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Research Report

Neonatal sensory deprivation promotes development ofabsence seizures in adult rats with genetic predispositionto epilepsy

Evgenia Sitnikova⁎

Institute of Higher Nervous Activity and Neurophysiology RAS, Butlerova str., 5A, 117485 Moscow, Russia

A R T I C L E I N F O

⁎ Fax: +7 499 743 00 56.E-mail address: jenia-s@mail.ru.Abbreviations: IS stage, intermediate sleep

wave spindles

0006-8993/$ – see front matter © 2011 Elsevidoi:10.1016/j.brainres.2010.12.067

A B S T R A C T

Article history:Accepted 21 December 2010Available online 29 December 2010

Absence epilepsy has age-related onset. In a WAG/Rij rat genetic model, absence seizuresappear after puberty and they are increased with age. It is known that (1) epileptic activity inWAG/Rij rats is initiated at the perioral area in the somatosensory cortex; (2) sensorydeprivation, i.e., whisker trimming during the critical period of development, could enhanceexcitatory activity in the somatosensory cortex. It is hypothesized that the cortex maybecome more excitable after neonatal vibrissae removal, and this may precipitate absenceseizures in adult rats. We found that whisker trimming during the first postnatal weekscaused more rapid development of EEG seizure activity in adult WAG/Rij rats. Epilepticdischarges in the trimmed rats were more numerous (vs control), showed longer durationand often appeared in desynchronized and drowsy EEG. The number of absence-likespindle-shaped EEG events (spike–wave spindles) in the whisker-trimmed rats was higherthan in control, especially during the intermediate sleep state. An age-dependent increaseof intermediate sleep state was found in the trimmed rats, but not in the intact animals. Wediscuss epigenetic factors that can modulate absence epilepsy in genetically prone subjects.

© 2011 Elsevier B.V. All rights reserved.

Keywords:WAG/Rij ratSomatosensory cortexAbsence epilepsySpike–wave dischargeSpike–wave spindleIntermediate sleep stage

1. Introduction

Epilepsy is a neurological disorder associated with abnormallyhigh neuronal excitability. Normally, excitatory/inhibitorybalance is controlled by a homeostatic mechanism, which iscapable of preventing the state of neuronal hyperexcitabilityand precludes epileptic discharges (Hartmann et al., 2008). It isknown that the efficacy of excitatory/inhibitory processeswithin neural circuits can be modulated by sensory experi-ence. There are several sensitive periods during the earlyontogenesis, when abnormal sensory experience may irre-

stage; SmI, somatosensor

er B.V. All rights reserved

versibly change cortical excitability (Lee et al., 2009). Forexample, the somatosensory cortex (SmI) in adult rats, whosevibrissae were trimmed during the first 3 postnatal weeks (i.e.,during a critical period for experience-dependent neuronalplasticity), displayed a stronger excitation and the lack ofinhibitory responses (e.g., Simons and Land, 1987; Sitnikova,2000; Shoykhet et al., 2005). It is remarkable that the SmI inrats with a genetic predisposition to absence epilepsy, e.g.,WAG/Rij rat strain, is characterized by synaptic hyperexcit-ability (D'Antuono et al., 2006). Furthermore, the SmI in WAG/Rij rats is known to be primarily involved in triggering of

y cortex; SWD, spike–wave discharges; SWsp (SW-spindles), spike–

.

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epileptic discharges (Meeren et al., 2002; van Luijtelaar andSitnikova, 2006). Based on these facts, we hypothesize thatneonatal sensory deprivation of the whisker system willstrengthen the excitatory activity in the SmI and this willprecipitate absence seizures in genetically prone subjects. Thepresent study examines the long-term effect of whiskertrimming during early postnatal ontogenesis on the develop-ment of spontaneous epilepsy in adult rats.

