig. 6A), the peak of the correlogram remained stable atapproximately −95 ms indicating a fixed delay between the genera-tion of OA waves in the thalamus and the involvement of the cortex.Following the activation of the thalamus, the fixed delay faded awayand the position of the peak in the correlogram changed from sweepto sweep. These variations in the synchrony of the thalamocorticalsystem were probably elicited by the changes of the firing pattern ofthalamic neurons depicted by the auto-correlograms of thalamicextracellular unit recordings before and after the stimulation of thePPT (Fig. 5B) and LC (Fig. 6B). In both cases the activation through PPT/LC stimulation was accompanied by a diminution in the probability ofburst firing from ~40% to 20%.

The mechanism, which may account for the decreased amplitudeof the thalamic EEG component of OA and for the desynchronizationof OA between the thalamus and the cortex following the activation ofcholinergic and noradrenergic modulatory systems, could be based onthe spike timing of thalamic neurons relative to the phase of the OA.

. A) Top panel depicts a cortical field EEG recording together with thalamic field EEG ands-correlogram between cortical and thalamic field EEG is presented in the bottom panel.-triggered auto-correlograms of thalamic unit recording and histograms of inter-spiketo-correlogram from oscillatory to tonic discharge, and reduces the probability of burst

Fig. 7.Disruption of thalamic firing patterns during OA following stimulation of PPT. A) Rawwaveforms of the ten isolated units from amultiunit recording. Individual units are color-coded. B) Spike histogram and field potential before and after stimulation. Same color-code for units as in panel A. C) Fraction of spikes per unit during the negative (blue) and thepositive (red) phases of the field potential before and after stimulation. Stimulation of PPT abolished phase preference of thalamic spikes.

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Since activities of both PPT and LC neurons depolarize TC relayneurons and cause switching from a synchronous bursting pattern to atonic desynchronized discharge, we further investigated the phases ofspikes relative to the OA oscillation in the thalamus.

Frommultiunit recordings in the thalamic VPL nucleus, we extractedindividual units based on their extracellular action potential waveform(Fig. 7A). We counted every occurrence during the positive or negativephases of OA for each putative single unit (Fig. 7B). The probability offiring for 10 isolated units obtained from extracellular recordings in theVPL nucleus during OA, before and after the stimulation of the PPT, isdepicted in Fig. 7C. During the OA the vast majority of units dischargedduring thenegative phase of theOA (average probability forfiringon thenegative half-cyclewasp=0.74). The depolarization of thalamic neuronsand the change in the firing pattern from bursting to tonic firingdescribed above decreased the probability of firing during the negativephase of theOA to 0.51, a value similar to the average probability offiringduring the positive phase (0.49). Thus, activation of neuromodulatorysystems indeed disrupted the grouping of thalamic action potentials bythe OA oscillatory pattern at the single unit level.

Discussion

The present study reports some major findings regarding themechanisms of generation of OA induced by kindling and the effects ofmodulatory systems on OA. First, we showed a continuous increase inthe incidence of spontaneous burst firing of TC neurons from VPLnucleus during cortical kindling. Second, we found that the cortex and

thalamus are sequentially involved in the generation of seizures inkindled cats. As previously reported in anesthetized animals (Steriadeand Contreras, 1995; Polack and Charpier, 2006) cortical paroxysmalactivities precede thalamic activities during the AS. Third during OA,thalamic field potential deflections precede cortical discharges.Fourth, despite the fact that PPT/LC stimulation showed a strongactivating effect when applied during naturally occurring SWS,activation via PPT/LC during OA did not succeed in stopping theparoxysmal discharges. However, it diminished the amplitude of OA atthalamic but not at cortical level without changing the meanfrequency of the OA. Finally, we determined that the synchronybetween cortical and thalamic oscillations was impaired followingPPT/LC stimulation as TC neurons changed their firing pattern frombursting to tonic and the fixed phase-lock between cellular activitiesand field oscillation was lost. Overall, our study suggests that thethalamus contributes to the generation of OA but is not playing anexclusive role.

OA represent a spontaneous paroxysmal event with a rhythmicactivity at ~2 Hz that follows the AS induced by electrical corticalstimulation. OA has never before been observed following acuteelectrical stimulation of the cerebral cortex under barbiturate,ketamine–xylazine or urethane anesthesia; although these manipula-tions elicit seizures (reviewed in Timofeev and Steriade, 2004). Hence,it is reasonable to presume that neuromodulatory systems may playan important role in the generation of OA.

