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Autonomic Neuroscience: Basic and Clinical 168 (2012) 72–81

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Differential responses of the insular cortex gyri to autonomic challenges☆

Paul M. Macey a,b, Paula Wu c, Rajesh Kumar c, Jennifer A. Ogren b, Heidi L. Richardson c,Mary A. Woo a, Ronald M. Harper b,c,⁎a UCLA School of Nursing, University of California at Los Angeles, Los Angeles, CA 90095, USAb Brain Research Institute, David Geffen School of Medicine at UCLA, University of California at Los Angeles, Los Angeles, CA 90095, USAc Department of Neurobiology, David Geffen School of Medicine at UCLA, University of California at Los Angeles, Los Angeles, CA 90095, USA

☆ Grants: financial support was provided by the Nat011230 (PMM), HD-22695 (RMH) and NR-009116 (MA⁎ Corresponding author at: Distinguished Professor o

Neurobiology, David Geffen School of Medicine at UCLAAngeles, Los Angeles, CA 90095-1763, USA. Tel.: +1 312224.

E-mail address: rharper@ucla.edu (R.M. Harper).

1566-0702/$ – see front matter © 2012 Published by Eldoi:10.1016/j.autneu.2012.01.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 November 2011Received in revised form 15 January 2012Accepted 24 January 2012

Keywords:Functional magnetic resonance imagingAutonomic nervous systemFunctional neuroanatomyValsalva maneuverHand gripCold pressor

Determining insular functional topography is essential for assessing autonomic consequences of neural inju-ry. We examined that topography in the five major insular cortex gyri to three autonomic challenges, theValsalva, hand grip, and foot cold pressor, using functional magnetic resonance imaging (fMRI) procedures.Fifty-seven healthy subjects (age±std: 47±9 years) performed four 18 s Valsalva maneuvers (30 mm Hgload pressure), four hand grip challenges (16 s at 80% effort), and a foot cold pressor (60 s, 4 °C), with fMRIscans recorded every 2 s. Signal trends were compared across gyri using repeated measures ANOVA. Signifi-cantly (Pb0.05) higher signals in left anterior versus posterior gyri appeared during Valsalva strain, and inthe first 4 s of recovery. The right anterior gyri showed sustained higher signals up to 2 s post-challenge, rel-ative to posterior gyri, with sub-gyral differentiation. Left anterior gyri signals were higher than posteriorareas during the hand grip challenge. All right anterior gyri showed increased signals over posterior up to12 s post-challenge, with decline in the most-anterior gyrus from 10 to 24 s during recovery. The left threeanterior gyri showed relatively lower signals only during the 90 s recovery of the cold pressor, while thetwo most-anterior right gyri signals increased only during the stimulus. More-differentiated representationof autonomic signals appear in the anterior right insula for the Valsalva maneuver, a bilateral, more-posterior signal representation for hand grip, and preferentially right-sided, anterior–posterior representa-tion for the cold pressor. The functional organization of the insular cortex is gyri-specific to unique autonomicchallenges.

© 2012 Published by Elsevier B.V.

1. Introduction

The insular cortex serves autonomic regulatory functions via pro-jections to visceral, thalamic, brainstem and limbic areas, as indicatedby rodent, primate and human studies (Mufson and Mesulam, 1984;Allen et al., 1991; Yasui et al., 1991; Cerliani et al., 2011; Jakab etal.). The insula receives input from multiple cortical areas involvedwith emotional, cognitive, and sensori-motor functions, many ofwhich can trigger sympathetic and parasympathetic changes. Howev-er, the topographic organization for autonomic regulation of thehuman insula has only partially been outlined. An anterior–posteriororganization for certain aspects of autonomic regulation is presentboth in rodents and humans (Oppenheimer and Cechetto, 1990;Yasui et al., 1991; Goswami et al., 2011), but functional boundaries

ional Institutes of Health, NR-W).f Neurobiology, Department of, University of California at Los0 825 5303; fax: +1 310 825

sevier B.V.

in humans remain unclear. The human insular cortex shows consis-tent gyral subdivisions, and the anatomical and cellular distinctionsof these subregions suggest that the functional neuroanatomy of au-tonomic control may also differ across gyri (Ture et al., 1999;Naidich et al., 2004; Afif et al., 2008; Anderson et al., 2009). The pre-sent study addresses the question of whether these gyri show distinctfunctional roles to different autonomic challenges.