Absence seizures in WAG/Rij rats develop spontaneouslyand they are accompanied by spike–wave discharges (SWD) inthe electroencephalogram (EEG) (Coenen and van Luijtelaar,1987). Young adult WAG/Rij rats (up to 2 months old) do notexpress epileptic discharges. Only immature forms of SWDcan be recorded at this age. The number of SWD graduallyincreases between 3 and 6 months of age in parallel with thegradual changes of EEG properties of seizure activity, i.e., anincrease in frequency and duration. In WAG/Rij rats, SWD aremost clearly expressed at the age of 7–8 months, and becomeeven more pronounced in elder rats (Coenen and vanLuijtelaar, 1987; van Luijtelaar and Bikbaev, 2007). Consideringthese literature data, we examine development of absence-like electroencephalographic patterns in WAG/Rij rats be-tween 5 and 8 months of age.

It is well known that absence epilepsy has very closerelationship with sleep. In human patients, absence seizuresoften occur during periods of decreased vigilance, e.g. intransition periods between wakefulness and non-REM sleep(refs in Dinner and Lüders, 2001). Typical absences with pure3 Hz spike–wave discharges appear during somnolence andsleep stage 1 (Halász et al., 2002). Also in WAG/Rij rats, SWDpredominantly occur during slow-wave sleep and passivewakefulness, and they tend to prevail in transitional states(Drinkenburg et al., 1991). In addition to that, epileptic activityin WAG/Rij rats associates with disturbances of sleep-controlling mechanisms. More specifically, WAG/Rij rats differfrom non-epileptic rats by a shorter REM-sleep (van Luijtelaarand Bikbaev, 2007) and longer periods of intermediate sleep (IS)stage (Gandolfo et al., 1990). The IS stage characterizestransition from deep non-REM sleep to wakefulness or to REMsleep, and it can be distinguished in EEG by the simultaneouspresence of high-voltage spindle activity in the fronto-parietalcortical areas and hippocampal theta activity (Gottesmann,1996). Mechanisms of IS stage are not well understood. There isan indication that amore pronounced expression of IS stage inWAG/Rij rats might correlate with genetic predisposition toabsence epilepsy (Drinkenburg et al., 1993). We assume thatepigenetic factors that presumably influence absence seizureswould also affect IS stage. In general, the current paperhighlights experience-dependent mechanisms that governabsence epilepsy in genetically prone subjects.

Fig. 1 – Electroencephalographic expression of SWD asrecorded in an 8-month old control WAG/Rij rat. SWD moreoften appeared in the desynchronized (a) and drowsy EEG (b)and, occasionally, in slow-wave sleep EEG (c).

2. Results

2.1. The incidence of SWD

SWD appeared in EEG as a sequence of stereotypic elements:well-pronounced sharp negative spikes followed by smallerwaves (Fig. 1). SWDmet the criteria provided by van Luijtelaarand Coenen (1986) (see Experimental procedures for details).

Control rats at the age of 5 months displayed very fewSWD. Seizures were detected in 3 out of 7 animals, and theother 4 subjects did not display any SWD. At the same age, allrats in the treated group expressed SWD (from 1 to 28, inaverage 25.1±21.6, Table 1), and the number of SWD wassignificantly higher than that in the control group [one-wayANOVA, factor ‘group’: F(1.11)=6.1, p<0.05]. The incidence ofepileptic discharges increased with age in both groups. Thenumber of SWD in 5-month old rats was significantly lowerthan in 8-month old animals [factor ‘age’ F(1.22)=6.8, p<0.05].Whisker-trimmed rats showed more SWD than the controlrats [factor ‘group’ was significant F(1.22)=4.9, p<0.05].

Table 1 – The influence of neonatal whisker trimming on the incidence of SWD and SW-spindles in adult animals(Mean±S.D.).

Age(months)

Number of episodes (per rat) Duration (s) Cumulative timea (s)

Control(n=7 rats)

Whisker-trimmed(n=7 rats)

Control(n=7 rats)

Whisker-trimmed(n=7 rats)

Control(n=7 rats)

Whisker-trimmed(n=7 rats)

SWD 5 3.0±5.3 25.1±21.6c 3.8±0.9 4.7±1.1 34±21 117±908 29.3±33.6b 55.0±36.3b,c 6.3±3.0 b 8.4±3.4b,c 225±247 521±387

SW-spindles 5 5.7±4.7 21.8±5.7 2.1±0.2 2.0±1.1 16.6±9.8 46.7±15.98 5.8±3.5 17.7±7.7 2.8±0.8b 3.1±0.6b 18.9±12.0 48.4±21.5

a Total sum of SWD/SW-spindle interval durations.b Significant differences between 5- and 8-month old rats in the trimmed group (within-subject design, p<0.005).c Significant differences between trimmed and control rats (p<0.005).

