Sleep Medicine 13 (2012) 252–262

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Sleep Medicine

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Original Article

Physiologic autonomic arousal heralds motor manifestations of seizuresin nocturnal frontal lobe epilepsy: Implications for pathophysiology

Giovanna Calandra-Buonaura a,⇑, Nicola Toschi b, Federica Provini a, Ivan Corazza c, Francesca Bisulli a,Giorgio Barletta a, Stefano Vandi a, Pasquale Montagna a,�, Maria Guerrisi b, Paolo Tinuper a,Pietro Cortelli a

a IRCCS, Istituto delle Scienze Neurologiche, University of Bologna, Bologna, Italyb Medical Physics Section, Faculty of Medicine, University of Rome ‘‘Tor Vergata’’, Rome, Italyc Cardiovascular Department, University of Bologna, Bologna, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Available online 15 February 2012

Keywords:Nocturnal frontal lobe epilepsyPhase of transitory activationHeart rate variabilityWavelet transformSeizure motor onsetAutonomic arousal

1389-9457/$ - see front matter � 2012 Elsevier B.V. Adoi:10.1016/j.sleep.2011.11.007

Abbreviations: ADNFLE, autosomal dominant nocAO, arousal onset; AP, arousal period; preAP, pre arotonic seizures; CAN, central autonomic network; CPGECG, electrocardiogram; EEG, electroencephalogram;electro-oculogram; HF, high frequency; HFnu, high frHR, heart rate; HRV, HR variability; HS, hyperkineticLFnu, low frequency in normalized units; MRI, MnAChR, nicotinic acetylcholine receptor; NFLE, noctNFLS, nocturnal frontal lobe seizures; NREM, nonparoxysmal arousals; PAT, phase of transitory activament; RRi, RR interval; SEM, standard error of mean;period; preSP, pre seizure period; VLF, vervideopolysomnography.⇑ Corresponding author. Address: IRCCS, Istituto

University of Bologna, via Ugo Foscolo 7, 40120512092772; fax: +39 0512092947.

E-mail address: giovanna.calandra@unibo.it (G. Ca� Prematurely deceased on 9th December 2010.

Objective: This study describes changes in heart rate (HR) and HR variability (HRV) related to clinicalonset of seizures in nocturnal frontal lobe epilepsy (NFLE) in order to determine whether signs of auto-nomic activation precede onset of seizure motor manifestations, which was selected as seizure onset(SO). Further, to clarify the nature (epileptic or physiologic) of the changes in autonomic cardiac controlpresumed to precede SO, time-dependent variations in HR and HRV related to physiological corticalarousals associated with motor activity (phases of transitory activation, PAT) were also investigated.Methods: HR and HRV spectral power, quantified by means of wavelet transform, were analyzed in rela-tion to the onset of motor manifestations in 45 NFLE seizures and 45 PAT derived from whole night video-polysomnographic recordings of ten patients and of ten control subjects, respectively.Results: Analysis of HRV showed a shift of sympathetic/parasympathetic cardiac control toward a sym-pathetic predominance in the 10 s immediately preceding SO, while changes in HR were evident onlyone second before SO. This sympathetic activation was not associated with a sleep-wake transition orchanges in respiratory activity, both of which occurred concurrently with SO. Similar changes in HRand HRV were observed in the 10 s before the motor and electroencephalographic onset of PAT.Conclusions: Our study demonstrates that a similar autonomic activation precedes the motor manifesta-tions of NFLE seizures and physiological arousal. This autonomic activation could represent part of thearousal response, which could be implicated in the occurrence of both seizure and arousal motormanifestations.

� 2012 Elsevier B.V. All rights reserved.

ll rights reserved.

turnal frontal lobe epilepsy;usal period; ATS, asymmetrics, central pattern generators;EMG, electromyogram; EOG,

equency in normalized units;seizures; LF, low frequency;

agnetic Resonance Imaging;urnal frontal lobe epilepsy;

rapid eye movement; PA,tion; REM, rapid eye move-SO, seizure onset; SP, seizurey low frequency; VPSG,

delle Scienze Neurologiche,3 Bologna, Italy. Tel.: +39

landra-Buonaura).

1. Introduction

Nocturnal frontal lobe epilepsy (NFLE) is characterized by aspectrum of sleep-related paroxysmal motor manifestations of var-iable duration and complexity [1–4]. NFLE can occur either sporad-ically or as an autosomal dominant form (ADNFLE) [5], the latterassociated with mutations in the genes encoding for subunits ofthe neuronal nicotinic acetylcholine receptors (nAChR) [6]. Scalpelectroencephalogram (EEG) and neuroradiological investigationsare usually poorly informative in NFLE [4,7]. As a consequence, dis-tinguishing nocturnal frontal lobe seizures (NFLS) from othersleep-related non-epileptic paroxysmal phenomena presentingwith similar motor features, i.e., disorders of arousal [8], remainsarduous [9–11]. A triggering role of the arousal system, which isresponsible for the rhythmic oscillations of cerebral and autonomicactivity during sleep, has been hypothesized for both conditions

G. Calandra-Buonaura et al. / Sleep Medicine 13 (2012) 252–262 253

[12,13]. An association between arousal fluctuations and NFLS hasbeen observed [4,14], but the time-dependent relationship be-tween arousals and seizures, and the features of the arousal (phys-iological or pathological) associated with NFLS, have not beenanalyzed with objective methods providing both quantitative andtemporal information. During sleep, the arousal response was pos-tulated to follow a hierarchic pattern, starting with signs of auto-nomic activation followed by EEG changes like delta bursts andk-complexes and ending in delayed cortical activation for stimuliof greater intensity [15,16].

However, when changes in autonomic cardiac control precedeclinical seizure onset in NFLE, they could not only reflect the arou-sal, but also be the consequence of the epileptic discharge affectingthe cerebral areas of the central autonomic network (CAN) devotedto the sympathetic and parasympathetic control of the heart [17].Heart rate (HR) variations during NFLS have been reported [4], buttheir possible relationship with clinical seizure onset has not yetbeen analyzed, even though recordings with intracerebral elec-trodes clearly demonstrated that NFLS epileptic discharges couldarise from regions of the CAN (prefrontal cortex, temporal lobe,and insula) involved in cardiovascular modulation [18–20].

