Original Article

Reduced insula habituation associatedwith amplification of trigeminalbrainstem input in migraine

Jeungchan Lee1, Richard L Lin1,2, Ronald G Garcia1,3,4,Jieun Kim5, Hyungjun Kim5, Marco L Loggia1, Ishtiaq Mawla1,Ajay D Wasan6, Robert R Edwards7, Bruce R Rosen1,Nouchine Hadjikhani1 and Vitaly Napadow1,7

AbstractBackground: Impaired sensory processing in migraine can reflect diminished habituation, increased activation, or evenincreased gain or amplification of activity from the primary synapse in the brainstem to higher cortical/subcortical brainregions.Methods: We scanned 16 episodic migraine (interictal) and 16 healthy controls (cross-sectional study), and evaluatedbrain response to innocuous air-puff stimulation over the right forehead in the ophthalmic nerve (V1) trigeminal territory.We further evaluated habituation, and cortical/subcortical amplification relative to spinal trigeminal nucleus (Sp5)activation.Results: Migraine subjects showed greater amplification from Sp5 to the posterior insula and hypothalamus. In addition,while controls showed habituation to repetitive sensory stimulation in all activated cortical regions (e.g. the bilateralposterior insula and secondary somatosensory cortices), for migraine subjects, habituation was not found in the posteriorinsula. Moreover, in migraine, the habituation slope was correlated with the amplification ratio in the posterior insula andsecondary somatosensory cortex, i.e. greater amplification was associated with reduced habituation in these regions.Conclusions: These findings suggest that in episodic migraine, amplified information processing from spinal trigeminalrelay nuclei is linked to an impaired habituation response. This phenomenon was localized in the posterior insula, high-lighting the important role of this structure in mechanisms supporting altered sensory processing in episodic migraine.

KeywordsMigraine, spinal trigeminal nucleus, posterior insula cortex, amplification, habituation

Date received: 21 January 2016; revised: 25 May 2016; accepted: 23 July 2016

Introduction

Migraine is a neurovascular disorder characterized byaltered neural processing in the central nervous system(1,2). Importantly, hyperalgesia, allodynia, and impairedhabituation have been commonly reported in migrainepatients, even during the interictal phase (betweenattacks) (3,4). While most migraine neuroimaging studiesassessing altered neural processing (e.g., impaired habitu-ation) have focused on cortical responses and usedevoked potentials and electrophysiological methods,functional MRI may be more appropriate to evaluatelonger duration stimuli and deeper brain structures,including the brainstem. In fact, increased gain or amp-lification of activity from the primary synapse in the

1Martinos Center for Biomedical Imaging, Department of Radiology,Massachusetts General Hospital, Harvard Medical School, Boston,MA, USA2Harvard-MIT Division of Health Sciences and Technology, HarvardMedical School, Boston, MA, USA3Neurovascular Science Group, Fundacion Cardiovascular de Colombia,Floridablanca, Santander, Colombia4Connors Center for Women’s Health and Gender Biology, Brigham andWomen’s Hospital, Harvard Medical School, Boston, MA, USA5Clinical Research Division, Korea Institute of Oriental Medicine,Daejeon, Korea6Department of Anesthesiology, University of Pittsburgh Medical Center,Pittsburgh, PA, USA7Department of Anesthesiology, Perioperative and Pain Medicine,Brigham and Women’s Hospital, Harvard Medical School, Boston,MA, USA

Corresponding author:

Jeungchan Lee, PhD Martinos Center for Biomedical Imaging, CNY149-2301, 13th St. Charlestown, MA 02129 USA.Email: aeiouphy@nmr.mgh.harvard.edu

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DOI: 10.1177/0333102416665223

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brainstem to cortical/subcortical brain regions has notbeen assessed in migraine.

Altered sensory processing can occur on multiplelevels of the central nervous system. For instance, amp-lification of peripherally generated signaling can occurat the level of the first synapse in the spinal cord, higherup in cortical or subcortical regions, or both. No stu-dies, however, have explicitly evaluated amplification ofactivation focusing on both the brainstem and cortical/subcortical brain regions in migraine, particularly withfunctional MRI (fMRI).

Habituation is a neurophysiological phenomenoncharacterized by a reduced response to repeated sensorystimulation (5). Neural habituation is considered protect-ive to the organism, as it shields the brain from excessiveinformation processing and limits energy consumption(6). For instance, habituation is critical for learning, inorder to discriminate relevant stimuli and focus on select-ive properties of external stimuli (5). In migraine,impaired habituation to not just pain but also non-nox-ious sensory stimuli of various modalities (visual, audi-tory, olfactory, somatosensory) has been commonlyreported, mainly using electrophysiological approaches(7,8). For instance, the late component of nociceptivelaser-evoked potentials, which may be generated fromthe deep insular and anterior cingulate cortices, demon-strates reduced habituation in migraine patients (9–11).FMRI neuroimaging has also assessed habituation inthe brain to repeating blocks of sensory stimulation(12,13). Additionally, a recent study found that migrainepatients do not report decreasing intensity ratings forrepeated painful stimuli, and when such ratings areused to guide the fMRI analysis, pain-evoked activityin the insular and anterior cingulate cortices similarlydemonstrates a lack of habituation (14).