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Statistical data of age-related changes of SWD are shown inFig. 2A. In the control group, 6 out of 7 rats showed an age-related increase in the number of SWD (in one rat SWD were

Fig. 2 – The number of SWD (A) showed substantial inter-individalmost all subjects. Very few SWD appeared during sleep (B). SW(mean±standard deviations). Asterisks are significant differencepost-hoc LSD test for 2-ways ANOVA.

absent). Also in the trimmed group, in 6 out of 7 rats thenumber of SWD increased in a period between 5 and 8 monthsof age. The one particular rat, in which the number of SWD

ual variations, but it was progressively increased with age inD preferably occurred in desynchronized and drowsy EEGs between the trimmed and control rats, according to the

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decreased with age, was an exception. At the age of 5 months,whisker-trimmed animals showed as many SWD as 8-monthold control rats (Table 1). This may suggest that whiskertrimming accelerates age-related increase of SWD and,therefore, SWD in the trimmed animals appeared earlierthan in the control group. Despite this, SWD in 5-month oldtrimmed rats were not fully blown, at least duration andcumulative seizure time were lower than that in 8-month oldcontrol rats (Table 1).

2.2. Duration of SWD

SWDduration values did not shownormal distribution neitherin the control, nor in the trimmed group (Kolmogorov–Smirnovtest for normality, p<0.01 in both groups, Fig. 3A). In thecontrol and trimmed rats, distributions of SWD durationshowed two peaks ~4 s and ~6–8 s. SWD whose durationwas <6 s were referred to as short-lasting events, and >6s-long-lasting.

As long as the test for normality was failed, thestatistical analysis of SWD durations was performed usinga non-parametric Mann–Whitney test. It was found thatduration of SWD increased with age in the control group(p<0.002) as well as in the whisker-trimmed group(p<0.00001, Table 1). In the whisker-trimmed group, SWDlasted significantly longer as compared to the age-matched

Fig. 3 – Histograms of duration distribution. Durations of SWD (Anormality) and showed two peaks, around 3–4 s and >6 s (corresSW-spindles (B) were normally distributed.

control. At the age of 5 months, the difference in the meanduration of SWD between two groups was about 1 s (Z=−1.9,p<0.05, Table 1), and, in 8-month animals, this difference waseven higher, i.e., about 2 s with the higher level of significance(Z=−7.0, p<0.0001).

SWD duration showed remarkable age-related changes. Inthe control 5-month old rats (Fig. 3A, left plot, white bars), allSWD were classified as short-lasting (peak durations in 4–5 s).Eight-month old animals (Fig. 3A, right plot, white bars)expressed both short- and long-lasting SWD (peak durationsin ~4 and ~7 s correspondingly). In the control group, theamount of short-lasting SWD in the older rats increased ascompared to the younger rats.

Young whisker-trimmed rats (5 months old) exhibited bothshort- and long-lasting SWD, in opposite to the control rats, inwhich only short-lasting SWD were detected at the same age(Fig. 3A, gray distributions). At the age of 8 months, SWDduration distribution formed a ‘plateau’-like broad peak from8 to 13 s, corresponding to the long-lasting SWD. Theproportion of long-lasting SWD in the trimmed groupincreased with age.

2.3. Phenomenology and incidence of SW-spindles

In addition to the genuine SWD, we examined pro-epilepticEEG events called as spike–wave spindles (SWsp, Fig. 4). SWsp

) were not normally distributed (Kolmogorov–Smirnov test forpondingly, short- and long-lasting SWD). Duration values of

Fig. 4 – Examples of SW-spindles as recorded during differentEEG states (8-month old control WAG/Rij rat). SW-spindlesconsisted of sharp negativewaveswith characteristic waxingand waning appearance (spindle-shaped) and includedfragments of spike–wave complexes. In desynchronized EEG(bottom plate), SW-spindles were preceded by relatively longperiods of EEG desynchronization. In sleep EEG background(top plate) and intermediate sleep state (middle plate),SW-spindles were preceded by very short periods ofdesynchronization.