Both in humans and in experimental animals, repetitive seizureactivity as it occurs during kindling or status epilepticus results in a

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major network reorganization (Coulter and DeLorenzo, 1999; Sloviter,1999; Ben-Ari, 2001). A multitude of structural and functionalsynaptic changes have been shown to follow repetitive seizures,such as reorganization of excitatory axons (Sutula et al., 1989), alteredfunction of excitatory neurotransmitter receptors (Turner and Wheal,1991; Lothman et al., 1995), changes in GABAA receptor-mediatedinhibition (Gibbs et al., 1997; Cossart et al., 2001), reduced thalamicvolume (Margerison and Corsellis, 1966; DeCarli et al., 1998), etc.

Much less is known about the changes in intrinsic neuronalproperties. A study in which status epilepticus induced by pilocarpineprogressed to a chronic epileptic condition resembling humantemporal lobe epilepsy (Turski et al., 1983) suggested that suchalterations may be highly significant, and a more recent study found amarked upregulationof intrinsic neuronal bursting associatedwith thedevelopment of temporal lobe epilepsy (Sanabria et al., 2001).Although almost all pyramidal neurons in the hippocampal CA1 areaare regular spiking cells in normal conditions (Jensen et al.,1994), ~50%of CA1 pyramidal cells in hippocampal tissue removed from rats thatexperienced repetitive seizures were found to be intrinsically burst-firing cells. Moreover, the upregulation of intrinsic bursting seemed toresult from the de novo appearance of Ca2+-dependent bursting that isnot ordinarily seen in this class of principal hippocampal neurons(Azouz et al.,1996). The density of T-type Ca2+ currents, but not of otherpharmacologically isolated Ca2+ current types, was upregulated in CA1pyramidal neurons after status epilepticus (Su et al., 2002). Recentstudies reported increased T-type calcium currents in thalamicneurons, whichmay underlie the bursts of actionpotentials participat-ing in SW activity (Bertram et al., 2001; Su et al., 2002), and alsoincreased thalamic T-type calcium channel gene expression in apilocarpine model of epilepsy (Graef et al., 2006). Such intrinsicchanges of the T-type calcium channel may account for the increasedbursting discharge pattern of TC neurons during epileptogenesis(Fig. 1) and may be associated with the development of spontaneousrecurrent seizures in the corticothalamic system.

TC neurons function in two different modes: either tonicallydischarging at depolarized membrane potentials or bursting athyperpolarized levels of the membrane potentials (Llinas and Jahnsen,1982; Steriade and Llinas, 1988). Neuromodulators can depolarize TCcells and switch the intrinsic activity pattern from bursting to tonicfiring (McCormick, 1992a,b). TC neurons operate under the control ofmodulatory systems originating in the brainstem and basal forebrain(Steriade andMcCarley, 2005). The direct PPT projection toTC neuronsin felines and primates (Pare et al., 1988; Steriade and Llinas, 1988)produces a powerful excitation at their targets. The muscariniccomponent of this prolonged depolarization is associated with ablock of K+ currents (McCormick, 1992a,b) and an increase in inputresistance (Curro Dossi et al., 1991), which explains the increasedresponsiveness of TC neurons during brain-activated states of wakingand REM sleep (Glenn and Steriade, 1982). Norepinephrine released in

Fig. 8. Proposed model for thalamocortical interactions during acute seizures/afterdischastructures (cortex, reticular nucleus, and thalamus) cooperate to generate the EEG rhythms.and thus strongly inhibit thalamus. Once released from reticular neuron's inhibition durpropagates to the cortex. However, when stimulating PPT/LC during OA the persistenceoscillations. Asterisks mark the leading structure.

the thalamus by locus coeruleus neurons depolarizes both thalamic REneurons, and TC neurons, and enhances a hyperpolarization-activatedcation current through β-adrenoreceptors (McCormick and Pape,1990a).

We tested here the effect of PPT/LC stimulation on the expressionof OA, since REM sleep is the sleep stage most resistant to thepropagation of epileptic EEG activities based on the clinical experience(Shouse et al., 2000), whereas SWS is considered to facilitate theepileptiform discharges (Gigli et al., 1992; Dinner 2002; Niedermeyerand Lopes da Silva, 2005). In our parallel study we demonstrated thatOA are absent during REM sleep (Nita et al., 2008-this issue).Cholinergic pontomesencephalic neurons discharge during waking,decrease firing during SWS, and increase firing during REM sleep andfacilitate cortical activation through nicotinic and muscarinic recep-tors (Curro Dossi et al., 1991; McCormick, 1992b). LC neurons typicallydischarge during active waking, decrease their firing during SWS andare silent during REM sleep (McCarley and Hobson,1975; Rudolph andAntkowiak, 2004). In our experiments, the stimulation of thesestructures induced activated EEG pattern tonic firing of TC neurons,and waked up animal. However, neither of them was able to blockcompletely the occurrence of OA. This may suggest that low-thresholdthalamic mechanisms are not essential for the generation of corticalOA.