The human insular cortex usually contains five major gyri, withadditional, smaller gyral areas in some subjects (Ture et al., 1999;Naidich et al., 2004). The major gyri lie in a dorsal–ventral orienta-tion, with three anterior gyri referred to as the “short” gyri, andtwo posterior gyri termed the “long” gyri. The short gyri consist ofthe “anterior,” “mid,” and “posterior” short gyri (ASG, MSG andPSG, respectively; Fig. 1). A small minority of people do not have anMSG. Two additional minor gyri lie within the anterior insula, name-ly the transverse and accessory insular gyri. All five anterior gyriconverge at the insular “apex,” the most-ventral portion of thesuperficial insular cortex (Ture et al., 1999; Naidich et al., 2004).The long gyri consist of the anterior and posterior long insular gyri(ALG and PLG, respectively). Both the cellular anatomy and connec-tivity differ across insular gyri (Jacobs et al., 2001; Elston, 2002;Elston and Rockland, 2002; Elston, 2003; Elston et al., 2005;

Fig. 1. Subregions of the anterior and posterior insular cortex. The PLG, ALG, PSG, MSG,and ASG are shown overlaid on the average of 57 healthy control subjects' anatomicalscans (normalized to MNI space, left side, sagittal slice at−38 mm). Gyri: ASG=anter-ior short gyrus, MSG=mid short gyrus, PSG=posterior short gyrus, ALG=anteriorlong gyrus, PLG=posterior long gyrus.

73P.M. Macey et al. / Autonomic Neuroscience: Basic and Clinical 168 (2012) 72–81

Anderson et al., 2009), supporting the concept of functional distinc-tions across these subregions.

Imaging and stroke studies in humans confirm lateralized and an-terior–posterior functional organization in the insular cortices. Theright anterior insular cortex, which encompasses the three shortgyri, shows functional magnetic resonance imaging (fMRI) signal in-creases to autonomic challenges (King et al., 1999; Wong et al.,2007). Other functions also show localization; a somatotopic organi-zation for sensory processing exists over the insula with projectionsto neighboring areas (Bjornsdotter et al., 2009; Mazzola et al.,2009), and some forms of pain representation lie principally withinMSG (Afif et al., 2008), although the dorsal posterior insula is respon-sive to cold and other temperature stimuli, with a somatotopic repre-sentation (Craig et al., 2000; Maihofner et al., 2002; Brooks et al.,2005; Hua le et al., 2005; Henderson et al., 2007). However, thesehuman studies have not resolved the topography of function to thelevel of gyri.

Refining our knowledge of the functional organization within theinsula would benefit understanding of autonomic control circuitry,as well as help with clinical interpretation of the consequences ofdamage in insular subregions. Initially, the pathology of principal con-cern was stroke (Oppenheimer et al., 1992; Oppenheimer, 1993;Oppenheimer et al., 1996), but the revelation of significant insular in-jury in heart failure (Woo et al., 2003) and obstructive sleep apnea(Macey et al., 2008) mandate clarification of topographical organiza-tion of function. Our objective was to evaluate differences in neuralresponses across the gyri of the left and right insular cortices to auto-nomic stimuli, the Valsalva maneuver, hand grip, and cold pressor(Mancia et al., 1978; Victor et al., 1987; Denq et al., 1998), usingfMRI. These three autonomic challenges elicit autonomic responsesin conjunction with differing sensory stimuli and volitional action.The Valsalva maneuver consists of both a sympathetic and para-sympathetic component, whereas the hand grip is a voluntarily-initiated, non-painful sympathetic challenge, and the cold pressorchallenge is a sensory-initiated sympathetic challenge with a moder-ate pain component.

