Original Article

Cephalalgia 32(13) 9981004 ! International Headache Society 2012 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0333102412453955 cep.sagepub.com

Habituation of the nociceptive blink reflex in episodic and chronic cluster headache
Dagny Holle1, Silke Zillessen1, Charly Gaul1, Steffen Naegel1, Holger Kaube2, Hans-Christoph Diener1, Zaza Katsarava1 and Mark Obermann1

Abstract Background: Analysis of habituation patterns in patients with primary headache disorders allows the detection of changes to the excitability level of the trigeminal nociceptive system. Previous studies demonstrated a habituation deficit to painful stimuli in migraine and it was suggested that similar observations could be made in cluster headache (CH). Methods: Habituation of the nociceptive blink reflex (nBR) (R2 response) was studied in 66 CH patients (18 episodic CH inside bout, 28 episodic CH outside bout, 20 chronic CH) as well as in 30 healthy controls in a case-control study design. Results: Habituation behaviour was similar in CH and healthy controls as well as in CH subtypes. No side-to-side differences of habituation between headache side and non-headache side were detected. Conclusion: Our results did not detect an altered habituation in CH patients. Despite clinical similarities, migraine and CH seem not to share the same pathophysiological mechanisms in this regard. Keywords Cluster headache, habituation, trigeminal nociception, nociceptive blink reflex
Date received: 23 November 2011; revised: 31 May 2012; accepted: 14 June 2012

Introduction
Habituation is dened as a process of reduced responses to repeated stimulations over time. It is thought to be a form of non-associative learning and represents a basal expression of nervous system plasticity (1). Habituation seems to be a general phenomenon in both humans and animals, which can be observed in all sensory modalities. In regard to pain processing, it helps to keep a healthy balance between nociceptive and anti-nociceptive mechanisms. Lack of habituation is a common observation in several neurological diseases, including migraine (26) and Parkinsons disease (7). A habituation decit was shown even for rst-degree relatives of migraineurs and, therefore, this might be an endophenotypic marker for pre-symptomatic migraine (6). Cluster headache (CH) and migraine share several clinical characteristics and the prevalence of migraine in patients with CH is increased compared to the general population. This overlap of clinical presentation led to the assumption of a similar pathophysiological pathway involved in these two primary headache disorders. Up to now, there are only few studies investigating habituation pattern in CH. Previous research results reported a habituation decit even more pronounced than in migraine using the regular blink reex (8). In contrast, studies using event-related potentials did not detect a lack of habituation in CH (9,10). Due to these conicting results, habituation pattern in CH remains to be determined. Investigation of the nociceptive blink reex (nBR) allows a direct exploration of the functional state of the trigeminal nociceptive system at the brain-stem level (11). In contrast to the classical innocuous blink reex (BR), the nBR is elicited by a stimulation electrode that predominantly stimulates cutaneous supercial nociceptive Ad-bres without depolarizing

1 2

Department of Neurology, University of Duisburg-Essen, Germany Interdisciplinary Pain Center, University of Freiburg, Germany

Corresponding author: Dagny Holle, Department of Neurology, University Duisburg-Essen, Hufelandstrasse 55, 45147 Essen, Germany. Email: dagny.holle@uk-essen.de

Holle et al. non-nociceptive Ab-bres in deeper layers of the skin (12). It sensitively reects changes in trigeminal activity (13). Analysis of habituation patterns of the nBR R2 response has evolved to be an important electrophysiological tool to assess central pain processing mechanisms. Recently, we investigated trigeminal nociception in CH using the nBR and observed a distinct side shift within central pain processing between the headache side and the non-headache side, predominantly in CH inside bout (14). This analysis did not address the question of habituation of consecutive response behaviour. Here we present a secondary analysis of the data in order re-evaluate the habituation of the nBR R2 response in episodic CH inside bout, outside bout and chronic CH in comparison to normal habituation in healthy controls. Due to the strict unilaterality of CH pain, the question of side dierences between headache side and non-headache side was illuminated.

999
All smokers (episodic CH inside bout: 78%; episodic CH outside bout: 79%; chronic CH 50%; healthy controls: 13%) were instructed not to smoke at least four hours before examination. All subjects were not allowed to drink caeine-containing beverages or any alcohol at the day of study participation.