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consisted of sharp negative waves with fragments of spike–wave complexes. In opposite to SWD, SWsp were character-ized by a waxing–waning morphology (gradual elevation anddescending of amplitude), and they were shorter in duration.SWsp roughly corresponded to the ‘spiky phenomena’ de-scribed by Drinkenburg et al. (1993) in WAG/Rij rats.

Inter-individual variability of the number of SW-spindleswas large in both groups. It was found that whisker trimmingaffected the incidence of SW-spindles. The number of SW-spindles in the trimmed group was significantly higher ascompared to the control (factor ‘group’, F(1.22)=38.4, p<0.0001,Table 1). Also total SW-spindle time in the deprived groupexceeded the corresponding control values more than twice(Table 1). The number of SW-spindles did not show anysignificant changes with age (Fig. 5A).

Durations of SW-spindles were normally distributed(Fig. 3B), therefore, ANOVA was used for the statisticalanalysis. Duration of SW-spindles slightly, but significantlyincreased with age in both groups (factor ‘age’ F(1.22)=9.6,p<0.005, Table 1).

2.4. State-dependent occurrence of SWD and SW-spindlesin EEG

All episodes of SWD and SW-spindles were subdivided in fourgroups based on the character of preceding EEG activity:desynchronized, drowsiness, slow-wave sleep and intermedi-ate sleep (IS) stage. EEG intervals ~10 s before the onset ofSWD/SW-spindle were visually classified as (1) ‘desynchro-nized EEG’, (2) ‘drowsiness’, i.e., the state when low-voltagefast activity was interrupted by high-voltage and slow waves,(3) ‘slow-wave sleep’ characterized by slow-wave activity andsleep spindles and (4) ‘intermediate state of sleep’ (IS) whichappeared in transition from the slow-wave sleep to eitherREM-sleep or drowsiness (criteria for determining the IS aregiven in Gottesmann, 1996). Briefly, the IS was characterizedby the simultaneous presence of high-voltage spindle activityin the fronto-parietal derivations and theta rhythm in theoccipital EEG (Fig. 1, middle plate). We scored the number of ISepisodes and measured their duration.

In the whisker-trimmed rats of both ages, SW-spindleswere found during intermediate sleep stage more often thanin the control (Fig. 5B, for both ages p's<0.05 as defined withpost-hoc LSD test for 2-way ANOVA). IS stage seems to befavorable for the occurrence of SW-spindles in the trimmedanimals. On the other hand, an increased duration of IS stagein deprived rats might be associated with a higher amount ofSW-spindles (see next Section 2.5).

A vast majority of SWD appeared during EEG desynchro-nization and drowsiness (Figs. 1 and 2B). SWD were absentduring REM sleep. In 5-month old rats, SWD were not presentduring slow-wave sleep and during IS stage in both groups(Fig. 2B). The older, 8-month old animals, expressed just a fewSWD during these two stages. Trimmed rats at the age of5 months showed significantly more SWD in desynchronizedand drowsy EEG states as compared to the control subjects[Fig. 2B, left plot, p<0.05 post-hoc analysis for ANOVA: factor‘group’: F(1.48)=13.1, p<0.001; factor ‘EEG state’: F(3.48)=6.2,p<0.005; factor interaction F(3.48)=4.3, p<0.05]. Also at the ageof 8 months, the whisker-trimmed group differs from thecontrol by a significantly higher amount of SWD during thedesynchronized EEG state [Fig. 2B, right plot, p<0.05 post-hocanalysis of ANOVA: factor ‘group’: F(1.48)=6.1, p<0.05; factor‘EEG state’: F(3.48)=6.9, p<0.001]. The number of SWD duringslow-wave sleep in the whisker-trimmed group did not differfrom the control level.

In general, whisker trimming promotes epileptic activityduring desynchronized EEG state and during drowsiness, butnot during slow-wave sleep.

2.5. Intermediate stage of sleep

Table 2 presents the statistical data characterizing IS stage. Inthe control group, parameters of the IS stage in 5-month oldanimals did not differ from that in 8-month old subjects. The

Fig. 5 – The number of SW-spindles (A) did not change with age. In the whisker-trimmed group, SW-spindles were morenumerous as compared to the control. (B) Mean number of SW-spindles (±standard deviations) in respect to the EEGbackground state. Asterisks are significant differences between the trimmed and control rats, according to the pos-hoc LSD testfor 2-ways ANOVA.