2. Methods

2.1. Subjects

We studied 57 healthy adults (age±std: 47.0±9.1 years, range:31–66 years; 37 males, 20 females). Subjects did not have a historyof cerebrovascular disease, myocardial infarctions, heart failure, neu-rological disorders, or mental illness, and were not taking cardiovas-cular or psychotropic medications. Subjects were recruited from the

general Los Angeles area, and did not weigh more than 125 kg orhave any metallic or electronic implants; the latter two issues areMRI scanner contraindications. All subjects provided informed con-sent in writing, and the research protocol was approved by the Insti-tutional Review Board of UCLA.

2.2. Valsalva maneuver

The Valsalva maneuver, a common test of autonomic function(Taylor, 1996), was performed in a sequence of four 18 s exhalationsagainst a closed glottis, spaced one minute apart, incurring a targetintra-thoracic pressure of 30 mm Hg (Taylor, 1996). The challengeelicits a sequence of sympathetic and parasympathetic responses,which are accompanied by a pattern of heart rate and blood pressurechanges corresponding to adaptations to thoracic and cardiovascularsequelae introduced by the maneuver (Taylor, 1996; Denq et al.,1998). A light signal was used to indicate onset of the challenge forthe Valsalva effort to the subject. Upon seeing the light signal, sub-jects were instructed to take a breath and exhale against a resistance,maintaining a target pressure. A second light was illuminated whenthe subject achieved 30 mm Hg pressure. Subjects practiced theValsalva maneuver prior to scanning.

2.3. Hand grip

The static hand grip is a voluntarily-initiated autonomic challengethat elicits increased heart rate andmuscle sympathetic nerve activity(Mancia et al., 1978; Mark et al., 1985). The test involved squeezingan MR-compatible pressurized bag connected to a pressure sensorwith instructions to maintain a short (16 s) test at a subjective 80%of maximum grip strength. Four repeated tests were performed,with an initial baseline of 100 s preceding the first test, and subse-quent hand grip periods separated by 1 min, and terminated with a100 s recovery period. Subjects practiced the hand grip prior toscanning.

2.4. Cold pressor

The cold pressor challenge involved immersing the right foot up tothe ankle in cold water (4 °C) for a 1 min period, preceded by a 2 minbaseline, and followed by a 2 min recovery period. Two investigatorslifted the foot into a basin with cold water that covered the ankle atthe start of the challenge, and removed it 60 s later. Each movementlasted less than 4 s (2 fMRI volumes), and signal artifact was presentduring periods of movement. The task elicits an increase in sympa-thetic activity and blood pressure, and pain if near-freezing water isused (Victor et al., 1987). The subjects verbally reported moderatelevels of pain at the 4 °C temperature, although there was variationin subjects' perceptions.

2.5. Physiologic Signals

Cardiac, load pressure and indicator signals (e.g., light on/off)were recorded with an analog-to-digital acquisition system (instru-Net INET-100B, GWI Instruments, Inc., Somerville, MA). Heart ratewas assessed using an MRI-compatible pulse oximeter (Nonin MedicalInc., Plymouth, MN). The sensor was placed on the right index fingerthroughout the scan, and heart rate was calculated from the raw oxim-etry signal acquired at 1 kHz using custom peak-detection softwarefollowed by expert review. Expiratory pressure was measured via tub-ing connected to a pressure sensor (Omega Engineering Inc., Stamford,CT) outside the scanner. Patient cue signals were simultaneouslyrecorded. Signals were aligned to the MRI scans, and data correspond-ing to the fMRI signals extracted.

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2.6. MRI scanning

Functional MRI scans were acquired using a 3.0 T scanner(Siemens Magneton Tim-Trio, Erlangen, Germany) while subjectslay supine. A foam pad was placed on either side of the head to mini-mize movement. We collected whole-brain images with the blood-oxygen level dependent (BOLD) contrast every two seconds (repetitiontime [TR]=2000 ms; echo time [TE]=30ms; flip angle=90º; matrixsize 64×64; field-of-view 220×220 mm; slice thickness=4.5 mm).The spatial resolution was based on achieving whole-brain coveragewith the fastest possible acquisition time. Two high resolution, T1-weighted anatomical images were also acquired with a magnetizationprepared rapid acquisition gradient echo sequence (TR=2200 ms;TE=2.2 ms; inversion time=900 ms; flip angle=9º; matrix size256×256; field-of-view 230×230 mm; slice thickness=1.0 mm).Field map data consisting of phase andmagnitude images were collect-ed using the standard Siemens protocol, to allow for correction ofdistortions due to field inhomogeneities.