The primary aim of our study was to describe time-dependentmodifications of HR and autonomic cardiac control related to mo-tor onset of NFLS to determine whether signs of autonomic activa-tion precede motor manifestations of seizures.

Further, to clarify the nature (epileptic or physiologic) of auto-nomic changes presumed to precede clinical seizure onset, we alsoinvestigated time-dependent variations in HR and sympathetic andparasympathetic cardiac control preceding motor onset of physio-logical cortical arousal [21] associated with motor activity (phasesof transitory activation, PAT) [22].

Autonomic cardiac control was studied through spectral analy-sis of HR variability (HRV) [23] by means of wavelet transform, atime-variant spectral analysis technique that has been successfullyemployed for time-dependent frequency analysis in several clinicalconditions [24–27].

2. Methods

2.1. Patients and control subjects

Patients referred to the Epilepsy and Sleep Centers of theDepartment of Neurological Sciences, University of Bologna foran evocative history of NFLE underwent a preliminary evaluationincluding full neurological examination, routine EEG during wake-fulness, brain Magnetic Resonance Imaging (MRI), and a wholenight video-polysomnography (VPSG). Inclusion criteria to enterthe study were a clear-cut diagnosis of NFLE documented by inves-tigations and the VPSG recording of at least two seizures meetingthe criteria for seizure selection described below. Cardiac, endo-crine, metabolic, renal diseases, and sleep-related breathing disor-ders were also excluded by history-taking and appropriateinvestigations.

All patients were drug-free at the time of VPSG except for anti-epileptic drugs that, if present, were reduced or withdrawn twoweeks before VPSG.

Potential control subjects were recruited after exclusionthrough a structured interview of any current or past medicalproblem, personal or family history of epilepsy or sleep disorders,or excessive caffeine or alcohol consumption. Further, control sub-jects were required to be drug-free and have a regular lifestyle aswell as sleep-wake schedule. Inclusion in the study was only con-firmed when a whole night VPSG recording showed normal sleepefficiency (P85%) and sleep structure and absence of sleep-relatedbreathing and movement disorders.

Written informed consent was obtained before entering thestudy, which was conducted in conformity with the principles ofthe Declaration of Helsinki and approved by the local ethicalcommittee.

2.2. Nocturnal sleep recording

Patients and control subjects underwent a whole night (from11 pm to 7 am) digitally recorded VPSG (XLTEK Connex™� EEG/PSG, sampling rate 256 Hz and NEUROFAX EEG-1200 Pro� NIHONKOHDEN, sampling rate 500 Hz) comprising standard bipolar scalpEEG (according to the International 10-20 System), surface rightand left electro-oculogram (EOG), electrocardiogram (ECG, from astandard D2 lead), microphone (taped on the antero-lateral partof the neck), thoracic and abdominal respirograms (strain-gauge),and continuous audio-visual acquisition. Electromyogram (EMG)of mylohyoid muscle, right and left tibialis anterior muscles, andof at least two other limb muscles was also included in order to de-tect seizure or arousal motor onset.

VPSG recordings were then visually scored following the Amer-ican Academy of Sleep Medicine criteria [28].

2.3. Criteria for seizure selection and analysis

VPSG recordings of patients were reviewed independently bythree examiners (GCB; FP; PT) and paroxysmal motor events wereselected for analysis only if a consensus was reached that they fullymatched one of these three patterns typical of NFLS: paroxysmalarousals (PA), asymmetric tonic seizures (ATS), or hyperkinetic sei-zures (HS) [4,10].

Only NFLS without ECG signal artifacts and in which the firstmotor manifestation could be clearly detected on the EMG signalwere included. In addition, seizures were only selected if they oc-curred after at least 5 min of seizure-free sleep and at least 90 safter the end of another cortical arousal or another brief motorphenomenon that did not cause awakening. This criterion wasadopted to avoid the confounding effect of persisting HR changesdue to seizures or other phenomena preceding the event underanalysis [29].

For each NFLS sleep stage of occurrence, clinical classification,onset, and duration were established.

Seizure onset (SO) was defined as the first visible increase inEMG signal amplitude (of any degree or duration and of one ormore recorded muscles) coinciding with the first clinical sign ofthe seizure detected by visual analysis of video recording. Seizuresin which the first change of EMG signal was preceded by other clin-ical manifestations (movements or eye opening) were excluded.

Seizure duration was calculated from SO to the end of the clin-ical epileptic manifestations.

EEG tracings were reviewed to assess any epileptic abnormali-ties during both ictal and interictal periods.

2.4. Criteria for arousal selection and analysis

VPSG recordings of control subjects were screened for presenceof cortical arousals [21] fulfilling criteria of PAT, defined as anacceleration of the background EEG activity with appearance of al-pha or beta activity, associated with a concomitant increase inEMG signal amplitude, muscular artifacts, and tachycardia [22].Only arousals in which the occurrence of fast EEG activity coin-cided with the onset of an increase in EMG signal amplitude ofone recorded muscle were analyzed. Further, in analogy with sei-zure-selection criteria, arousals were selected only when precededby at least 90 s of sleep free from additional cortical arousal orother movement. These arousal selection criteria were specificallydesigned in order to be able to compare events (arousals and

Fig. 1. Three seizure-related periods were selected to describe time-dependentchanges in heart rate and heart rate variability components (e.g., seizure 3 [NREM3] of patient 2): basal period (Basal), from second 60 to second 30 before motoronset of seizure (seizure onset, SO identified with second zero), during which nomovements occurred and autonomic conditions appeared stationary compared toprevious epochs of sleep; pre seizure period (preSP), 10 s interval immediatelypreceding SO; seizure period (SP), from SO to the highest change in HR associatedwith clinical epileptic manifestations (e.g., from 0 to 11 s after SO).