Afferent sensory inputs (tactile, thermosensory, andnociceptive signals) from the face are transmitted to thetrigeminal sensory complex in the brainstem throughthe afferent trigeminal nerve, then to the thalamus(lateral and medial nuclei) and primary somatosensorycortex (SI, face area). This pathway is known as the pri-mary somatosensory trigeminal pathway (15-18). Thetrigeminal sensory complex in the brainstem consists ofthe main sensory nucleus and spinal trigeminal nucleus(Sp5). The main sensory nucleus (rostral portion ofthe trigeminal sensory complex) is activated by low-threshold tactile (e.g., touch and pressure, Ab mediatedafference) stimulation. Sp5 is also activated by Ab-mediated tactile (touch and pressure) afference,mainly in the oralis subdivision, while the caudalis sub-division is known to mainly process Ad-/C-mediatednociceptive and thermosensory afference (15-18).In addition, while multiple fMRI studies have demon-strated Sp5 activation in response to nociceptive stimu-lation in patients with trigeminal tractotomy (16),

trigeminal neuropathy (19), migraine (20), as well asin healthy adults (21), few studies have evaluated thetrigeminal sensory complex response to non-painfulstimulation.

In this study, we applied fMRI to investigate twoaspects of tactile sensory processing in episodicmigraine: impaired habituation and cortical/subcorticalamplification of the brainstem response to non-painful ophthalmic nerve somatosensory input. Wehypothesized that interictal migraine patients woulddemonstrate reduced habituation in multiple stimulus-activated brain regions, which would be associated withincreased amplification relative to activation at Sp5, thesite of the primary brainstem synapse.

Methods

All studies were performed at the Martinos Center forBiomedical Imaging in Boston, Massachusetts. Theexperimental procedure was approved by the PartnersHuman Research Committee (approval number:2005P-000466), and research was performed in accord-ance with the principles of the Declaration of Helsinki.All participants in the study were fully informed of theprocedure and provided written informed consent.

Demographics and clinical characterization

Migraine diagnosis was based on the classification of theInternational Headache Society (Headache ClassificationCommittee of the International Headache Society, 2004).Inclusion criteria allowed for the enrollment of episodicmigraine patients with 2–15 episodes per month.Exclusion criteria included other neurological or majorpsychiatric disorders. Sixteen migraine patients (MIG, 15females, 35.8! 13.4 years old, mean! SD) and sixteensex/age-matched healthy controls (HC, 15 females,36.0! 13.7 years old, p¼ 0.96) participated in thiscross-sectional study (22). In migraine patients, clin-ical data such as episodes per month (times), presenceof aura, migraine duration (years), lateralization ofmigraine attack (right, left, or variable right/left), andspatial extent (frontal, temporal, occipital, vertex, or acombination) were collected. All MIG patients werescanned interictally (i.e. between episodes). Subjectswere asked on the day of the scan when their precedingictal event took place (PRE_I: the number of daysbetween the previous migraine episode and the scanvisit), and were followed up by phone regarding thesubsequent migraine episode, after the scan visit(NEXT_I: the number of days between the scan visitand the subsequent migraine episode). We then calcu-lated the ‘‘interictal phase’’, defined for the day the sub-ject was scanned (see equation 1), to take into accountindividual patients’ attack-to-attack cycle, which resulted

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in a metric scaled from 0 (immediately after the lastattack) to 100 (immediately preceding the next attack).

INTERICTAL PHASE ¼ PRE I

PRE I þ NEXT I$ 100

ð1Þ

Air-puff stimulation

To investigate the stimulus-related brain response inmigraine patients and healthy controls, the outlet ofMR-compatible air tubing (inner diameter¼ 12mm)was positioned over the right supraorbital region ofthe forehead, approximately 2 cm above the medialaspect of the eyebrow. This location targeted theophthalmic (V1) spinal trigeminal nerve branch(Figure 1(a)). The tubing was passed through theMR-scanner penetration panel and connected to anair compressor controller (AIRSTIM, San DiegoInstruments, Inc., CA, USA). Non-painful air-puffstimulation (80 Psi, 5Hz) was delivered through thetubing to the subjects using a block fMRI design (14seconds ON and 20 seconds OFF, 11 repetitions, a totalof 370 seconds (Figure 1(b)), see below for more detail).We chose a stimulus frequency, 5 Hz, on the same orderbut higher than that reported in previous studiesdemonstrating reduced habituation (e.g. 3Hz somato-sensory stimulus (23)). After the fMRI experiment,the intensity of air-puff sensations during the scan(i.e. psychometric outcome) was rated by subjectson a numerical rating scale (NRS) of 0 to 10 (0: nosensation, 10: pain detection threshold, i.e. on theverge of painful sensation). A two-tailed Student’st-test (unpaired, SPSS v. 10.0.7, Chicago, IL, USA)was used to evaluate the difference between MIG andHC, significant at p< 0.05.

MRI data collection

This fMRI study was performed using a 3.0T scanner(Trio, Siemens Medical, Germany) with a 12-channelhead coil. Subjects were asked to lay supine in the scan-ner with their eyes closed while staying alert and awake.They were also asked to focus on the air-puff stimula-tion and remain still during structural and functionalscan runs, which were assisted by padding the headinside the head coil. Earplugs were provided to attenu-ate noise during data collection.