2.7. MRI data preprocessing

All anatomical scans were inspected to ensure the absence of vis-ible pathology. For each fMRI series, the global signal was calculatedand the images realigned to account for head motion. Subjects withlarge changes in global BOLD signal, or who moved more than4 mm in any direction were not included in the study. Each fMRI se-ries was linearly detrended to account for signal drift (but not globaleffects), and corrected for field inhomogeneities (Hutton et al., 2002),spatially normalized, and smoothed (8 mm Gaussian filter), andmean time trends from each voxel were calculated across all subjects,as well as the challenge-means across each of the four Valsalva andhand grip periods (the cold pressor only had one repeat and so wasnot averaged). A mean image of all subjects' spatially normalized, an-atomic scans was created. Software used included the statistical para-metric mapping package, SPM8 (Wellcome Department of CognitiveNeurology, UK; www.fil.ion.ucl.ac.uk/spm), MRIcron (Rorden et al.,2007), and MATLAB-based custom software.

2.8. Region of interest (ROI) tracing

The five major gyral regions in the insular cortex, the ASG, MSG,PSG, ALG and PLG, were outlined on the mean anatomical imagewith MRIcron software, using the anatomical descriptions by Ture etal. (1999) and Naidich et al. (2004), as shown in Fig. 1. Individualtracing would be more accurate for identifying the gyral differentia-tion on anatomical scans. However, the fMRI data are at a muchlower spatial resolution (voxel volume of 53 mm3 versus 0.8 mm3

for the anatomical scans), and the BOLD effect itself, which is thebasis for assessing neuronal responses, is diffuse, so the advantageof individual tracing would be minimal. The three main gyri of the an-terior insula, the ASG, MSG, and PSG, make up the convex surface ofthe structure, and are visible on the sagittal and axial views of themean anatomical image. The accessory and transverse gyri, twoother gyri in the anterior insula, are difficult to visualize (Naidich etal., 2004), and were not visible on the mean anatomical image.Thus, in our tracing of the ASG, we included the entire most-anterior portion of the insula, which incorporated the accessory andtransverse gyri. The posterior gyri (ALG and PLG) were easily visibleon sagittal as well as axial sections of the anatomical volume.

2.9. Statistical analysis

Repeated measures ANOVA (RMANOVA), implemented with themixed linear model procedure “proc mixed” in SAS software (Littellet al., 1996), was used to identify periods of significant responserelative to baseline, during the Valsalva, hand grip, and cold pressor

periods and subsequent recovery. We modeled the fMRI responsesas a function of scan period. Significance was first assessed at theglobal level (Pb0.05), as per the Tukey–Fisher criterion for multiplecomparisons; for significant models, the time-points of significant re-sponses were identified. To avoid potential confounds due to globalvascular effects, we focused on relative changes between gyri, andpresent graphical visualizations with respect to the PLG, as the poste-rior insula typically responds less than anterior regions in response toautonomic stimuli (King et al., 1999). The restriction of only assessingdifferences rather than absolute responses results from the inherentrelative nature of the BOLD-based fMRI technique.

3. Results

3.1. Heart rate responses

The heart rate patterns to the different challenges are shown inFig. 2, with heart rate showing significant changes during and afterall three tasks (RMANOVA, Pb0.05; time-points of significant changerelative to baseline indicated above right-hand graphs in Fig. 2). Tran-sient heart rate increases appeared in the initial phase of the Valsalvamaneuver, followed by a steady rise in the second phase, then a thirdtransient increase upon release of strain, followed by a decline belowbaseline, and a slow return to baseline. The hand grip elicited an ini-tial heart rate spike, peaking at 5 s into the task, followed by a sus-tained elevated heart rate, which upon release, gradually returnedto baseline. The cold pressor response showed a sustained heartrate increase, with an undershoot and gradual return to baseline fol-lowing removal of the cold stimulus.