254 G. Calandra-Buonaura et al. / Sleep Medicine 13 (2012) 252–262

seizures) with similar degrees of activation sharing analogous tem-poral references. Arousal onset (AO) was defined as the first visibleincrease in EMG signal amplitude of at least one recorded muscle(mylohyoid muscle included). For each arousal, sleep phase ofoccurrence and duration were annotated and computed. Arousalduration was calculated from AO to return to sleep [21] and wasrequired to fall between 3 and 30 s [30].

2.5. HR analysis

HR was calculated by measuring the interval between two con-secutive R-waves of QRS complexes (RRi) in the ECG trace. RRi ser-ies were digitally identified and automatically calculated for theentire night recorded during VPSG by means of Vision Analyzersoftware (version 1.05-Brain Products). Visual control of RRi seriesallowed erroneous R waves and missed detection to be corrected.Ectopic beats were deleted from the resulting RRi series and re-placed by a virtual beat by interpolating adjacent R waves as rec-ommended [23].

2.6. HRV spectral analysis

Spectral analysis of HRV was performed in order to assess sei-zure- and arousal-related changes in autonomic cardiac control.The power spectrum of HRV comprises high-frequency compo-nents (HF: 0.15–0.4 Hz), reflecting parasympathetic outflow andbreathing activity, low frequency components (LF: 0.04–0.15 Hz),mediated mainly by sympathetic activity, and very low frequencycomponents (VLF: 0–0.04 Hz) whose physiological correlates arenot fully understood. Even if the interpretation of the LF compo-nent as a pure marker of sympathetic activity is still debated[31], the LF/HF ratio is widely used as a reliable indicator of sym-pathetic and parasympathetic outflow balance [32].

Several mathematical approaches are commonly employed inHRV analysis and, among these, Fast Fourier Transform and autore-gressive spectral analysis are the most common. In this study a dis-crete wavelet transform was chosen in order to (a) overcomelimitations imposed by classical spectral analysis such as signalstationarity and (b) provide information about the time-dependentevolution of signal frequency content [24].

Analysis of each seizure and arousal was conducted off-lineover a period of 20 consecutive minutes using Mathematica� 7.0.A 20 min truncation length was chosen based on the observationthat time frequency decompositions for the same time window ob-tained by applying wavelet transform to the full signal (wholenight recording) yielded superimposable time–frequency spectraeven in the VLF domain (data not shown). RRi series were resam-pled at 10 Hz using cubic spline interpolation, following the heu-ristic rationale of resampling at approximately 10 times the bandof the fastest regulatory mechanism of interest (which is assumedto be active beat-to-beat) [33].

A multiresolution analysis was then performed choosing theDaubechies-16 form as mother wavelet to guarantee a steep fall-off at the boundaries of the mother wavelet spectrum and a closematch between the full width at half maximum boundaries of di-lated wavelet spectra and the HF and LF band transition frequen-cies. A family of basis functions was built by dilation andtranslation of the mother wavelet. The similarity between the sig-nal and these basis functions was estimated through coefficientscomputed by convolving the original signal with the basis func-tions in the time domain. The squared level specific amplitudecoefficients were summed across appropriate decomposition levelsto compute total band powers in bands of interest (VLF: 0.01–0.04,LF: 0.04–0.15, HF: 0.15–0.42). Band transitions/edges were esti-mated as the points of half-maximum spectral power amplitudesof the level-specific mother wavelet dilations. This approach was

preferred to the traditional one of quoting the central frequencyof the dilated wavelets to take into account the shape of themother wavelet spectrum, hence better estimating the bands re-lated to the coefficients resulting from wavelet transform compu-tation. Band specific powers were then filtered using a linearrecursive filter in the time domain (exponential filtering witha = 0.1) to reduce noise.

2.7. Data and selection of analysis periods

Spectral components of HRV were measured as absolute valuesof power (seconds squared), as normalized units (LFnu and HFnu)representing the percentage of LF and HF components in relation tothe total power minus the VLF component, and as LF/HF ratio. Nor-malized units and ratio were calculated to better represent themodulation exerted by the sympathetic and parasympatheticbranches of the autonomic nervous system and to disclose the ef-fect on the values of LF and HF components of a change in absolutemagnitude of total HRV. The VLF component was excluded fromthe analysis due to its uncertain physiological meaning.

To describe the temporal evolution of HR (RRi) and HRV (LF, HF,LFnu, HFnu, and LF/HF) changes related to seizures, the followinganalysis periods were selected (Fig. 1):

(1) Basal period (Basal): from second 60 to second 30 before SO(identified as second zero) during which no movementsoccurred and autonomic conditions, evaluated by LFnu,HFnu, and LF/HF ratio assessed with wavelet transform,appeared stationary compared to previous epochs of sleep;

(2) Pre seizure period (preSP): 10 s interval immediately pre-ceding SO;

(3) Seizure period (SP): from SO to the point of the largestchange in HR compared to Basal values associated with clin-ical epileptic manifestations. SP could cover the entire sei-zure duration or correspond to the first part of the seizureif a progressive return of HR to Basal values started beforeseizure end.

G. Calandra-Buonaura et al. / Sleep Medicine 13 (2012) 252–262 255

Periods of arousal analysis included: (1) Basal period (Basal):from second 60 to second 30 before AO (identified as second zero);(2) Pre arousal period (preAP): 10 s interval immediately precedingAO; (3) Arousal period (AP): from AO to the point of the largestchange in HR compared to Basal values associated with arousal.

2.8. Statistical analysis

Data are reported as means ± standard error of means (SEM). Inorder to work at a time resolution of 1s, the mean value of eachsecond of the 10 Hz sequences of resampled data was calculatedfor the parameters evaluated (RRi, LF, HF, LFnu, HFnu, LF/HF) andused for statistical analysis. Logarithmic transformation was ap-plied to non-normally distributed data (RRi and HRV spectra com-ponents) to obtain normally distributed data.