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trimmed animals, in opposite, displayed age-related elevationof the IS stage: a higher amount of IS episodes, a longerduration of each episode and an increased cumulative time ofIS stage were found in the older rats as compared to theyounger rats (5 months old).

At the age of 5 months, characteristics of IS stage in thetrimmed group did not differ from the control. Differencesbetween the whisker-trimmed and control rats becamesignificant at the older age. In 8-month old trimmed rats,both cumulative time of the IS stage and number of ISepisodes were higher than in age-matched control rats.

Table 2 – Characteristics of the intermediate stage of sleep (Mea

Age(months)

Number of episodes (per rat) Mean d

Control Whisker-trimmed Control

5 3.1±2.5 1.8±1.2 10.9±8.98 2.5±1.8 7.2±3.3a,b 12.0±5.9

a Significant differences between 5- and 8-month old rats in the trimmeb Significant differences between trimmed and control rats at the age of

In the control group, the number of SW-spindles during ISstage showed positive correlations with the number of ISepisodes (Spearman rank correlations were R=0.75 in 5-monthold rats and R=0.83 in 8-month old, both p's<0.05). Also, thenumber of SW-spindles during IS stage correlatedwith the totalduration of IS stage (in younger rats R=0.79, in older rats R=0.80,p's<0.05). No correlations were found between the above-mentioned parameters in the whisker-trimmed animals. It isapparent that if the control subject (but not whisker-trimmed)had a higher amount of IS sleep, it also expressed the highernumber of SW-spindles during IS irrespectively from its age.

n±S.D.).

uration of a singleepisode (s)

Total duration (s)

Whisker-trimmed Control Whisker-trimmed

7.0±9.1 49.4±52.8 22.3±32.815.8±2.9a 35.7±34.2 116.8±58.4a,b

d group (within-subject design, p<0.005).8 months (p<0.005).

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In summary, within-subject analysis indicates that the ISstage significantly increased with age only in the whisker-trimmed animals, but not in the control rats. Between-subjecttest demonstrates significantly more pronounced IS stage inwhisker-trimmed subjects (vs control) at the age of 8 months,i.e., noteworthy that at this age animals also expressedintense seizure activity. Correlation analysis shows that age-related increase of IS stage in the trimmed group did notfacilitate the occurrence of the SW-spindles during thisparticular stage. In other words, the number of SW-spindlesduring IS stage in young animals was already high and it wasnot increased further with age.

1 Ih current controls cellular excitability and it is closely linkedto neuronal hyperexcitability during pathological conditions,such as epilepsy [more details in Strauss et al. (2004)].

3. Discussion

Ourdata clearly indicate thatwhisker trimmingduring the first3 weeks of life enhances the incidence of SWD and SW-spindles in adultWAG/Rij rats.Wewere focused in thewhiskersystem, because it provides afferent input to the epileptic zonein the SmI (Meeren et al., 2002; Sitnikova and van Luijtelaar,2004). It is known that neonatal whisker trimming causespermanent changes of neuronal activity and enhances excit-ability of neurons in the SmI (Shoykhet et al., 2005; Breton andStuart, 2009), and this can lead to hyperactivation of theepileptic cortical zone and promote epileptic activity in WAG/Rij rats. Our results show that absence seizures in thewhisker-trimmed rats appeared earlier than in the control group. At theage of 5 months, whisker-trimmed animals showed as manySWD as 8-month old control rats, however, EEG parameters ofseizures remained immature.