Structural MRI data were collected using aT1-weighted MP-RAGE pulse sequence (TR¼ 2530 ms,TE¼ 1.64ms, flip angle¼ 7', FOV¼ 256( 256 mm2,176 axial slices, voxel size¼ 1( 1( 1 mm3), andwhole-brain functional MRI data were acquired usinga T2*-weighted blood oxygenation level dependent(BOLD) pulse sequence with increased matrix size forimproved in-plane spatial resolution to increase sensi-tivity for brainstem nuclei with small cross-sectionalarea (TR¼ 2500ms, TE¼ 30ms, flip angle¼ 90',FOV¼ 220( 220 mm2, matrix¼ 84( 84, 43 axialslices, slice thickness¼ 2.62mm, gap¼ 0.5mm, voxelsize¼ 2.62( 2.62( 3.12 mm3).

We corrected for cardiorespiratory-related physio-logical artifacts in fMRI data, which is critical forbrainstem analysis (24). Peripheral physiological datawere acquired using the Powerlab system (ML880,ADInstruments Inc., Colorado Springs, CO) at a400Hz sampling rate. Electrocardiogram (ECG) datawere filtered via an MR-compatible monitor (InVivoMagnitude CV, Invivo Research Inc., Orlando,Florida) designed to minimize radiofrequency (RF)and gradient switching artifacts generated during theMRI scan. Heartbeat annotation was performed tolocalize R-peaks using custom-made MATLAB (TheMathWorks Inc., MA, USA) scripts. Respiration datawere collected using an MR-compatible belt system

Block design: 11 air-puff stimulation blocks

ON: 14 sec OFF: 20 sec

(a) (b)

0

Stimulus site

R

14 34 48 370

Time [seconds]

Figure 1. Innocuous somatosensory (air-puff) stimulation, which was used for ROI selection. (a) Right forehead (V1, ophthalmic nerveterritory) was stimulated. (b) In a block design (air pressure¼ 80 Psi with 5 Hz, 11 repetitions, 14 second stimulation and 20 second rest).

Lee et al. 3

constructed in-house, based on the system devised byBinks et al. (25) similar to our previous studies (12,26,27).

MRI data preprocessing

Data preprocessing was performed using FSL (FMRIBSoftware Library, http://www.fsl.fmrib.ox.ac.uk/fsl/),AFNI (Analysis of Functional NeuroImages, http://afni.nimh.nih.gov/afni), and FreeSurfer (http://surfer.nmr.mgh.harvard.edu). FMRI data were corrected forcardiorespiratory physiological artifacts using the retro-spective image correction algorithm (RETROICOR)(24). Head motion was corrected using FSL-MCFLIRT (28). FMRI data were smoothed in space(Gaussian kernel with FWHM¼ 5mm) and time (highpass cutoff¼ 0.0147Hz, consistent with twice the dutycycle of our block design) domains to increase SNR andto remove MR signal drift, respectively. Structural MRIdata were registered to fMRI data (FREESURFER-BBREGISTER) (29), allowing for co-registration ofboth to the standard space (MNI152 template) usingnon-linear warping (FSL-FNIRT).

MRI data analysis

A first-level general linear model (GLM) was used toestimate brain response to air-puff stimulation using asingle regressor for the air-puff stimulation blockdesign, convolved with the canonical hemodynamicresponse function (Double-Gamma). The results ofthis analysis (i.e., the parameter estimate and its vari-ance) were transformed into standard space (MNI152),and passed up to a combined group analysis usingFMRIB’s Local Analysis of Mixed Effects (FLAME1þ 2, cluster-corrected for multiple comparisons,Z¼ 2.3, p< 0.05). A combined activation map wasobtained for the purpose of identifying regions of inter-est (ROIs), which were subsequently used for groupcomparisons. Both groups were combined in order tomaximize SNR for brainstem response and to defineROIs with unbiased localization for the subsequent cor-tical/sub-cortical amplification and habituation ana-lyses (see below), which were the focus of this study.ROIs identified from the combined group activationmap included a ponto-medullary cluster consistentwith Sp5 (localized based on brainstem atlas (30)),important for the amplification analysis.

For activated brain regions, we then evaluated thecortical/subcortical amplification of Sp5 activation.Our metric was similar to that used by other studiesinvestigating central amplification at higher levels ofthe central nervous system, e.g. the cortical (i.e. theprimary somatosensory cortex)/peripheral (i.e. themedian nerve) amplification ratio using sensory nerveaction potentials was calculated for Carpal Tunnel

Syndrome patients (31). For our analysis, a 3mmdiameter sphere mask was centered on the peak voxelfor each ROI, and the mean percent signal changeof activated voxels was calculated. For mathematicalstability, the amplification ratio was calculated as aninverted ‘Sp5 ROI activity / cortical/subcortical ROIactivity’, due to the fact that Sp5 signal was morelikely to approach nil in some subjects. This ratio wascontrasted between MIG and HC using a two-tailedStudent’s t-test (unpaired, SPSS v. 10.0.7, Chicago,IL, USA), significant at p< 0.05. For easier interpret-ability, visual plots are provided with the more intuitive‘cortical/subcortical / Sp5’ ratio.