To assess time-dependent changes of HR and HRV related toNFLS and to arousals, a general linear univariate model (factorialANOVA) was initially computed with the parameter of interest(RRi, LF, HF, LFnu, HFnu, and LF/HF) as the dependent variableand patient or control subject and period as fixed factors (groupanalysis). Bonferroni’s correction was used for multiple compari-sons. Nature of interaction between factors was explored by meansof interaction graphs.

Single subject analysis was then performed for patients andcontrol subjects, selecting the seizures and the arousals of each pa-tient and control subject, respectively, with the parameter of inter-est (RRi, LF, HF, LFnu, HFnu, and LF/HF) as the dependent variableand seizure or arousal and period as fixed factors. The same modelwas also applied selecting seizures or arousals characterized by thesame sleep phase of occurrence and seizures with the same ictalfeatures.

Finally, in order to better describe time dependent evolution ofHR and sympathetic/parasympathetic balance before seizure andarousal, a general linear model for repeated measures was applied,comparing RRi, LF, HF, and LF/HF values during each 10 s immedi-ately preceding SO or AO and the first 4 s (second zero included) ofthe SP or AP to mean value of the basal period (simple contrast)(time as within-factor, using 15 s as levels). Each patient or controlsubject was considered as between-factor to evaluate interaction.

Table 1Demographic, clinical and interictal electrophysiological characteristics of the 10 patients

Pt Sex Age at NFLS onset (years) Age at VPSG (years) NFLS frequency at VPSGnight

1 F 5 23 >5

2 F 14 23 >5

3 M 14 34 >54 M 14 41 1–3,

1–2 nights/week5 M 3 20 2–5

6 M 8 19 2–3

7 F 9 23 >5

8 M 6 12 >5

9 F 21 35 1–2

10 M 7 23 >5

Pt, patient; F, female; M, male; NFLS, nocturnal frontal lobe seizures; VPSG, video-polysolamotrigine; CZP, clonazepam; R, reduced; B, bilateral; L, left; R, right.

The degree of HR and HRV changes between seizures and arous-als was not compared directly since remarkable inter-individualvariability in the magnitude of arousal-related cardiac and auto-nomic response has been observed in control subjects even whenconfounders like age, time of the night, and sleep stage were con-trolled [16].

All statistical analyses were performed with SPSS-PASW (Pre-dictive Analytics Software) version 18 and statistical significancewas set at p 6 0.05.

3. Results

3.1. Patients

Ten patients were included in the study (six males; mean ± SEMage at VPSG = 25.3 ± 2.8 years) (Table 1). Age at seizure onset ran-ged from 3 to 21 years (mean ± SEM age = 10.1 ± 1.7 years). Fourpatients (patients 1, 3, 7, 8) had a positive family history for para-somnias and two (patients 1 and 9) for sleep talking [8]. History-taking disclosed sleep enuresis in two patients (patients 3 and 6)and febrile seizures in one (patient 1). Neurological examinationand brain MRI were normal in all patients. Interictal EEG showedclear epileptic abnormalities only in one patient (patient 2) duringwakefulness and in two patients during sleep (patients 2 and 6)(Table 1).

Seizure frequency at the time of VPSG was reported as high inmost patients. Eight patients were under antiepileptic drugs thatwere reduced in seven and withdrawn in one (Table 1).

Two years after VPSG, patient 8 underwent evaluation for surgi-cal treatment of drug-resistant seizures at the C. Munari epilepsysurgery center in Milan. The epileptogenic zone was localized inthe right cingulate gyrus through stereo-EEG recordings and subse-quently removed with resective micro-surgery with a seizure-freeoutcome of four years.

3.2. Seizures

Forty-five seizures (12 PA, 12 ATS, and 21 HS) arising fromNREM sleep (mean ± SEM duration of PA = 8.2 ± 1.6 s; ATS= 36.9 ± 6.4 s; HS = 34.9 ± 6.8 s) were analyzed (seizure number

included in the study.

/ AEDs at VPSG (mg/die)

Interictal EEG during sleep (VPSG)

CBZ 800; TPM 50R: CBZ 400; TPM 50

–

CBZ 1000; LTG 300R: CBZ 600; LTG 200

Posterior vertex andB frontal spikes

– –– –

CBZ 1000; CLB 20R: CBZ 800

–

CBZ 800Withdrawn

R and L fronto-temporal spikes and sharpwaves

CBZ 1000; CZP 1.5R: CBZ 400

Posterior vertex andR parietal theta rhythmic activity

CBZ 600; LTG 50R: CBZ 400

B fronto-temporal theta activity

CBZ 600; LTG 200R: CBZ 200

–

CBZ 800; CLB 20R: CBZ 400

–

mnography; AEDs, antiepileptic drugs; CBZ, carbamazepine; TPM, topiramate; LTG,

Table 2Clinical and electrophysiological features of the 45 seizures analyzed.

Pt Seizuresrecorded(seizuresanalyzed)

Seizureduration(mean ± SEMs)

Sleep phase ofoccurrence

EEG activity and timelag before SO (s)

NREM2 NREM3

1 10 HS (7) 23 ± 0.8 3 4 K-complex or slowwaves (63)Diffuse delta rhythmicactivity (67)Fast, low voltagediffuse activity (0)

2 4 ATS (4) 22 ± 1.7 2 2 Fast, low voltagediffuse activity (0)

3 17 PA (8) 8.1 ± 2.3 2 6 K-complex or a slowwave (61)

4 4 PA (3) 9.7 ± 1.2 – 3 K-complex or a slowwave (62)

5 3 HS (2) 34.5 ± 7.5 2 – L fronto-temporalflattening (0)

6 8 HS (3),>5 PA

106 ± 12.1 2 1 K-complex (61)Fast, low voltagediffuse activity (<1)

7 12 ATS(7), >5 PA

37 ± 5 6 1 Posterior vertex and Rcentro-parietal thetarhythmic activity (0)

8 9 HS (7),>5 PA

18.4 ± 0.65 3 4 B centro-fronto-parietal thetarhythmic activity (0)

9 1 PA (1)1 ATS (1)

596

1 1 K-complex (62 s)Diffuse flattening (0)

10 3 HS (2),1 PA

28 ± 7 2 – K-complex (62 s)Diffuse flattening (0)

Pt, patient; PA, paroxysmal arousals; ATS, asymmetric tonic seizures; HS, hyperki-netic seizures; SEM, standard error of mean; NREM, non rapid eye movement; B,bilateral; L, left; R, right; SO, seizure onset; 0 = coincided with SO.