It is important that whisker deafferentation in our experi-ments was performed during a short critical period for thedevelopment of the whisker system, and this manipulationhad a long-term effect promoting development of seizures.Vibrissal deprivation after the critical period, i.e., in adultanimals, may have an opposite effect. For example, acutedeactivation of the major sensory pathway from the whiskerpad (n. infraorbitalis) with 2% Novocain in 6–8-month oldWAG/Rij rats is known to suppress the incidence of SWD (Abbasovaet al., 2010). In these experiments, authors blocked excitatoryinput from whiskers to the SmI and thus prevented excessiveexcitation of the cortical epileptic source and effectivelyreduced the incidence of EEG seizures. Similar results werefound in our previous study, in which SWD were temporallyreduced after local administration of 2% lidocaine directly tothe SmI (Sitnikova and van Luijtelaar, 2004). In the currentstudy we used the opposite manipulation, sensory depriva-tion, that seems to shift excitatory–inhibitory balance in theSmI towards excitation, and this could be a precondition forthe development of more severe epileptic activity.

3.1. Age-related changes of SWD

Our study demonstrates that the number of SWD increaseswith age. Similar age-related elevation of absence epilepticdischarges has been reported in other rat strains with absenceepilepsy: in GAERS, whose genotype and phenotype are veryclose to WAG/Rij rats (Carçak et al., 2008), and in Fischer 344rats that do not have genetic affinity with WAG/Rij rat strain

(Buzsáki et al., 1988). We show that duration of SWD is alsoincreased with age. This associates with the presence of long-lasting SWD in the older subjects. In the control group, long-lasting SWD were found in 8-month old rats and they wereabsent in younger animals (5 months old). In the trimmedgroup, long-lasting SWD were already present in 5-month oldrats. These facts, taken together, lead us to a conclusion aboutmore rapid development of seizure activity in the sensorydeprived animals in comparison to the intact rats.

3.2. Epigenetic control of absence epilepsy

It is well accepted that absence epilepsy is genetically prede-termined and it is characterized by age-dependent expression.Based on our findings, we can add that age-related changes ofabsence epilepsy can bemodified by early sensory experience. Inother words, absence epilepsy is governed by experience-dependent mechanisms. Our conclusion is in line with theearlier findings, demonstrating that absence seizures inWAG/Rijare sensitive to environmental manipulations of Schridde et al.(2006). In particular, neonatal handling andmaternal deprivationof rat pups during the first threeweeks resulted in a pronouncedreduction of absence seizures in adulthood. It is also importantthat long-term reduction of absence seizures in neonatallydeprived or handled rats was linked with an enhancement ofthe hyperpolarization-activated cation current (Ih)1 in the SmI(Schriddeet al., 2006). This suggests that theSmI is endowedwithintrinsic ion channel mechanisms that could mediate environ-mental influences on the development of seizures in geneticallyprone subjects.

At least two factors may contribute to pathogeneticprocesses in the epileptic source in the SmI: 1) an impairmentof intrinsic properties of neurons, includingmembrane and ionchannels (i.e., channelopathy) (Strauss et al., 2004; Schridde etal., 2006; Klein et al., 2004); and 2) a deficiency of inhibition(Luhmann et al., 1995). Inasmuch as absence epilepsy has astrong genetic component, some of the abovementionedabnormalities could be linked to genotype. Our study demon-strates that environmental epigenetic factors could influencephenotypic expressionof geneticallypredeterminedpathology.

3.3. Sleep-related mechanisms of SWD, intermediatesleep state

The present study demonstrates that SWD rarely occur duringslow-wave sleep. This agrees with findings in GAERS, dem-onstrating very few SWD during slow-wave sleep (Pinaultet al., 2006); but slightly disagrees with the earlier data inWAG/Rij rats, suggesting that sleep is themost favorable statefor SWD to occur (Drinkenburg et al., 1991). The latter ideaoriginates from the feline penicillin model of P. Gloor(reviewed by Kostopoulos, 2000), who showed that normalsleep spindle oscillations could give rise to the spike–wave EEGpattern, when cortex entered hyperexcitable state. Morerecent investigations define the intimate relationship be-tween EEG arousal phenomena and spike–wave paroxysms.

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Spontaneous spike–wave seizures are often associated witharousal-dependent phasic events preceded by K-complexes(e.g., Halász and Kelemen, 2009). Perhaps, epileptic dischargesappear in thalamo-cortical circuitry as abnormal hypersyn-chronous neuronal bursts during the waking state. Thesebursts are unusual for healthy subjects and could result fromgenetically predetermined channelopathies or other distur-bances in cortical and brainstem arousal systems.