Electrophysiolgical settings
Two planar concentric electrodes (FA Walter Graphtek GmbH, Lu beck, Germany; http://www.walter-graphtek.com) were attached to the skin 10 mm above the entry zone of the supraorbital nerve approximately 2 cm apart. Stimulation was applied in the following setting: triple pulse, monopolar square wave, duration 0.5 ms, pulse interval 5 ms, interstimulus interval 1218 seconds, pseudo-randomized, intensity for stimulation two times the individual pain threshold. Individual pain thresholds were determined, with two ascending and descending sequences of successive current intensities between 0.2 and 2 mA in 0.2 mA intensity steps. One block of 15 stimuli was applied to the headache and nonheadache sides in CH patients, or to the right and left head sides in healthy controls. Two randomly selected stimulation sequences, headache/non-headache or non-headache/headache were used in CH patients, and right/left or left/right in healthy controls, to avoid possible bias. The nBR was recorded using surface electrodes placed infraorbitally referenced to the ipsilateral orbital rim. Recording parameters were bandwith 1 Hz1 kHz, sampling rate 2.5 kHz, sweep length 300 ms (1401 plus, Signal, Cambridge Electronic Design, Cambridge, UK). Signal analysis was performed by an investigator blinded to the diagnosis and study design. The rst sweep of each block was excluded from further analysis to avoid contamination by a startle response. The area under the curve (AUC) of the R2 response was analysed in each sweep oine after demeaning, rectication and averaging between 27 and 87 ms. Each block was analysed separately. Mean values for each block were calculated. The regression coecient for each block (habituation), as well as the mean regression coecient (MRC), for each stimulation side (headache vs. non-headache) were calculated. MRC < 0 means habituation, MRC > 0 means augmentation.

Material and methods

The study was approved by the local ethics committee. Written informed consent was obtained from all patients and healthy controls prior to study inclusion. Patients were recruited from a tertiary headache centre between January 2009 and February 2010. The study design, as well as demographic and clinical characteristics of CH patients, was reported elsewhere (14). Briey, 66 patients with CH according to the ICHD-II (International Classication of Headache Diseases, second edition) classication (code 3.1.) were investigated in a case-control study design. Forty-six patients suered from episodic CH (code 3.1.1.). Eighteen of these were inside bout at the time of recording (mean age: 43 12 years; range: 1960 years) with a mean attack frequency of 1.2 1.0 per day; 28 were outside bout at the time of recording (mean age: 42 11 years; range: 1967 years). The remaining 20 patients had chronic CH (code 3.1.2.) (mean age: 47 12 years; range 1965 years) with a mean attack frequency of 3.1 2.7. None of the patients had suered from an attack within the last four hours prior to measurement. All patients had no concomitant migraine and a negative family history for migraine (rst-degree relatives). Cluster-related acute and prophylactic medication was maintained and prescribed independently from study participation according to current therapy guidelines. Additionally, the nBR habituation was investigated in 30 healthy controls (mean age: 44 14 years; range 2068 years) who did not suer from any primary headache disorder and had a negative family history for headache disorders.

Statistical analysis
Analysis of covariance (ANOVA) was used for the analysis of regression coecients (i.e. habituation) of CH patients and healthy subjects. The within-subject factor was side (headache side vs. non-headache side in CH patients and right vs. left side in healthy controls); between-subject factor was disease (episodic CH

1000
inside bout vs. episodic CH outside bout vs. chronic CH vs. healthy controls) . Bonferroni correction was used for post hoc analysis. The level of signicance was set to p < 0.05. Pearsons correlation was used to nd correlations between regression coecients and time from last attack. All statistics were calculated with SPSS 16 (SPSS Inc., Chicago, IL, USA).

Cephalalgia 32(13) lines are shown in Figure 1. Exemplary traces of single rectied nBR responses for each group are shown in Figure 2.