We also explored habituation across the multiple air-puff stimulation blocks. This analysis used an add-itional single subject level GLM, in which each of the11 stimulation blocks was modeled as a separate regres-sor, thereby assessing activation for each individualstimulus block (similar to our previous fMRI habitu-ation study (12)). Parameter estimates and variances foreach of the 11 stimulation blocks were then trans-formed into standard space for the ROI analysis. Thehabituation slope (first-order linear regression) of per-cent-change across the 11 stimulation blocks was calcu-lated for the same ROIs identified above. Habituation(a linear decrease of activation over stimuli) wasdefined by passing two criteria, significant at p< 0.05:(1) a significant linear correlation (i.e. Pearson’s r-coef-ficient), and (2) a linearly-decreasing slope significantlydifferent from nil. Additionally, for each ROI,slopes were contrasted between MIG and HC usingan unpaired, two-tailed Student’s t-test, significantat p< 0.05.

We also investigated whether the amplification ratiowas correlated with the habituation slope within spe-cific brain regions, thereby more closely linking theseneurophysiological outcomes. Additionally, the clinicalrelevance of these brain responses was evaluated bycalculating the correlation between activation, the amp-lification ratio (for regions showing amplification) andthe habituation slope (for regions showing habituationin HC but not MIG), and the interictal phase(as defined above). Following testing for normal distri-bution using a Kolmogorov-Smirnov test, a Pearson’scorrelation coefficient, r, was calculated, significant atp< 0.05.

Results

Demographic and clinical characterization of the16 enrolled MIG subjects is available in Table 1.There were no missing data, and every subject com-pleted this study. Out of the 16 MIG subjects, sevenreported the presence of aura. MIG subjects withaura did not differ from others in terms of episodes

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per month (with aura¼ 5.9! 2.1 episodes/month, mean!SD, without aura¼ 5.9! 3.1 episodes/month, p¼ 0.98),migraine duration (with aura¼ 14.3! 15.1 years, withoutaura¼ 15.4! 12.0 years, p¼ 0.87), or interictal phase(with aura¼ 66.4! 21.6, without aura¼ 60.5! 31.9,p¼ 0.68).

All subjects tolerated the air-puff procedures andstimulation intensity, which was rated as non-painful(below 10 on the 0–10 NRS) by all subjects. Therewere no group differences in ratings of air-puff stimu-lation intensity (Table 1).

Brain response to innocuous somatosensoryophthalmic nerve stimulation

No subjects were removed from analysis due to exces-sive head motion (criteria:> 2mm TR-to-TR transla-tion), and displacement did not differ between groups(RMS displacement: MIG¼ 0.11! 0.09mm, mean!SD, HC¼ 0.11! 0.05mm, p¼ 0.91). Bilateral activa-tion in the brainstem included pontomedullary junction

locations compatible with Sp5 (consistent with thehuman brainstem atlas (30)) and, more anteriorly, pon-tine nuclei (Figure 2(a)). Periaqueductal gray (PAG)activation was found in the midbrain (Figure 2(b)).Cortical activation was noted in the bilateral secondarysomatosensory cortex (SII), bilateral posterior insula(pINS), and bilateral supramarginal gyrus (SMG).Subcortical activation was noted in the left hypothal-amus (Hyp), right putamen, and right caudate nucleus(Figure 2(b)). No group differences for activationresponse to air-puff stimulation were found withinSp5 or any of the cortical/subcortical brain regionsnoted above (Table 2).

Cortical/subcortical amplification of Sp5 activation

We then calculated the amplification ratios for the cor-tical/subcortical regions noted above relative to Sp5activation, and contrasted MIG versus HC groups.A greater amplification ratio was found for MIG com-pared to HC in two regions: the contralateral posteriorinsula cortex and hypothalamus (Figure 3, Table 3).Differences between groups in amplification were notseen for other ROIs (Table 3).

Habituation to repeated blocks of ophthalmicnerve somatosensory stimulation

Neural response habituation was defined by explicitcriteria for the fMRI signal (see Methods). For HC,habituation was noted for all activated cortical regions(bilateral SII, bilateral pINS, bilateral SMG), and puta-men. For MIG, habituation was noted for bilateral SIIand SMG. Directly contrasting MIG and HC, wefound a significantly different slope magnitude for theright posterior insula (Figure 4, Table 3). Specifically,for HC, the insula response linearly decreased over time(consistent with habituation criteria), whereas for MIG,the habituation criteria were not met.

Associations between different brain activitymetrics and links to clinical measures

For MIG, we found a correlation between thehabituation slope and amplification ratio in the rightpINS (r¼ 0.54, p¼ 0.03, Figure 5(a)) and right SII(r¼ 0.56, p¼ 0.02, with outlier removed: r¼ 0.58,p¼ 0.02, Figure 5(b)), i.e. since a more negative habitu-ation slope reflects greater habituation, greater ampli-fication was associated with reduced habituation inthese regions.