According to the present data, the early whisker trimmingaffects state-related dynamics of SWD and enhances theintermediate sleep stage. Sensory deprived rats express moreSWD during a desynchronized EEG state, and show a tendencytowards elevation of SWD during drowsiness, but not duringslow-wave sleep. It is known that sleep homeostasis mecha-nisms, which govern transitions between wakefulness andnon-REM sleep, are impaired in subjects with absence epilepsy(e.g., Halász et al., 2002). Also in WAG/Rij rats, the duration ofsleep cycle and the duration of REM sleep are shorter than innon-epileptic ACI rats (van Luijtelaar and Bikbaev, 2007). It ispossible that sensory deprivation might indirectly affect sleephomeostasis and, therefore, create favorable conditions forthe occurrence of SWD during desynchronized EEG state.

Our paper also demonstrates that trimmed subjects, inopposite to untreated rats, display more pronounced IS stage.Interestingly, parameters of IS stage in the control group are thesame in 5- and 8-month old rats. This fits well with theobservations of Kirov and Moyanova (2002) in Wistar rats, whodid not show any differences in respect to the IS stage between 4and 11-month old rats. Age-dependent changes were only notedin rats later in life, i.e., at the age of 23months, when IS stageappeared to be shorter than in the younger rats. In ourexperiments, development of IS stage in the untreated WAG/Rijrats does not distinguish from that in Wistar rats (Kirov andMoyanova, 2002), but it remarkably differs from that in thewhisker-trimmed rats. A drastic increase of IS stage in thetrimmed rats between 5 and 8 months of life is accompaniedby a progressive increase of seizure activity. During IS stage,the forebrain is known to receive the lowest activatinginfluences from the brainstem, and this state correspondsto a physiological cerveau isolé-like preparation consequent toboth forebrain deactivation and disinhibition (Gottesmann,1996). More frequent and longer periods of IS stage in thewhisker-trimmed rats might result from some abnormalitiesin the reticular activating system.

3.4. Phenomenology of SW-spindles

In general, the electroencephalographic pattern of SW-spindlesis similar to ‘high voltage spiky phenomena’ described by Drinken-burg et al. (1993) inWAG/Rij rats: ‘high amplitude, at least twice thebackground EEG, frontal, symmetrical, 8–14-Hz sharp phenomena,lasting at least 1 s’ (p. 780, Drinkenburg et al., 1993). Basically,SWspmeet this description, however, our detection criteria aremainly related to the EEG waveform, but not to the duration(nevertheless, the vastmajority of SWsp last >1 s, in average, 2–3 s). In fact, SW-spindles combine SWD- and spindle-likefeatures i.e., they express spike–wave elements (similar toSWD) and have waxing–winning morphology (similar tospindles). SW-spindles could either represent a hypersynchro-nous spindle activity or an abortive form of SWD. SW-spindles

might be relevant to ‘type 2’ sleep spindles, which are recentlydescribed in WAG/Rij rats (Sitnikova et al., 2009). These ‘type 2’sleep spindles exhibit an abnormal time–frequency profile andconstitute ~10% of all spindles. We can speculate that EEGphenotype of SW-spindlesmeet ‘type 2’ sleep spindles, and thishypothesis could be addressed in the future.

It has long been accepted that SWD and spindles share thesame thalamo-cortical mechanism (refs in Kostopoulos, 2000).This point of viewwas also acknowledged by Drinkenburg et al.(1993), who considered ‘spiky phenomena’ as a transitory EEGwaveform between normal spindle activity and SWD. Ourrecent studies in WAG/Rij rats cast doubts about the fictionalrelationship between sleep spindles and SWD (reviewed inSitnikova, 2010). The current results imply a dual nature of SW-spindles. Similar to SWD, the incidence of SW-spindles areaffectedbyepigenetic factors.At least,whisker trimmingduringthe first postnatal weeks promotes the generation of SW-spindles in adult animals. However, unlike SWD, the incidenceof SW-spindles doesnot changewith age, and in that sense SW-spindles seem to be resistant to aging processes.