3.2. fMRI responses

All three challenges elicited fMRI signal responses that differed be-tween gyri (Figs. 3–5; mean values in Tables 1–3 of the supplementa-ry materials). Subject movement rarely exceeded 1 mm in anydirection, and the few larger shifts (2–3 mm range) were always tran-sient, i.e., one or two volumes only. The signal differences in the topgraphs of each figure illustrate a general pattern of higher activity inthe anterior, relative to the posterior gyri. Note that the lower panelsreflect the raw signal, which is a combination of regional and globaleffects, the latter consisting primarily of changes in cerebral bloodvolume. The upper panels can be assumed to reflect differences onlyin regional effects, and therefore, indirectly, neuronal activity. Thus,the upper panels reflect gyral differentiation, and the lower panels re-flect the magnitude of regional responses superimposed on changesin cerebral blood volume. Left and right differences are shown forthe only non-lateralized challenge, the Valsalva maneuver (Fig. 6). Asynthesis of the major findings is shown in Table 1, with in-depthdescriptions below.

3.2.1. Valsalva maneuverSignificant fMRI signal differences emerged between gyri in both

left and right insulae during the second phase of the Valsalva(Fig. 3). In the left insula, the ASG showed an elevated response rela-tive to other gyri throughout the strain period (time 4–18 s), with theMSG also showing a sustained, higher response relative to more pos-terior gyri, as did the PSG, relative to the ALG and PLG (e.g., at 16 sinto the challenge, ASG>MSG by 0.27%, MSG>PSG by 0.14%, andPSG>PLG by 0.36%; Table 1 of Supplementary material). Upon re-lease (Phase III), the ALG showed a briefly higher response than thePLG (20 s), followed by a return toward baseline by all gyri. Theright insula showed matching responses in the ASG and MSG, whichwere also similar to those in the PSG. All right short gyri showedsustained higher signals relative to the long gyri.

Fig. 2. Physiologic responses to autonomic challenges. Heart rate responses to the autonomic challenges are shown across the series and averaged over repeated challenges(Valsalva and hand grip) with time-points of significant deviations from baseline denoted above the right-hand traces (RMANOVA, Pb0.05; key at top). Gray shading denotes challengeperiods. Mean heart rates are shown for each challenge, and for the Valsalva maneuver, expiratory pressure is also shown, and for hand grip pressure, change from rest is shown.

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Signals in all gyri were greater on the right than the left during thestrain phase (Fig. 6, which reflects left–right differences between rawsignals as shown in the lower panels of Fig. 3). The right ASG showeda transient higher signal at onset of the maneuver (Phase I, time

0–4 s), whereas responses in all other gyri were higher throughoutthe later strain period (phase II, time 6–18 s), with peak differencesat 8 s (PSG, 0.26% higher on right; PLG, 0.31% higher) and 10 s(MSG, 0.32% higher; ALG, 0.29% higher).

Fig. 3. fMRI signal responses across insular cortex gyri, averaged over four 18-second Valsalva maneuvers in 57 subjects. The top graphs illustrate signal differences between gyri,relative to the PLG, and indicate inter-gyral differences in neural responses. The middle section illustrates time-points of statistically significant differences between gyri (Pb0.05,RMANOVA), and the bottom graph illustrates the raw signals relative to baseline, which reflect a combination of regional and global effects (the global raw signal patterns relateprimarily to changes in cerebral blood volume). Mean values are reported in Table 1 of the Supplementary material. All traces are mean±SE. Gyri: ASG=anterior short gyrus,MSG=mid short gyrus, PSG=posterior short gyrus, ALG=anterior long gyrus, PLG=posterior long gyrus.fMRI signal responses across insular cortex gyri, averaged over four 18-secondValsalvamaneuvers in 57 subjects. The top graphs illustrate signal differences between gyri, relative to the PLG, and indicate inter-gyral differences inneural responses. Themiddlesection illustrates time-points of statistically significant differences between gyri (Pb0.05, RMANOVA), and the bottom graph illustrates the raw signals relative to baseline,which reflect acombination of regional and global effects (the global raw signal patterns relate primarily to changes in cerebral blood volume). Mean values are reported in Table 1 of the Supplementarymaterial. All traces are mean±SE. Gyri: ASG=anterior short gyrus, MSG=mid short gyrus, PSG=posterior short gyrus, ALG=anterior long gyrus, PLG=posterior long gyrus.