We also found a positive correlation between theinterictal phase and fMRI signal response in the right,ipsilateral Sp5 for MIG (r¼ 0.58, p¼ 0.02, Figure 6).Thus, MIG subjects relatively closer to their next attack

Table 1. Demographics, psychophysics, and clinical measures.

Healthycontrols(HC,n¼ 16)

Migrainepatients(MIG,n¼ 16)

P-value(HC vs.MIG)

Demographics

Age (years old) 36.0! 13.7 35.8! 13.4 0.96

No. of females 15 15 1.00

Psychophysics

Intensity of airpuffsensation (0–10)

3.31! 1.74 3.63! 1.75 0.62

Clinical measures

Interictal phase (0–100) n/a 63.09! 27.18 n/a

Migraine duration (years) n/a 14.91! 13.00 n/a

Episodes permonth (times)

n/a 5.88! 2.63 n/a

Migraine side

Right n/a N¼ 6 n/a

Left n/a N¼ 3 n/a

Right/left (variable) n/a N¼ 7 n/a

Migraine spatial extent

Frontal n/a N¼ 5 n/a

Temporal n/a N¼ 6 n/a

Occipital n/a N¼ 5 n/a

Vertex n/a N¼ 1 n/a

Whole head n/a N¼ 3 n/a

Migraine with aura n/a N¼ 7 n/a

Data are shown as mean! SD. Interictal phase¼ ratio between preced-ing (¼0) and subsequent (¼100) attacks from experiment visit. n/a ¼ notapplicable.

Lee et al. 5

demonstrated greater Sp5 activation. Kolmogorov-Smirnov testing confirmed that all variables for signifi-cant correlation tests were normally distributed (Sp5:p¼ 0.32, i.e. data consistent with normal distribution;interictal phase: p¼ 0.45; amplification ratio, rightpINS: p¼ 0.35, right SII: p¼ 0.20; habituation slope,right pINS: p¼ 0.89, right SII: p¼ 0.77).

Discussion

Our study found that compared to HC, MIG subjectsshowed enhanced amplification of Sp5 activation andreduced habituation in the posterior insula.Amplification and habituation were correlated, suggest-ing a shared pathophysiology and highlighting theimportant role of the posterior insula for mechanisms

supporting altered sensory processing in episodicmigraine.

Amplification or increased gain for incoming affer-ence can occur at the level of the primary synapse in thespinal cord (or the brainstem for orofacial inputs),higher up in the cortical or subcortical regions, orboth. Thus, for migraine, while amplification can existat the peripheral trigeminal nerve receptor, trigeminalganglion, or Sp5 in the pontomedullary junction, wespecifically investigated amplification from activationoccurring at Sp5 up to higher order regions in thebrain. Compared to HC, MIG demonstrated amplifica-tion in two regions: pINS (significant in left/contralat-eral and trending in right pINS) and the hypothalamus.The posterior insula cortex, which receives Sp5 affer-ence via the thalamus, is an important region for pain

Hyp

(a)

(b)

R

Obex +17 mm

pINS

PAG

Caud SMG

10–6

10–4

10–2

1

+

Z= 21 mmZ= 18 mm

SII

Z= 36 mm

R

Z= –7 mm Z= –2 mm Z= 10 mm p-value

Put

Sp5 Sp5

Figure 2. Brainstem response to somatosensory stimulation. (a) Activation was found in the bilateral spinal trigeminal nuclei (Sp5),consistent with the Duvernoy brainstem atlas (a, left) (30), and overlaid on the MNI-space template (A, middle), rotated for consistencywith the Duvernoy brainstem atlas. The right panel of (a) shows the level of the axial slice in green (Obex þ 17 mm). (b) Group map ofthe cortical/subcortical/midbrain response to innocuous air-puff stimulation from all subjects also showed significant activation incortical areas (the bilateral posterior insula, pINS; the bilateral secondary somatosensory cortex, SII; and the bilateral supramarginalgyrus, SMG), in subcortical regions (hypothalamus, Hyp; right putamen, Put; and right caudate nucleus, Caud), and in a midbrain region(periaqueductal gray, PAG). Both group maps, (a) and (b), were cluster-corrected for multiple comparisons (Z¼ 2.3, p< 0.05).

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and interoceptive processing (32,33), and has beenlinked with migraine pathophysiology (34). However,this region also responds to innocuous somatosensorystimulation, as evident in our study and many others,and invasive intracortical stimulation of pINS com-monly produces generalized somatosensory (e.g. tin-gling, vibrating, numbness, warmth) and, when moredorsal, even painful sensations (35). Future studiesshould evaluate if greater amplification of innocuoussomatosensory input to pINS in both ictal and inter-ictal states of episodic migraine are linked to allodyniain these subjects. However, we should note that MIGdid not rate the air-puff stimulation as painful, support-ing the contention that interictal episodic migrainepatients may not show allodynia (36).