4. Experimental procedures

4.1. Animals

Experiments in WAG/Rij rats were performed at Institute ofHigherNervousActivity andNeurophysiology (ratswereobtainedfrom the Department of Biological Psychology, Radboud Univer-sity Nijmegen, the Netherlands). All rats were kept under a light–dark regime (12–12 h with light off at 19:00) with food and wateravailable ad libitum. All experimental procedures were approvedby the animal ethics committee of the Institute ofHigherNervousActivity and Neurophysiology.

Aweek before delivery, pregnant female ratswere placed inindividual cages and kept alone. The day of delivery wasdenoted as PN1. The number of pups per litterwas reduced to 6or 8, and the number of females and males per litter was thesame: 3/3 or 4/4. Thewhole litterswere assigned as treated andcontrol group (‘between-litter design’). In the present study,wepreferred whole-litter in order to avoid social disturbances,which are known to affect early development. In ‘half-litter’experiments, when control littermates are reared togetherwith whisker-trimmed subjects, physiological maturation andtiming of the early forms of behavior in control rats are gettingcloser to the trimmed littermates and significantly differ fromthat obtained in the control pups reared in ‘whole-litter’conditions (Shishelova and Raevskiĭ, 2009).

In the treated group (three litters), all whiskers weretrimmed daily from PN2 to PN20. During this period, thecontrol group (three litters) was subjected to a sham trimming,i.e., gentle mechanic stimulation of the surface of whiskerpads with blunt ends of scissors.

In deprived animals, whiskers were rapidly re-grown afterthe trimming and reached their normal length in approxi-mately a month after the end of the trimming procedure.

At the age of 130–140 days, two or three males from eachlitter were randomly selected for the electroencephalographicexamination. EEG analysis was performed in seven rats fromthe treated group and in seven control animals.

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4.2. Surgery

Animals were equipped with EEG recording electrodes. Stereo-tactic surgery was performed under chloralhydrate anesthesia(325mg/kg, 4% solution in 0.9% NaCl). Electrodes were secured tothe skull using stainless steel screws (shaft length=2.0 mm, headdiameter=2.0 mm, shaft diameter=0.8 mm). Active electrodeswere placed epidurally in the frontal cortex (AP 2; L 2.5), parietalarea (somatosensory cortex, AP −2; L 6) and occipital cortex (AP−5; L 4). All coordinates are given in mm relative to the bregma.EEG was recorded monopolarly with respect to a referenceelectrode over the cerebellum. Electrodes were permanentlyfixed to the skull with methyl methacrylate monomer (Vertex™)together with three additional anchoring screws.

4.3. EEG recording and analysis

EEGwas recorded twoweeks after the surgery (recovery period),when animals were 5 months old. Next EEG recordings weremade in the same rats at the age of 8–9 months. EEG wasrecorded in freelymovingrats. TheEEGrecording sessions lasted20–24 h starting between 4–5 pm (the beginning of dark period).EEG signals were fed into a multi-channel differential amplifiervia a swivel contact, band-pass filtered between 1 and 500 Hz,digitized with 526 samples/s/channel and stored on hard disk.

SWD were detected off-line using the criteria of vanLuijtelaar and Coenen (1986). Briefly, SWD appeared in thefrontal and parietal EEGs as synchronous trains of repetitive 7–10 Hz discharges (~ 8 Hz in average), whose amplitude was atleast two times higher than background and duration waslonger than 1 s.

The number of SWD and SW-spindles were counted overthe period of 6 h during the dark phase of the dark–light cycle(from 2-nd to 8-th hour of dark period). Durations of SWD andSWsp were also measured.

Numerical data were tested for normality using theKolmogorov–Smirnov test. If the hypothesis of normal distribu-tion was rejected (p<0.05 for D value), we used a non-parametric Mann–Whitney test (p<0.05) in order to accessage-related changes within group and differences betweengroups. Statistical analysis of the normally distributed datawas performed using ANOVA (within-subject factor ‘age’ withtwo levels, 5 and 8 months; and between-subjects factor‘group’ for the control and whisker-trimmed rats).

Acknowledgments

This study was supported by a grant from the by RussianFoundation for Basic Research (RFBR, N 09-04-01302). Isincerely appreciate the suggestions and support of Prof.Vladimir V. Raevsky. Technical assistance of Elizaveta Ruts-kova is gratefully acknowledged.

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