256 G. Calandra-Buonaura et al. / Sleep Medicine 13 (2012) 252–262

per patient varied from two to eight) (Table 2). EEG recordingsfailed to disclose a clear epileptic discharge before SO in all pa-tients. EEG activity was mainly characterized by an abrupt sleep-wake transition coinciding with SO, usually preceded by a K-com-plex or a burst of slow-waves. A diffuse flattening of backgroundactivity, a fast low voltage diffuse activity, and a focal or diffusetheta/delta rhythmic activity were the most frequent EEG abnor-malities observed coinciding with or preceding SO (Table 2).

Respiratory activity, as monitored by strain-gauge, was regularbefore SO in all seizures, whereas irregular breathing often startedafter SO.

3.3. Control subjects and arousals

Ten healthy control subjects (seven males; mean ± SEMage = 35.7 ± 2.9 years) were included in the study and 45 arousalsarising from NREM sleep (NREM2 = 22 arousals; NREM3 = 23arousals; mean ± SEM duration = 17.8 ± 1.1 s) were analyzed (arou-sal number per control subjects varied from two to eight).

Motor activity at arousal onset was mainly characterized by anincrease in EMG signal amplitude of mylohyoid muscle alone or bya limb or head movement. A K complex or a delta burst usuallypreceded motor arousal onset as well as transition from sleep to al-pha EEG activity. Respiratory activity was checked to be regular be-fore AO.

3.4. Time-dependent changes in HR and HRV

3.4.1. HR and HRV changes related to seizures: group analysisGroup analysis indicated a significant effect (p 6 0.0005) of both

patient and period on all the parameters evaluated (RRi, LF, HF,LFnu, HFnu, LF/HF) and a significant interaction between patientand period effects (p 6 0.05). Compared to Basal, RRi did not

significantly change during the preSP (p = 0.415), whereas a signif-icant tachycardia (RRi decrease) was observed during the SP com-pared to both Basal (p < 0.0005) and preSP (p < 0.0005) (Fig. 2A).

The analysis of HRV spectral components demonstrated thatduring the preSP LF absolute values significantly increased com-pared to Basal (p < 0.0005), whereas HF absolute values did not sig-nificantly change (p = 0.895) (Fig. 2A). A significant increase inLFnu (p < 0.0005) (Fig. 3) and LF/HF (p < 0.0005) and a significantdecrease of HFnu (p < 0.0005) were also observed, indicating a shiftof sympathetic/parasympathetic balance toward a sympatheticpredominance during this period. This pattern of variation alsoencompassed the SP, which presented a further increase in LFnu(Fig. 3) and LF/HF and a decrease of HF and HFnu (Fig. 3) that sig-nificantly differed from both Basal (p < 0.0005) and preSP(p < 0.05), while LF absolute values significantly increased with re-spect to the Basal period only (p < 0.0005) (Fig. 2A) (Supplementarydata_1).

Results of group analysis were reproduced for all parametersregardless of sleep phase of occurrence (23 seizures NREM2; 22seizures NREM3).

3.4.2. HR and HRV changes and seizure featuresAnalysis of time-dependent variations of HR and HRV spectral

components during the preSP yielded the same results regardlessof seizure features (Fig. 2). HR and HF absolute values did not sig-nificantly change compared to the Basal, whereas a significant in-crease in the LF component was observed (p < 0.0005). During theSP a significant tachycardia (p < 0.0005) occurred compared to Ba-sal, associated with a significant increase in LF absolute values anda decrease trend of HF absolute values that reached statistical sig-nificance during ATS and HS only (Fig. 2). The temporal change insympathetic/parasympathetic balance was independent of seizuretype, with a significant increase of LFnu and LF/HF and a significantdecrease of HFnu during the preSP and the SP as resulted from thegroup analysis (Supplementary data_1).

3.4.3. HR and HRV changes related to seizures: single subject analysisSingle subject analysis showed a significant period effect

(p < 0.05) on the parameters evaluated (RRi, LF, HF, LFnu, HFnu,LF/HF) in all patients, while seizure effect and interaction betweenseizure and period effects varied among patients and parameters.

In accordance with the results of group analysis, RRi remainedunchanged during the preSP and significantly decreased in the SPin the ten patients analyzed (Fig. 3A). Similarly, analysis of HRVdemonstrated a significant sympathetic activation (increase ofLFnu and LF/HF) in both the preSP and SP in all patients (Fig. 3Band C) (Supplementary data_2 and data_3). The significant increasein LFnu and LF/HF in the preSP was due to a significant increase inLF absolute values in all patients, while HF absolute values re-mained unchanged in four patients, significantly increased in two(patients 5 and 9), and decreased in the other four (patients 1, 2,4, and 10).

Interaction graphs of seizure and period confirmed the increasetrend in LFnu and LF/HF in all 45 seizures during the SP and in 43out of 45 seizures during the preSP. For the remaining two seizures(seizure 2 of patient 6 and seizure 3 of patient 7), mean LFnu andLF/HF Basal values were compared to the mean LFnu and LF/HF Ba-sal values of the other seizures of the same patient and were foundto be significantly higher (p < 0.05).