The hypothalamus also demonstrated amplification,relative to Sp5 activity, in MIG. The importance of thehypothalamus in migraine pathophysiology has beenpreviously reviewed (37), and an altered response inthis area is consistent with commonly reported, non-pain symptoms of migraine, including disturbances insleep, arousal, and homeostatic/autonomic functioning.Specifically, this region has been hypothesized to playan important role as a trigger of migraine attacks, par-ticularly in patients who can sense oncoming attacksfrom altered arousal and fatigue (38). Moreover,increased excitation (or decreased inhibition) of the

hypothalamus (39,40) has been reported to triggermigraine attacks, and significant hypothalamic activa-tion has been observed during migraine episodes andmaintained in the postictal state (41). Thus, amplifica-tion of sensory stimuli in the hypothalamus may pro-duce alterations of autonomic and other homeostaticfunctions associated with oncoming attacks.

Reduced habituation has been noted as one of themost consistently reported interictal neurophysiologicalphenomena in episodic migraine (8). Habituation isdefined as a diminished response to repeated stimula-tion and is noted in both behavioral and neurophysio-logical domains (5). It is thought to be protective ofexcessive information processing and energy consump-tion (6). Our study demonstrated clear cortical (e.g. SII,pINS, SMG) and even subcortical (putamen) habitu-ation to innocuous somatosensory stimuli in HC. Incontrast, for MIG, habituation was noted only in bilat-eral SII, which is known to process physical aspects ofsomatosensory stimuli (42,43). Additionally, a directcontrast found a significantly different (less negative)habituation slope in pINS, highlighting the reducedhabituation of activation in this brain region.Stankewitz et al. also noted a lack of fMRI activityhabituation for episodic migraine in response to nasaltrigemino-nociceptive (ammonia) stimuli, which wasfound in the anterior insula and anterior mid-cingulate

Table 2. Brain response to air-puff stimulation.

Location (MNI, mm) Z-scoreP-value(HC vs.MIG)Side

Size(mm3) X Y Z

Obex(mm) Z-score HC MIG

Cortical areasPosterior/middle

insula (pINS)L 14152 )36 )28 10 6.09 2.49! 2.05 3.04! 1.96 0.21

R 10872 42 )20 6 6.00 2.59! 2.01 3.42! 1.93 0.24

Secondary somatosensorycortex (SII)

L 14152 )64 )22 22 4.58 1.39! 1.28 1.48! 1.87 0.87

R 10872 58 )28 14 4.62 2.60! 2.70 3.07! 3.25 0.66

Supramarginalgyrus (SMG)

L 14152 )64 )36 34 4.35 1.07! 1.03 0.84! 1.13 0.56

R 10872 62 )40 38 3.71 1.15! 1.32 1.12! 1.67 0.95

Subcortical areasHypothalamus (Hyp) L 408 )2 )4 )8 4.26 1.10! 0.98 1.02! 1.39 0.84

Caudate nucleus (Caud) R 272 20 )4 22 3.50 0.84! 1.16 0.72! 1.10 0.77

Putamen (Put) R 136 24 0 10 2.96 0.44! 1.17 0.38! 0.84 0.87

BrainstemSpinal trigeminal

nucleus (Sp5)L 88 )10 )34 )42 þ19 3.37 0.70! 1.14 0.62! 0.94 0.83

R 160 12 )36 )40 þ21 3.00 0.56! 1.12 0.60! 0.96 0.92

Pontine nucleus L 8 )12 )26 )44 þ17 2.47 0.26! 0.91 0.85! 1.16 0.12

R 72 10 )20 )44 þ17 3.47 1.02! 1.48 0.69! 1.03 0.47

Periaqueductal gray (PAG) – 184 0 )32 )12 þ49 3.81 0.78! 0.77 0.73! 1.20 0.88

Data are shown as mean! SD. HC¼Healthy controls; MIG¼Migraine patients; L¼ Left side (contralateral to stimulation); R¼Right side (ipsilateralto stimulation). Distance from obex was estimated after rotating the brainstem image into the upright position.

Lee et al. 7

cortices (14). However, in the same study, migraine sub-jects demonstrated marked habituation to an innocu-ous olfactory stimulus. As our study found reducedhabituation for an innocuous somatosensory trigeminalstimulus, this effect may be modality-dependent.Furthermore, in our study the habituation slope wascorrelated with the amplification ratio in pINS forMIG, i.e. greater amplification was directly associatedwith reduced habituation. These results highlight thealtered stimulus-response physiology for pINS duringthe interictal phase of migraine.

Our study found significant activation in response totrigeminal nerve somatosensory stimulation in bilateralSp5 (oralis subnucleus). Interestingly, Stankweitz et al.(20) demonstrated a positive correlation between Sp5activity and time to next ictal event, suggesting that Sp5response to nociceptive stimulation may be an import-ant predictor of an upcoming migraine attack. In ourstudy, we similarly observed a positive correlationbetween Sp5 activation and the interictal phase,

though not specifically with time to subsequent attack(r¼)0.33, p¼ 0.21). While the two parameters differ inthat the interictal phase is a relative measure, andrelated to individual patients’ attack-to-attack cycles,the relationship with Sp5 activity was similar, suchthat activation is higher when the next attack is immi-nent. Moreover, our results suggest that the associationbetween Sp5 activation and subsequent attack onset isnot only for nociceptive processing, but also extends toinnocuous somatosensory processing in Sp5. Also,bilateral Sp5 activation may have been due to the spa-tially broad distribution of our air-puff stimuli and theclose proximity of the air-puff target region to the facemidline, perhaps leading to stimulation of cutaneousreceptors on the opposite side of the facial midline.