Discussion
Our results show no habituation decit in chronic and episodic CH inside and outside bout compared with healthy controls. Furthermore, no signicant side differences between headache side and non-headache side were detected in all groups. Subgroup analysis did not reveal dierences of habituation responses comparing patients with CH in dierent stages of disease (inside bout vs. outside bout vs. chronic). Our results contradict prior data by Perotta et al., who investigated the habituation behaviour of the classical BR (R2 and R3 component) in 27 CH patients with inside bout, 22 migraineurs and 20 healthy controls. Results showed a signicant habituation decit in CH patients compared with healthy controls, which was even more pronounced than in migraine patients. The authors suggested that abnormal suprasegmental modulation of the BR might be responsible for this observation (8). One explanation for these diering results might rest in the dierent electrophysiolgical methods used in the two studies, notably the classical BR and the nBR, as they involve varying reex arches. First of all, BR and nBR are elicited by activation of dierent populations of

Results
On all recordings, the R2 component of the nBR was clearly identied and there was no R1 component. In CH patients, nBR R2 responses habituated with repetitive stimulation (mean regression coecient: episodic CH inside bout [MRC headache side 2.2, MRC non-headache side 1.2; p 1.0]; episodic CH outside bout [MRC headache side 1.4, MRC nonheadache side 1.2; p 1.0]; chronic CH [MRC headache side 1.4, MRC non-headache side 1.4, p 1.0]). No signicant dierences between headache and non-headache side stimulation were detected. No signicant correlation between R2 component habituation and time from last attack was detected. In healthy controls, similar habituation was observed (MRC right-sided stimulation 1.4, MRC left-sided stimulation 1.8; p 1.0). There were no signicant dierence of nBR response comparing CH and healthy controls (ANOVA: factor disease degrees of freedom (d.f.) 7; F 0.754; p 0.626). Group mean regression

(A) 10 5 0 5 10

(C) 10 5 0 5 10

(B) 10 5 0 5 10

(D) 10 5 0 5 10

Figure 1. Habituation of the nociceptive blink reflex R2 response in cluster headache (CH). Dashed lines indicate individual regression lines (across 14 sweeps); bold lines indicate group mean regression lines. Dotted lines indicate the reference line (r 0). (A) Healthy controls; (B) episodic CH, inside bout; (C) episodic CH, outside bout; (D) chronic CH. R: slope coefficient. Slopes of only one side are shown: headache side in CH patients and right side in controls.

Holle et al.

1001
(A) (B)

SA

R2 responses sweep 15 sweep 10 sweep 6 sweep 2

(C)

(D)

Figure 2. Habituation pattern of typical single rectified nociceptive blink reflex responses. Sweep 2, 6, 10 and 15 R2 responses are displayed in (A) healthy controls; (B) episodic cluster headache (CH), outside bout; (C) episodic CH, inside bout; and (D) chronic CH. The first sweep of each block is excluded from further analysis to avoid contamination by startle response. No significant difference of R2 habituation was observed between the several groups. Traces of only one side are shown; headache side in CH patients and right side in healthy control. SA: stimulus artefact.

trigeminal bres. For elicitation of the nBR, nociceptive Ad-bres are stimulated selectively with little contamination (< 10%) of somatosensory Ab-bers (12), while in contrast, for BR elicitation, Ab-bers are stimulated on a larger scale (15), leading to activation of other parts of the somatosensory and central processing systems. The medullary interneurons of the nBR are predominantly located in the subnucleus caudalis of the trigeminal spinal tract nucleus (STN); in contrast, the BR involves interneurons located more rostrally at the level of the inferior olive, for example, at the subnucleus interpolaris (16). This anatomic dierence might contribute to the observed divergent results. Furthermore, one can hypothesise that noxious and innocuous trigeminal stimuli are modulated by dierent suprasegmental or segmental inuences.

Additionally, the stimulation mode used was dierent. Perotta el al. delivered the electrical stimuli in ve dierent frequencies (0.2, 0.3, 0.5, 0.7 and 1 Hz) (8), which all were much faster than in our experiment, where the interstimulus interval (ISI) was between 12 and 18 seconds. In contrast to the nBR, the classical BR does not habituate to ISI intervals as low as 15 s (4,7). Most likely, there is an inuence of the stimulation frequency on habituation, and an alteration might be more pronounced for higher stimulation frequencies, as already suggested by the authors themselves (8). In which regard an increase of stimulation frequency may have inuenced our results will be subject to future studies. However, our stimulation paradigm is very similar to the one used by Karsarava et al., who detected a signicant habituation decit