3.4.4. HR and HRV changes related to arousalsGroup analysis indicated a significant effect (p 6 0.0005) of both

subject and period on all the parameters evaluated (RRi, LF, HF,LFnu, HFnu, LF/HF) and a significant interaction between subjectand period effects (p 6 0.05). Compared to Basal, RRi significantlydecreased during the AP only (p 6 0.0005), while remaining

Fig. 2. Means ± standard error of means (SEM) of RR interval (RRi) (left y-axis), HF and LF absolute values (right y-axis) during the three defined periods (Basal, basal period;preSP, pre seizure period; SP, seizure period) evaluating (A) 45 seizures (group analysis), (B) 12 PA, paroxysmal arousals, (C) 12 ATS, asymmetric tonic seizures, and (D) 21 HS,hyperkinetic seizures. A significant autonomic activation, characterized by an increase in LF absolute values without a concomitant variation of the HF component (increase inLF/HF) and RRi was observed in the preSP in the group analysis, irrespective of seizure type. During the SP, a significant tachycardia (RRi decrease) was observed. Tachycardiawas associated with persistence of the LF increase and appearance of the HF decrease (reduction of parasympathetic activity) that reached statistical significance only ingroup analysis, ATS, and HS. A shift of sympathetic/parasympathetic balance toward sympathetic activation was observed during both the preSP and SP in the group analysisand irrespectively of seizure type. � indicates significant difference compared to Basal (p 6 0.05). SEM values <0.02 are not visible.

G. Calandra-Buonaura et al. / Sleep Medicine 13 (2012) 252–262 257

unchanged during the preAP (p = 0.483). During the preAP, HRVanalysis showed a significant increase of LF absolute values(p < 0.0005) and a significant decrease of HF absolute values com-pared to Basal (p = 0.004) (Fig. 4). A significant increase of LFnu(p < 0.0005) and LF/HF (p < 0.0005) and a significant decrease ofHFnu (p < 0.0005) were also observed, indicating a shift of sympa-thetic/parasympathetic balance toward a sympathetic predomi-nance during this period, in analogy with the observations in theseizure group. During the AP a significant tachycardia was associ-ated with a significant increase in LF, LF/HF, and LFnu (p < 0.0005),and a significant decrease in HF and HFnu (p < 0.0005) (Fig. 4). Theobserved increase of LF, LF/HF, and LFnu and the decrease of HFand HFnu were confirmed during preAP and the AP, regardless ofsleep phase of occurrence.

Single subject analysis confirmed that RRi significantly de-creased in all subjects only in the AP, while LF, LF/HF, and LFnu sig-nificantly increased and HFnu significantly decreased during boththe preAP and the AP in all subjects (p 6 0.05). Noticeable inter-subject variability was instead observed during both periods forthe HF component. In particular, during the preAP, HF componentsignificantly decreased in five subjects, significantly increased inone (subject 7), and did not significantly change in the other four(subjects 3, 4, 6, 8) (Supplementary data_4 and 5).

3.4.5. Second per second estimation of HR and HRV changes precedingseizure and arousal motor onset

The analysis of changes in RRi, LF, HF, and LF/HF during thewhole preSP (10 s) and the first 4 s of the SP compared to mean Ba-sal value showed a significant effect of time (p 6 0.005) and a sig-nificant interaction between time and patient effect (p 6 0.005).RRi began to decrease significantly 1 s before SO (p = 0.03). A

significant progressive increase trend of LF and LF/HF ratio com-pared to Basal was observed as early as 10 s before SO(p 6 0.0005), reaching a peak at SO (Fig. 5A and C), while HF signif-icantly decreased only after SO (Supplementary data_6). The onsetof the RRi decrease varied between patients from 1 s before to 1 safter SO, while an increase of LF/HF ratio, albeit of different degreeamong patients, started 10 s before SO in all patients (observedthrough interaction graphs), becoming progressively higher inthe following seconds.

Time-dependent evolution of changes in spectral componentsof HRV in a single seizure (e.g., seizure three of patient one) isshown in Fig. 6.

Analysis of time-dependent changes of HR and HRV beforearousal motor onset showed a pattern of variation similar to theone observed in seizures (Fig. 5B and D and Supplementarydata_7), characterized by a significant progressive increase of LFand LF/HF ratio compared to Basal starting 10 s before AO(p 6 0.0005), while RRi began to decrease significantly 1 s beforeAO (p 6 0.0005). On the contrary, HF component variation was dif-ferent before AO when compared to the period before SO, as HFabsolute values started to significantly decrease 8 s before AO(p = 0.01), with the higher statistical significance being reached3 s before AO (p 6 0.0005). However, while LF and LF/HF startedto increase 10 s before AO in all subjects, the onset and the direc-tion of HF changes varied between control subjects (observedthrough interaction graphs).

4. Discussion

This study was designed to investigate time-dependent varia-tions in HR and sympathetic and parasympathetic cardiac control

Fig. 3. Means ± standard error of means of (A) RR interval (RRi), (B) LF normalizedunits (LFnu), and (C) HF normalized units (HFnu) during the three defined periods(Basal, basal period; preSP, pre seizure period; SP, seizure period) evaluating allpatients (45 seizures) as well as each patient separately. The number of seizuresanalyzed varied between two and eight among patients. In all cases, time-dependent analysis of heart rate variability showed a shift of sympathetic/parasympathetic balance toward sympathetic activation (increase in LFnu anddecrease of HFnu) that started in the preSP and continued after seizure motor onset,while a significant tachycardia (decrease in RRi) was observed in the SP only. �indicates significant difference compared to Basal (p 6 0.05).

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associated with NFLS, focusing on the temporal relationship be-tween changes in autonomic cardiac control and clinical seizureonset. Since seizures in NFLE arise from sleep, the autonomic car-diac control preceding seizure motor onset is unlikely to be influ-enced by psychical stress or subjective symptoms like aura, butcould instead be modulated by two other main factors: (1) theautonomic response associated with arousal from sleep, whichhas been hypothesized to play a role in triggering seizures; and(2) the direct effect of the epileptic discharge involving areas ofthe CAN responsible for cardiovascular control.