76 P.M. Macey et al. / Autonomic Neuroscience: Basic and Clinical 168 (2012) 72–81

3.2.2. Hand grip challengeSignal differences appeared between gyri during and after the

hand grip challenge in the ipsilateral challenge (right) insula, but inthe left insula, differences in activation between gyri only appearedduring the challenge, and primarily during the initial 8 s (Fig. 4).The left insular responses showed distinct patterns in the short andlong gyri, with significant differences between the short gyri andthe long gyri, but no differences amongst short or long gyri. The

maximum relative amplitude difference was at 12 s after challengeonset between the MSG and PLG (MSG−PLG=0.24%). In the rightinsula, the short gyri each showed differences relative to the longgyri. However, unlike the left side, the MSG response was significantlyhigher than in the ASG, with the greatest amplitude difference at 8 s(MSG−ASG=0.17%). That time point also showed the greatest differ-ence between long and short gyri (MSG−PLG=0.29%, time=8 s). Theshort/long differences persisted for 12 s (time=28 s) after release, and

Fig. 4. fMRI signal responses across insular cortex gyri, averaged over four 16-second hand grip challenges in 57 subjects. The top graphs illustrate signal difference between gyri,relative to the PLG, and indicate gyral inter-differences in neural responses. The middle section illustrates time-points of statistically significant difference between gyri (Pb0.05,RMANOVA), and the bottom graph illustrates the raw signals relative to baseline, which reflect a combination of regional and global effects (the global raw signal patterns relateprimarily to changes in cerebral blood volume). Mean values are reported in Table 2 of the Supplementary material. All traces are mean±SE. Gyri: ASG=anterior short gyrus,MSG=mid short gyrus, PSG=posterior short gyrus, ALG=anterior long gyrus, PLG=posterior long gyrus.fMRI signal responses across insular cortex gyri, averaged over four16-second hand grip challenges in 57 subjects. The top graphs illustrate signal difference between gyri, relative to the PLG, and indicate gyral inter-differences in neural responses.The middle section illustrates time-points of statistically significant differences between gyri (Pb0.05, RMANOVA), and the bottom graph illustrates the raw signals relative to base-line, which reflect a combination of regional and global effects (the global raw signal patterns relate primarily to changes in cerebral blood volume). Mean values are reported inTable 2 of the Supplementary material. All traces are mean±SE. Gyri: ASG=anterior short gyrus, MSG=mid short gyrus, PSG=posterior short gyrus, ALG=anterior long gyrus,PLG=posterior long gyrus.

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the MSG−ASG differences continued until 24 s (time=40 s) after theend of the challenge, with the ASG showing an undershoot relative toall four other gyri.

3.2.3. Cold pressor challengeIn the ipsilateral-to-challenge (right) insula, BOLD fMRI differences

appeared between gyri solely during the foot cold pressor challenge,

while fMRI differences in the left insula appeared only after the chal-lenge (Fig. 5; Table 3 of Supplementary material). In the right side,the two anterior-most gyri (ASG andMSG) increased in a similar pat-tern, relative to the three more-posterior gyri (PSG, ALG and PLG).The maximum difference was at 14 s (ASG−ALG=0.51%), withmaximum differences between the ASG/MSG and other gyri ofapproximately 0.3% and higher throughout the challenge (time 4 s