1002
in migraine patients (2). Therefore, the paradigm used seems to be able to detect habituation pattern in headache. A dierent, very simple explanation of habituation observed by Perotta et al. might be that some of the investigated CH patients also possibly had migraine or rst relatives with migraine and were, therefore, prone to have a habituation decit. Dierent disease phases as well as drug therapy might also be signicant confounders responsible for the divergent study results. Our nding of normal habituation in CH is in line with studies on event-related potentials in this patient group (9,10). Evers et al. investigated visually evoked event-related potentials (ERPs) in 50 episodic CH patients inside bout and 11 chronic CH patients. There was no cognitive habituation decit in CH as has been detected in migraineurs. The authors hypothesised that pathophysiological mechanisms of mitochondrial energy metabolism and serotonergic pathways dysfunction, which are thought to contribute to a lack of habituation in migraine patients, do not play a relevant role in the pathophysiology of CH (10). Reduced habituation is a commonly observed phenomenon in migraine patients. Habituation decit was detected using several electrophysiological study methods, including nBR (2,5), classic BR (3,4), visually evoked potentials (17,18), auditory evoked potentials (18,19) and contingent negative variation (CNV) (20). A habituation decit was also shown in rst-degree relatives of migraineurs who were not aected by the disease (6). Interestingly, the nBR habituation decit is detected predominantly interictally. During the acute migraine attack, the habituation normalises and is comparable with that of healthy controls (2). Additionally, previous studies showed that nBR habituation in migraineurs increases with increasing attack frequency (5,6). Our observed CH patients, namely inside bout and chronic patients, were apparently more likely to be recorded in a closer temporal relationship to a headache attack, which might contribute to the observation of a normal habituation pattern in these CH subgroups. However, this does not explain regular habituation in CH outside bout. A regular habituation was also shown in hypnic headache, a rare primary headache disorder that shares some clinical features (e.g. nocturnal headache attacks) with CH (21). Therefore, a lack of habituation is not a general phenomenon in headache diseases in general but seems to be a more specic electrophysiological feature in distinct headache entities such as migraine. Habituation is a classic phenomenon of polysynaptic activity (22). There is only sparse information on the exact mechanisms associated with habituation. It is known that classical BR habituation is modulated by dopamine, as a lack of habituation was observed in Parkinsons disease, which was found to be reversible

Cephalalgia 32(13) under dopaminergic therapy (23). It remains to be determined if this pathway is also involved in the habituation of the nBR in primary headache disorders. Further hypotheses of habituation modulation include segmental control mechanisms, such as pre- or postsynaptic inhibition, homosynaptic depression and membrane desensitization (1,2428), but suprasegmental inuences mediated by descending modulatory nociceptive/anti-nociceptive pathways were also discussed as being involved. Previous functional neuroimaging studies showed an increased activity of the rostral anterior cingulate cortex (rACC) during habituation to pain (29,30), supporting its crucial role in regard to anti-nociceptive mechanisms in the habituation phenomenon. Schoenen et al. hypothesised that ceiling eects of neuronal activation might explain reduced habituation in migraineurs. According to this pathophysiological theory, migraine patients have a lower pre-activation level of the sensory cortex compared with healthy controls. After repetitive stimulation, migraineurs, therefore, have a larger range for activation up to the ceiling and thus lack proper habituation, whereas healthy controls reach the top of response activity faster and thus show appropriate habituation in response to painful potentially damaging stimuli (20). Our previous data already suggested that cortical excitability and especially eciency of inhibitory topdown circuits are not altered in CH (14). This might explain why habituation remains unaected in CH compared with migraine. Some important limitations of this study have to be addressed. Prophylactic and acute pain medication might inuence our results and account for dierences between CH and healthy controls. To what extent medication intake aects habituation and in what manner has not yet been investigated. Unfortunately, separation into smaller, medication-adjusted groups is virtually impossible because of the large number of dierent medications and medication combinations, which would lead to very small group sizes without enough statistical power. Nicotine intake has to be considered as additional confounder. There was a between-group dierence in smoking habits between CH and healthy controls. More patients than healthy controls were smokers, so an inuence of smoking on our results cannot be ruled out completely. To minimize its possible eects, all patients and healthy controls were instructed not to smoke for at least four hours before study participation. Studies on the eects of nicotine on nBR habituation are lacking. However, we did not determine any signicant correlation between smoking frequency and habituation behaviour, suggesting that its impact might be rather small. Additionally, in this study, habituation inside and outside bout was measured in dierent patients, which might inuence the study results signicantly. To conrm our data, a