4.1. HR and HRV changes related to seizures and PAT

We analyzed 45 seizures in 10 patients whose clinical and elec-trophysiological characteristics matched those reported in a largepopulation of NFLE patients [4] and 45 cortical arousals with fea-tures of PAT in 10 control subjects.

The first finding of our study was that consistent changes in HRare not evident before seizure motor onset. Time-dependent anal-ysis showed a slight statistically significant increase in HR only one

second before SO (p = 0.03), with differences among patients. A sig-nificant tachycardia was instead observed after seizure motor on-set, reaching values up to 140 beats per minute and confirmingprevious clinical observations of remarkable autonomic activationassociated with NFLS [4].

Despite the absence of significant changes in HR, spectral anal-ysis of HRV demonstrated a significant shift in autonomic cardiaccontrol toward a sympathetic predominance in the 10 s precedingmotor manifestation of seizures, with a further increase after SO.This autonomic activation was due to a significant increase in theLF component, mainly representing sympathetic activity, begin-ning before NFLS motor onset and persisting throughout the SP,while a significant decrease in the HF component, representingparasympathetic outflow and respiratory activity, was observedonly after SO. The increase in the LF component and the patternof sympathetic/parasympathetic balance variation preceding NFLSmotor onset were confirmed in all patients analyzed and in 43 outof 45 seizures, regardless of seizure features and despite differ-ences in HF component behavior. This autonomic activation inthe preSP was not associated with a sleep-wake transition orchanges in respiratory activity, both occurring concurrently withSO. The lack of information on epileptic discharge spread raisesthe question of the nature of this autonomic activation observedbefore NFLS motor onset. Is this sympathetic activation causedby an epileptic discharge involving areas of the CAN or is it theexpression of the arousal mechanism?

Analysis of HRV changes related to PAT provided additionalclues to the matter. Patterns of variation in autonomic cardiaccontrol before the motor onset of the 45 arousals evaluatedreproduced the features of the autonomic activation observed be-fore seizures, showing a shift of sympathetic/parasympatheticcontrol towards a sympathetic predominance in the 10 s immedi-ately preceding onset of arousal motor activity while HR changesbecame evident 1 s before AO. A single difference between sei-zures and arousals was found in the fact that the HF componentdecreased significantly before AO while remaining unchanged be-fore SO. However, the high inter-subject variability in HF compo-nent behavior in both control subjects and patients calls for acautious interpretation of these results. For example, while sym-pathetic activation has been constantly documented in associa-tion with spontaneous or induced arousal from sleep, thedirection of the parasympathetic response varied among studies[34].

A temporal pattern of sympathetic/parasympathetic balanceand motor changes similar to that observed in our study has beendemonstrated using wavelet analysis of HRV related to three typesof movements occurring during sleep and frequently associatedwith an arousal from sleep: periodic leg movements, isolated legmovements, and respiratory-related leg movements [26]. A weakbut significant increase in the LF component compared to basalvalues (from second 10 to second 7 before movement onset) wasseen to occur nearly 6 s prior to the movement onset of the threedifferent movement types, becoming progressively higher 3 s later,while a significant HR increase was detected only 1 s before move-ment onset. Autonomic activation also preceded EEG changes,which started 2 s before movement onset with prominent deltasynchronization, followed by a gradual progression to faster EEGfrequencies.

This temporal pattern of autonomic changes preceding arousalconfirms that autonomic activation may represent an early markerof brain arousability during sleep [15,16,35]. In addition, it indicatesthat the sympathetic activation observed before the onset of seizuremotor manifestations in our study may reflect the autonomic prep-aration that preceded the cortical and behavioral response to spon-taneous arousal, hence representing the expression of the arousalmechanism.

Fig. 4. Means ± standard error of means of (A) RR interval (RRi), (B) LF absolute values, (C) LF/HF ratio, and (D) HF absolute values related to 45 arousals (phases of transitoryactivation) during the three defined periods (Basal, basal period; preAP, pre arousal period; AP, arousal period). A significant decrease of RRi was observed, compared to Basal,during the AP only while a significant increase of LF/HF was observed during both the preAP and the AP. The increase of LF/HF was due to a significant increase in LF and asignificant decrease of HF absolute values. � indicates significant difference compared to Basal (p 6 0.05).

Fig. 5. Means ± standard error of means (SEM) of RR interval (RRi) (A and B) and LF/HF (C and D) during the 10 s immediately preceding motor manifestations of seizures(seizure onset, SO) and of arousals (arousal onset AO) (gray rhombs and squares) and the first four s after SO and AO (black rhombs and squares). A similar pattern of variationin autonomic cardiac control was observed before seizure and arousal motor onset: RRi significantly decreased 1 s before SO and AO while LF/HF ratio significantly increased10 s before SO and AO. � indicates a significant difference (p 6 0.05) compared to the mean value of Basal (white rhomb and square). SEM values of LF/HF <1.5 are not visible.

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4.2. Interpretation of time-dependent changes in autonomic cardiaccontrol: the role of arousal in NFLE pathophysiology

Additional electrophysiological [4,14,36] data point towards aninvolvement of arousal in NFLE pathophysiology. However, thesestudies [4,14,36] postulated an association between arousal andNFLS by means of visual inspection of EEG, which is a methodunsuitable to demonstrate a time-dependent relationship or toestablish if the arousal preceding NFLS has pathological or physio-logical features.

By evaluating the autonomic response, which is considered theprimary response of an arousing brain, using a time variant spec-tral analysis technique, our study provides further evidence to clar-ify the associations between arousal and seizures. This analysisclosely preceded by an autonomic arousal in a consistent mannerand this observation is similar to that noted in the physiologicalarousal.