We were not able to investigate completely the behavioral seizuressince the ethical limiting point of our experimentswas the occurrence ofbehavioral seizures in kindled cats. We predict that OA are preserved inisolated thalamus but complete isolation of the thalamus in vivo in non-anesthetized cats cannot be envisaged due to ethical considerations.However, in two experiments, we kindled cats that underwent corticaldeafferentation of the suprasylvian gyrus— an experimentalmodel thatwe currently use to study epilepsy related to cortical deafferentation(Nita et al., 2006, 2007). Congruently with experiments shown in Figs. 3and4, the amplitude of theOAwasmuchdiminished in the deafferentedcortex (data not shown). This observation further supports a contribu-tion of extracortical structures to the generation of OA. The experimentswithkindlingof undercut cortex arenot extensively discussedhere sinceit is difficult to distinguish between epileptogenesis induced bydeafferentation (Timofeev and Bazhenov, 2005) and epileptogenesisinduced by kindling.

Our results demonstrate an increase in the spontaneous burstfiring of thalamic neurons following kindling and differential roles forthe cortex and the thalamus in the generation of seizures and OAfollowing kindling (Fig. 8). The AS that follows cortical stimulationprobably has a cortical origin, and cortical volleys impinging on REinhibitory thalamic neurons induce strong IPSPs in TC neurons. As aresult, TC neurons become steadily hyperpolarized, and usually do notfire during cortically-generated AS. Cortical SW complexes precedethalamic SW activity. During OA, however, thalamic neurons are likelyreleased from steady inhibition imposed by reticular neurons, and may

rges (AS) and outlasting activities (OA). During natural states of vigilance, the threeDuring cortically-generated AS cortical neurons stimulate GABA-ergic reticular neuronsing the postictal depression thalamic neurons generate rhythmic OA, which furtherof unaltered OA at cortical level suggests that neocortex is able to self-sustain these

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fire bursts of action potentials in the frequency range of intrinsic deltaoscillation (1–4 Hz). Cortical drive synchronously activate RE neurons,which inhibit TC neurons and their intrinsic rebound (Grenier et al.,1998) drive cortex. Therefore, thalamic components of OA precede thecortical components. The involvement of the thalamus in OA generationis further supported by the fact that PPT/LC stimulation induces tonicfiring of TC neurons and decreases the amplitude of OA at thalamic butnot at cortical level. The persistence of OA at cortical level suggests thatneocortex is able to sustain these oscillations even in the absence of anactive contribution of the thalamus.

The mechanisms that underlie kindling phenomenon in thecorticothalamic circuits and contribute to the generation of OA mayalso account for the increased severity of human epileptic disordersfollowing repetitive seizures (Hauser et al., 1990, 1998; Hesdorffer et al.,1998). However, by difference with acute seizures, which despitepowerful and strongly synchronized events usually last for only tens ofseconds; OA last for hours and therefore they could induce long-lastingmodifications of synaptic or intrinsic properties, altering the thresholdfor the development of seizures. In humans, it was previously reportedthe occurrence of interictal slow delta activities in patients withepilepsy, and the presence of these electric activities was associatedwith more severe forms of epilepsy (Koutroumanidis et al., 2004;Hughes and Fino, 2004; Hughes et al., 2004). These long-lasting slowpatterns of synchronization in the cortical circuits were never describedpreviously in experimental animals; therefore, this model could bevaluable for studying themechanisms of epileptogenesis and for testingthe effect of potential newantiepileptic drugs at different sites inside thecorticothalamic circuit (Holmes, 2007).

Acknowledgments

We would like to thank Pierre Giguère for excellent technicalassistance. This study was supported by Canadian Institutes of HealthResearch (Grant MOP-37862, MOP-67175), NSERC of Canada (grant298475) and Savoy Foundation. I.T. is Canadian Institutes of HealthResearch scholar. F.F. acknowledges Terrence J. Sejnowski for con-tinued support.

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