Fig. 5. fMRI signal responses across insular cortex gyri, averaged over one 60 second cold pressor challenge in 57 subjects. The top graphs illustrate signal difference between gyri,relative to the PLG, and indicate inter-gyral differences in neural responses. The middle section illustrates time-points of statistically significant difference between gyri (Pb0.05,RMANOVA), and the bottom graph illustrates the raw signals relative to baseline, which reflect a combination of regional and global effects (the global raw signal patterns relateprimarily to changes in cerebral blood volume). Mean values are reported in Table 3 of the supplementary material. All traces are mean±SE. Gyri: ASG=anterior short gyrus,MSG=mid short gyrus, PSG=posterior short gyrus, ALG=anterior long gyrus, PLG=posterior long gyrus.fMRI signal responses across insular cortex gyri, averaged over one60 second cold pressor challenge in 57 subjects. The top graphs illustrate signal difference between gyri, relative to the PLG, and indicate inter-gyral differences in neural responses.The middle section illustrates time-points of statistically significant differences between gyri (Pb0.05, RMANOVA), and the bottom graph illustrates the raw signals relative to base-line, which reflect a combination of regional and global effects (the global raw signal patterns relate primarily to changes in cerebral blood volume). Mean values are reported inTable 3 of the supplementary material. All traces are mean±SE. Gyri: ASG=anterior short gyrus, MSG=mid short gyrus, PSG=posterior short gyrus, ALG=anterior long gyrus,PLG=posterior long gyrus.

78 P.M. Macey et al. / Autonomic Neuroscience: Basic and Clinical 168 (2012) 72–81

to 58 s). The left insula showed similar patterns across gyri duringthe cold pressor stimulus, with relative declines in the anterior rela-tive to the posterior gyri occurring up to 90 s after the challenge(time 150 s). From 56 s after the end of the cold pressor (time116 s), the MSG and PSG showed a significantly lower signal relativeto the other gyri. The greatest difference was 14 s after the removalof the stimulus (time=74 s, PLG−ASG=0.23%).

4. Discussion

The insular cortices showed unique responses to different auto-nomic challenges between anterior and posterior gyri, and betweenleft and right insulae, indicating that functional organization of pro-cessing related to autonomic challenges differs between gyri. The an-terior insular gyri were most responsive during the challenge periods

Fig. 6. Lateralization of Valsalva BOLD responses. The fMRI BOLD responses (mean±SE,N=57) to the Valsalva are shown on the right relative to the left side for each gyri,with all right gyri showing predominantly higher signals on the right over the left(Pb0.05, RMANOVA; “*”indicates time points of significant difference). These patternsreflect regional differences independent of global effects, and can be assumed to indi-cate neural activation. The ASG has transiently higher signals, whereas the other fourgyri showed sustained increases on right over left sides during the challenge period.All traces are mean±SE. Gyri: ASG=anterior short gyrus, MSG=mid short gyrus,PSG=posterior short gyrus, ALG=anterior long gyrus, PLG=posterior long gyrus.

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of all tasks, whereas the two posterior gyri (ALG and PLG) respondedsimilarly during all tasks, and always with similar, or lower magni-tude than the anterior gyral patterns. A remarkable characteristicwas the large transient onset and offset patterns to cold and Valsalvachallenges, principally in anterior gyri to cold, but in both anteriorand posterior gyri in Valsalva maneuvers. Signal patterns during

Table 1Simplified representation of direction of differences in gyral signal responses during and aftgyrus; LG: long gyri, including anterior long gyrus and posterior long gyrus.

Valsalva Recovery Hand grip

Task Task

Left ASG – ASG+MSG+PSG>MSG >LG>PSG>LG

Right ASG+MSG – MSG+PSG>PSG >ASG>LG >LG

recovery periods also help differentiate gyral responses. These find-ings are consistent with existing literature showing that the anteriorportion of the insular cortex principally serves sympathetic regula-tion; however, the data extend previous findings by demonstratingthat functional representation of signals to autonomic challengesdiffers across the individual insular gyri.

4.1. Valsalva

Responses in both the right and left insular cortices during thestrain phase of the Valsalva maneuver differed between gyri in ananterior–posterior order, with greater responses in the anterior gyri.This phase is characterized by a large and rapid increase in sympa-thetic activity, as reflected in the heart rate increase; thus, the findingconfirms the anterior location of autonomic control within the insula.However, while the right-sided anterior gyri (ASG, MSG and PSG)responded similarly, on the left activity was greatest in the ASG,followed by the MSG, which was greater than the PSG. Given the pref-erential left-sided parasympathetic dominance (Oppenheimer andCechetto, 1990), this finding suggests that sympathetic regulationon the left insula is greatest in the most-anterior gyri (ASG), whereasthe regulation is similar across all three anterior gyri on the right side.