Abnormalities of the arousal system, and in particular of thedorsal cholinergic arousal branch, whose projections reach the cor-tex through a thalamic relay, were demonstrated for the genetic

Fig. 6. (A) Polygraphic recording and (B) time frequency decomposition of heart rate variability (HRV) related to seizure 3 of patient 1 (hyperkinetic seizure; NREM3). Periodsof analysis (Basal, basal period; preSP, pre seizure period; SP, seizure period) are delimited by the dotted line. The power of HRV frequencies is plotted with a chromatic scalein logarithmic units (column on the right; dB, decibel; reference power [mean power in basal period over all bands]: 0.032 s2). (A) The first increase in electromyographic(EMG) activity related to the seizure was observed on the mylohyoid (Mylo) and the left deltoid (L Delt) muscles and identified with seizure onset (SO, second zero). Nearly 7 sbefore SO scalp electroencephalographic recording showed the appearance of a diffuse delta rhythmic activity, more evident over the fronto-centro-parietal regions, followedby a sleep wake transition coinciding with SO. A significant increase in heart rate appeared only after SO. (B) Time–frequency analysis of HRV showed an increase in lowfrequency (0.04–0.15 Hz) oscillations beginning nearly 16 s before SO and progressively increasing over time, becoming significantly higher compared to Basal during thepreSP and the SP. High frequency (0.15–0.42 Hz) power related to this seizure started to decrease in the preSP nearly 4 s before SO with a further decrease during the SP. Asignificant shift of sympathetic/parasympathetic control toward a sympathetic predominance was observed during both the preSP and the SP.

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form of NFLE and implicated in its pathophysiology [37,38].According to Montagna [13] an abnormal activation of this arousalsystem may also be involved in the pathophysiology of the spo-radic form of NFLE and may trigger the onset of the epileptic dis-charge by modulating cortical excitability.

The involvement of the arousal mechanism in the pathophysiol-ogy of NFLE is also suggested by clinical data documenting thecoexistence of arousal parasomnias in NFLE patients [4] and theirincreased frequency in patients’ relatives with respect to controls[39]. The complex and stereotyped motor features common toNFLS and disorders of arousal are hypothesized to be the conse-quence of the activation of ‘‘central pattern generators’’ (CPGs)whose activities are under cortical control and could be influencedby the arousal state [40,41]. A dysfunction in the arousal mecha-nism could activate the CPGs either directly or through activationof epileptogenic foci. The results of our study support the specula-tive hypothesis that the triggering role of the arousal on epilepticmotor manifestations is unlikely to be related to its intrinsic fea-tures, rather it may depend on its altered occurrence [13].

However, the relationship between arousal fluctuations andNFLS remains controversial. Studies exploring EEG activity withintracerebral recordings in NFLE patients demonstrated an associ-ation between epileptic discharges and increased arousal fluctua-tions where the latter appeared reduced after surgical treatmentof seizures, suggesting that epileptic discharges not detectablewith scalp EEG could be the primary effectors and not the conse-quence of the increase in arousal fluctuations [42,43]. Accordingto these findings, the sympathetic activation that constantly her-alded the motor manifestation of seizures in our study could be

alternatively interpreted as the first expression of the epileptic dis-charge involving areas of the CAN that modulate sympathetic arou-sal response. However, the two studies cited above [42,43]explored the relationship between the epileptic discharge andthe arousal only by means of a temporal correlation analysis anddid not take into account the autonomic component of the arousal.A previous study performed with intracranial EEG recordings dem-onstrated the absence of changes in HRV related to frontal seizuresoccurring without clinical manifestations, suggesting that epilepticdischarges localized in the frontal areas do not lead to detectablechanges in autonomic cardiac control [44]. Therefore, the availableliterature and the demonstration of a strict similarity of autonomicchanges preceding seizure and arousal motor onset, including theirtemporal features, make the epileptic nature of the autonomic acti-vation preceding seizure motor onset unlikely.

4.3. Sympathetic activation and sleep-related motor events

Irrespective of the nature (epileptic or physiologic) of the sym-pathetic activation preceding NFLS motor onset, our findings are ofinterest with regard to the role of the sympathetic nervous systemin the occurrence of sleep-related motor events. A sympatheticactivation has been demonstrated to precede different stereotypedsleep-related movements such as periodic limb movements ofsleep [26] and sleep bruxism [45] before evident EEG changes. Inaddition, a reduction of the sympathetic tone by means of cloni-dine, an agonist of inhibitory presynaptic alpha adrenergic recep-tors, was shown to be associated with reduced sleep bruxismepisodes [46].

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Thus, we could hypothesize that in NFLE a sudden increase insympathetic tone may facilitate the release of CPGs and the occur-rence of stereotyped minor motor events [47] or other sleep-re-lated movements [42]. This hypothesis could explain theobservation of an increased frequency of sleep bruxism in NFLE pa-tients when compared to controls [39] and the recording of a largeamount of minor motor events during sleep in these patients [47].

5. Conclusions

The present study provides the first description of time-depen-dent variations in HR and sympathetic/parasympathetic cardiaccontrol related to the onset of motor manifestations of NFLS. Sev-eral important conclusions emerge from the study: first, a shift ofautonomic balance toward a sympathetic prevalence closely pre-cedes NFLS motor manifestations when HR changes are not yet evi-dent; secondly, this autonomic activation resembles that observedbefore physiological arousal from sleep and could therefore repre-sent the autonomic expression of arousal, which in turn may facil-itate the occurrence of seizure motor manifestations; third,wavelet analysis is confirmed to be a sensitive technique to dis-close sudden variations of autonomic balance when HR analysisalone is uninformative.

Our study evaluating HR and HRV changes in relation to the on-set of motor manifestations of NFLS does not allow to analyse theinteraction between the brain regions, where the epileptic dis-charge arises and spreads, and the changes of the autonomic car-diac regulation observed.

Further studies with concomitant time-dependent analysis ofthe intracerebral EEG signals and HRV in the population of NFLEpatients considered for epilepsy surgery are needed to clarify thecomplex interactions between autonomic arousal, the epilepticdischarge, and the occurrence of seizure motor manifestations.

6. Financial support

None.

Conflict of Interest

The ICMJE Uniform Disclosure Form for Potential Conflicts ofInterest associated with this article can be viewed by clicking onthe following link: doi: 10.1016/j.sleep.2011.11.007.

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