In addition to Sp5, we also observed significantsignal increase in more anterior pontine nuclei, andhigher cortical and subcortical regions including basalganglia areas such as the caudate and putamen.The basal ganglia and cortical regions such pINS, SII,

Brain amplification of Sp5 activation

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S /

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Hyp

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p5 a

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4

(a)

(b)

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1.5

1

0.5

0

HC MIG

HC MIG

Figure 3. Cortical/subcortical amplification of Sp5 activation. Migraine patients showed a greater cortical (i.e. posterior insula, pINS;a) and subcortical (i.e. hypothalamus, Hyp; b) amplification ratio, relative to Sp5 activation. n.b. *p< 0.05, error bars denoted thestandard error of the mean.

8 Cephalalgia 0(0)

Tab

le3.

Am

plifi

cation

of

Sp5

activa

tion

and

habi

tuat

ion

tooph

thal

mic

nerv

eso

mat

ose

nsory

stim

ulat

ion.

Side

Am

plifi

cation

ratio

P-va

lue

(HC

vs.

MIG

)

HC

(lin

ear

fit)

MIG

(lin

ear

fit)

P-va

lue

(HC

vs.

MIG

)H

CM

IGSl

ope

r(P

)Sl

ope

r(P

)

Cort

ical

area

s

Post

erio

r/m

iddle

insu

la(p

INS)

L1.

02!

0.80

2.99!

0.50

<0.0

5)

0.0

3!

0.0

1*

)0.7

0(<

0.0

5)

)0.

02!

0.01

)0.

53(<

0.01

)0.

36

R1.

14!

0.83

3.02!

0.62

0.06

)0.0

5!

0.0

1**

)0.8

5(<

0.0

01)

)0.

00!

0.01

)0.

11(0

.76)

<0.0

5

Seco

ndar

yso

mat

ose

nsory

cort

ex(S

II)L

0.83!

0.72

0.47!

0.52

0.52

)0.0

8!

0.0

1**

)0.9

2(<

0.0

001))

0.0

6!

0.0

1**

**)

0.7

8(<

0.0

1)

0.55

R1.

85!

1.35

3.16!

1.94

0.23

)0.0

9!

0.0

2**

*)

0.9

3(<

0.0

001))

0.0

7!

0.0

2**

)0.8

5(<

0.0

01)

0.38

Supr

amar

gina

lgy

rus

(SM

G)

L0.

51!

0.38

0.26!

0.49

0.53

)0.0

5!

0.0

2*

)0.7

6(<

0.0

1)

)0.0

5!

0.0

2*

)0.6

3(<

0.0

5)

0.81

R0.

77!

0.56

1.04!

0.87

0.56

)0.0

6!

0.0

2*

)0.6

9(<

0.0

5)

)0.0

8!

0.0

2**

)0.7

7(<

0.0

1)

0.55

Subc

ort

ical

area

s

Hyp

oth

alam

us(H

yp)

L0.

93!

0.26

1.84!

0.40

<0.0

50.

00!

0.01

0.02

(0.9

4))

0.02!

0.01

)0.

28(0

.40)

0.34

Cau

dat

enu

cleu

s(C

aud)

R0.

59!

0.18

0.43!

0.19

0.47

)0.

01!

0.01

)0.

44(0

.18)

)0.

01!

0.01

)0.

33(0

.32)

0.99

Put

amen

(Put

)R

0.30!

0.23

0.20!

0.14

0.49

)0.0

3!

0.0

1*

)0.6

2(<

0.0

5)

)0.

02!

0.01

)0.

52(0

.10)

0.71

Bra

inst

em

Spin

altr

igem

inal

nucl

eus

(Sp5

)L

n/a

n/a

n/a

0.01!

0.02

0.45

(0.1

7)0.

00!

0.01

)0.

04(0

.90)

0.57

Rn/

an/

an/

a0.

00!

0.01

)0.

08(0

.81)

)0.

01!

0.01

)0.

29(0

.38)

0.79

Pont

ine

nucl

eus

Ln/

an/

an/

a0.

02!

0.02

0.34

(0.3

1)0.

01!

0.01

0.15

(0.6

5)0.

75

Rn/

an/

an/

a0.

02!

0.02

0.27

(0.4

2)0.

01!

0.01

0.19

(0.5

8)0.

64

Peri

aque

duc

talgr

ay(P

AG

)–

n/a

n/a

n/a

0.00!

0.01

0.02

(0.9

6))

0.01!

0.01

)0.

39(0

.24)

0.50

Dat

aar

esh

ow

nas

mea

n!

SEM

.H

abitua

tion

(bo

ldit

alici

zed)

def

ined

bya

nega

tive

slope

sign

ifica

ntly

diff

eren

tfr

om

nil,

and

sign

ifica

ntlin

ear

fit(r

-coef

ficie

nt).