Holle et al. longitudinal investigation with measurement of the same patients inside and outside bout is already planned. In summary, our data show normal habituation of the nBR R2 response to repetitive nociceptive stimuli in episodic as well as chronic CH. Dierent mechanisms, such as central sensitization, seem to play a more crucial role in the pathophysiology of this disease than alteration of habituation in CH. These results do not support the hypothesis that CH and migraine share the same pathophysiological mechanisms in regard to neuronal adaptation processes such as habituation, despite a considerable overlap in clinical characteristics. Funding
This research received no specic grant from any funding agency in the public, commercial, or not-for-prot sectors.

1003
2. Katsarava Z, Giffin N, Diener H-C and Kaube H. Abnormal habituation of nociceptive blink reflex in migraineevidence for increased excitability of trigeminal nociception. Cephalalgia 2003; 23(8): 814819. 3. de Tommaso M, Murasecco D, Libro G, et al. Modulation of trigeminal reflex excitability in migraine: effects of attention and habituation on the blink reflex. Int J Psychophysiol 2002; 44(3): 239249. 4. De Marinis M, Pujia A, Natale L, et al. Decreased habituation of the R2 component of the blink reflex in migraine patients. Clin Neurophysiol 2003; 114(5): 889893. 5. Di Clemente L, Coppola G, Magis D, et al. Nociceptive blink reflex and visual evoked potential habituations are correlated in migraine. Headache 2005; 45(10): 13881393. 6. Di Clemente L, Coppola G, Magis D, et al. Interictal habituation deficit of the nociceptive blink reflex: an endophenotypic marker for presymptomatic migraine? Brain 2007; 130(Pt 3): 765770. 7. Penders CA and Delwaide PJ. Blink reflex studies in patients with Parkinsonism before and during therapy. J Neurol Neurosurg Psychiatr 1971; 34(6): 674678. 8. Perrotta A, Serrao M, Sandrini G, et al. Reduced habituation of trigeminal reflexes in patients with episodic cluster headache during cluster period. Cephalalgia 2008; 28(9): 950959. 9. Evers S, Bauer B, Suhr B, et al. Cognitive processing in primary headache: a study on event-related potentials. Neurology 1997; 48(1): 108113. 10. Evers S, Bauer B, Suhr B, et al. Cognitive processing is involved in cluster headache but not in chronic paroxysmal hemicrania. Neurology 1999; 53(2): 357363. 11. Hopf HC. Topodiagnostic value of brain stem reflexes. Muscle Nerve 199; 17(5): 475484. 12. Kaube H, Katsarava Z, Ka ufer T, et al. A new method to increase nociception specificity of the human blink reflex. Clin Neurophysiol 2000; 111(3): 413416. 13. Katsarava Z, Lehnerdt G, Duda B, et al. Sensitization of trigeminal nociception specific for migraine but not pain of sinusitis. Neurology 2002; 59(9): 14501453. 14. Holle D, Gaul C, Zillessen S, et al. Lateralized central facilitation of trigeminal nociception in cluster headache. Neurology 2012; 78(13): 985992. 15. van Vliet JA, Vein AA, le Cessie S, et al. Reproducibility and feasibility of neurophysiological assessment of the sensory trigeminal system for future application to paroxysmal headaches. Cephalalgia 2002; 22(6): 474481. 16. Cruccu G, Iannetti GD, Marx JJ, et al. Brainstem reflex circuits revisited. Brain 2005; 128(Pt 2): 386394. 17. Afra J, Cecchini AP, De Pasqua V, et al. Visual evoked potentials during long periods of pattern-reversal stimulation in migraine. Brain 1998; 121(Pt 2): 233241. ndor PS and Schoenen J. 18. Afra J, Proietti Cecchini A, Sa Comparison of visual and auditory evoked cortical potentials in migraine patients between attacks. Clin Neurophysiol 2000; 111(6): 11241129. 19. Wang W, Wang GP, Ding XL and Wang YH. Personality and response to repeated visual stimulation in