HC¼

Hea

lthy

cont

rols

;M

IG¼

Mig

rain

epa

tien

ts;L¼

Left

side

(cont

rala

tera

lto

stim

ulat

ion)

;R¼

Rig

htsi

de

(ips

ilate

ralto

stim

ulat

ion)

;Sl

ope

:ch

ange

inac

tiva

tion

(%ch

ange

)pe

rst

imul

usbl

ock

;r¼

corr

elat

ion

coef

ficie

nt.H

abitua

tion

slope

isdiff

eren

tfr

om

nil:

*p<

0.05

,**

p<

0.01

,**

*p<

0.00

1,**

**p<

0.00

01.

Lee et al. 9

Habituation associated with amplification of Sp5 activation

Hab

ituat

ion

slop

e (p

INS

)H

abitu

atio

n sl

ope

(SII)

Amplification ratio (pINS/Sp5)

Amplification ratio (SII/Sp5)

0.1

(a)

(b)

0.05

0.15

0.1

0.05

0

–0.05

–0.1

–0.15

–0.2

–0.25

–0.3

0

R

R

Z= –2 mm

Z= 18 mm

0 5

r = 0.54, p = 0.03

r = 0.58, p = 0.02

10

–0.05

50 10

–0.1

Figure 5. Association between the cortical amplification ratio and the habituation slope. Greater amplification was correlated withimpaired habituation (a less negative habituation slope) in (a) the posterior insula and (b) the secondary somatosensory cortex.

Habituation to repeated trigeminal nerve stimulation

pIN

S a

ctiv

atio

n (%

-cha

nge) HC, slope= –0.05 (r = –0.58**)0.7

0.8

0.6

0.5

0.4

0.3

0.2HC

MIG

1 3 5Stimulus block

7 9 11

R

Z= –2 mm

0.1

0.0

Figure 4. Habituation to repeated stimulus blocks for regions activated by innocuous somatosensory stimulation. For healthycontrols (HC), habituation was noted for multiple activated cortical/subcortical regions, including the posterior insula (pINS). Incontrast, migraine (MIG) subjects demonstrated a significantly (p< 0.05) reduced linear habituation slope in the posterior insula. n.b.**p< 0.01, significant linear fit across blocks for healthy controls.

10 Cephalalgia 0(0)

and SMG are known to commonly activate in responseto painful and non-painful somatosensory stimulation(43–46), consistent with our innocuous air-puff stimu-lus. Basal ganglia areas, including the caudate andputamen, have been previously noted to show reducedactivation and gray matter volume in migraine patientswith more frequent attacks (46), though in our study,activation in these regions was not associated with theinterictal phase.

Several limitations in this study need to be men-tioned. Firstly, our time-variant analysis assessedlinear habituation. While other non-linear responsepatterns may indeed have occurred, it is important tonote that a linear contrast is still sensitive to non-lineartime-variant responses. Moreover, a linear contrast hasthe advantage of easier interpretability. Further, whileclinical measures did not differ between aura and non-aura MIG subgroups, our sample size did not allow fordirect comparisons of fMRI measures between thesesubgroups. As another limitation, non-noxious tactilestimuli typically activate Sp5-oralis and the main sen-sory nucleus of the trigeminal sensory complex. In ourstudy, the lack of activation in the main sensory nucleusmay have been due to the small cross-sectional area ofthis nucleus, and future studies using ultrahigh field(e.g. 7T) fMRI, with better SNR and spatial resolution,should be performed.

In conclusion, our study identified amplificationof the cortical and subcortical response, relative tobrainstem Sp5 activity, for somatosensory ophthalmicnerve stimulation. Furthermore, we linked this amplifi-cation to reduced habituation in several corticalbrain regions. Specifically, amplification was foundin the hypothalamus, and the posterior insulademonstrated correlated amplification and reducedhabituation – i.e. greater amplification was associatedwith reduced habituation. Our study thus highlights theimportant role of the posterior insula in mechanismssupporting altered sensory processing in episodicmigraine.

Article highlights

. Migraine subjects showed greater amplification of trigeminal tactile information transferred from the brain-stem to the posterior insula cortex.

. Migraine subjects also demonstrated significantly reduced habituation, which was correlated to greateramplification, in the posterior insula, a key region for sensory processing in episodic migraine.

. Neuronal amplification is evident within the central nervous system in migraine.

Acknowledgements

We would like to thank Helen S. Xu and Yazhuo Kong fortheir helpful comments on data analysis and Hanhee Jung forassistance in recruitment and scanning.

Declaration of conflicting interests

The authors declared no potential conflicts of interest withrespect to the research, authorship, and/or publication of thisarticle.

Funding

The authors disclosed receipt of the following financialsupport for the research, authorship, and/or publication of

this article: This work was supported by the NationalInstitutes of Health, National Center for Complementary andIntegrative Health [R01-AT007550, P01-AT006663]; NationalInstitute of Mental Health [R21-MH103468]; NationalInstitute of Arthritis and Musculoskeletal and Skin Diseases[R01-AR064367]; National Institute of Neurological Disordersand Stroke [R21-NS087472, R21-NS082926]; the NationalCenter for Research Resources [P41RR14075]; and theColombian Department of Science, Technology andInnovation [COLCIENCIAS, Grant No. 656664239871].

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0.25

Sp5 activation depends on interictal phase

Interictal phase

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Act

ivat

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(%-c

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0.1

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r = 0.58, p < 0.05

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