Conflicts of interest
Dagny Holle has received a research grant from Gru nenthal. Silke Zillessen reports nothing to disclose. Charly Gaul has received research grants and honoraria from Deutsche Gesellschaft fu r Muskelkranke, RouxProgram of the University of Halle-Wittenberg, MSD, Berlin Chemie, Medtronic and Bo hringer Ingelheim. Steen Naegel reports nothing to disclose Holger Kaube has received research grants and honoraria from MSD, Boston Scientic, St. Jude Medical, Medtronic Medical, Linde AG, Berlin-Chemie and Gru nenthal. Hans-Christoph Diener has received honoraria for participation in clinical trials, and has contributed to advisory boards or lectures for Addex Pharma, Allergan, Almirall, AstraZeneca, Bayer Vital, Berlin Chemie, Coherex Medical, CoLucid, Bo hringer Ingelheim, Bristol-Myers Squibb, GlaxoSmithKline, Gru nenthal, Janssen-Cilag, Lilly, La Roche, 3M Medica, Minster, MSD, Novartis, Johnson & Johnson, Pierre Fabre, Pzer, Schaper and Bru mmer, SanoAventis and Weber & Weber. He has received research support from Allergan, Almirall, AstraZeneca, Bayer, GalaxoSmith-Kline, Janssen-Cilag, and Pzer. Headache research at the Department of Neurology in Essen is supported by the German Research Council (DFG), the German Ministry of Education and Research (BMBF) and the European Union. Zaza Katsarava has received research grants and honoraria from Allergan, Bayer, Biogen and Merck, and is an advisory board member for Allergan. Headache research at the Department of Neurology in Essen is supported by the DFG, the BMBF and the European Union. Mark Obermann has received scientic grants by the German Federal Ministry of Education and Research BMBF 01EM 0513.

References
1. Thompson RF and Spencer WA. Habituation: a model phenomenon for the study of neuronal substrates of behavior. Psychol Rev 1966; 73(1): 1643.

1004
migraine and tension-type headaches. Cephalalgia 1999; 19(8): 718724. (discussion 697698). Schoenen J. Deficient habituation of evoked cortical potentials in migraine: a link between brain biology, behavior and trigeminovascular activation? Biomed Pharmacother 1996; 50(2): 7178. Holle D, Gaul C, Krebs S, et al. Nociceptive blink reflex and pain-related evoked potentials in hypnic headache. Cephalalgia 2011; 31(11): 11811188. Desmedt JE and Godaux E. Habituation of exteroceptive suppression and of exteroceptive reflexes in man as influenced by voluntary contraction. Brain Res 1976; 106(1): 2129. Basso MA, Powers AS and Evinger C. An explanation for reflex blink hyperexcitability in Parkinsons disease. I. Superior colliculus. J Neurosci 1996; 16(22): 73087317. Byrne JH. Analysis of synaptic depression contributing to habituation of gill-withdrawal reflex in Aplysia californica. J Neurophysiol 1982; 48(2): 431438. Carew TJ, Pinsker HM and Kandel ER. Long-term habituation of a defensive withdrawal reflex in aplysia. Science 1972; 175(4020): 451454.

Cephalalgia 32(13)
26. Christoffersen GR. Habituation: events in the history of its characterization and linkage to synaptic depression. A new proposed kinetic criterion for its identification. Prog Neurobiol 1997; 53(1): 4566. 27. Wiel DE, Wood ER and Weeks JC. Habituation of the proleg withdrawal reflex in Manduca sexta does not involve changes in motoneuron properties or depression at the sensorimotor synapse. Neurobiol Learn Mem 2001; 76(1): 5780. 28. Burrell BD, Sahley CL and Muller KJ. Non-associative learning and serotonin induce similar bi-directional changes in excitability of a neuron critical for learning in the medicinal leech. J Neurosci 2001; 21(4): 14011412. 29. Bingel U, Schoell E, Herken W, et al. Habituation to painful stimulation involves the antinociceptive system. Pain 2007; 131(12): 2130. 30. Bingel U, Herken W, Teutsch S and May A. Habituation to painful stimulation involves the antinociceptive systema 1-year follow-up of 10 participants. Pain 2008; 140(2): 393394.

20.

21.

22.

23.

24.

25.