4.2. Hand grip challenge

The strain phase of the hand grip challenge, which is associatedwith moderate sympathetic activity as reflected in the heart rate in-crease, elicited the greatest responses in the MSG and PSG. On theleft side, the ASG showed similar increases, whereas on the rightthe ASG increases were less than the MSG and PSG. The findings likelyreflect the motor as well as autonomic components of the task. In ameta-analysis of 1768 studies, including functional imaging experi-ments, motor function was predominately found in the PSG(Bamiou et al., 2003; Kurth et al., 2010), which is consistent withthe greater responses in the PSG over ASG in the present study.Since the sympathetic increase during hand grip is moderate (relativeto during the Valsalva maneuver), contributions to the ASG responsesrelated to sympathetic activation may have been dominated by themotor components. The MSG followed the same patterns as the PSGon left and right sides, suggesting motor roles for that area.

4.3. Cold pressor

The left ASG showed increased activation during only the initialperiod of the cold pressor challenge, possibly suggesting an initialnovel sensory processing role, or possibly the anticipation of pain(Ploghaus et al., 1999). The trend toward declining signals relativeto the PLG may reflect an increase in that area relative to other gyri,reflecting the right-foot temperature and nocioceptive stimulation(Craig et al., 2000; Brooks et al., 2005; Hua le et al., 2005;Henderson et al., 2007, 2011). This possibility is consistent with theraw signal pattern (bottom of Fig. 5) showing a signal increase acrossall gyri on the contralateral side. The right-sided increases in the

er challenges. ASG: anterior short gyrus; MSG: middle short gyrus; PSG: posterior short

Recovery Cold pressor Recovery

Task

– – LG

>ASG>MSG+PSG

MSG+PSG+LG ASG+MSG –

>ASG >PSG+LG

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anterior gyri (ASG and MSG) presumably reflect sympathetic activa-tion, which is not seen in the PSG. As with the left side, the ASG ini-tially rose to a peak before falling towards baseline throughout theone minute challenge period, perhaps reflecting a gradual adaptationto the new state. The uniformly-elevated MSG signal is consistentwith the pain aspect of the task (Brooks et al., 2005; Afif et al.,2008; Henderson et al., 2011). Thus, the cold pressor findings supporta right ASG role in sympathetic activity, a right MSG role in pain syn-thesis, and a left posterior insula role in sensory integration.

4.4. Lateralization of function across insula gyri

The sympathetic phase of the Valsalva was accompanied by higherright than left insular fMRI signals (Fig. 6), consistent with a preferen-tial role for sympathetic integration on that side. Lateralization offMRI responses during the Valsalva maneuver appeared across allgyri during phase II with sustained differences between the rightand left insulae appearing throughout the Valsalva breath hold in allgyri, except the ASG which showed only transiently higher signalsat the onset and offset of the challenge. Such lateralization is consis-tent with findings in rats and humans (Oppenheimer and Cechetto,1990; Oppenheimer et al., 1991; Oppenheimer and Hachinski, 1992;Oppenheimer et al., 1992, 1996). In resting-state fMRI studies, theright anterior insula also has stronger functional connectivity withautonomic regions, such as the brainstem, pons, and right thalamus,whereas left anterior regions do not show connectivity with the thal-amus at all (Cauda et al., 2011). The more prominent sympathetic roleof the right insula likely reflects those structural connections to auto-nomic control areas.

Asymmetry in the cold pressor and hand grip challenges could notbe directly evaluated, since the cold pressor challenge was onlyapplied to the right foot, and the hand grip performed only by theright hand.

4.5. Considerations

While the challenges are known to elicit sympathetic responses,we did not have access to nerve recordings in the scanner environ-ment, and heart rate as a measure of autonomic output reflects bothsympathetic and parasympathetic influences. Thus, the sympathetic-dominant phases of the challenges must be inferred from otherstudies.

Resolution of the fMRI scans in this study was limited to voxelsizes of 3.4×3.4×4.5 mm. For group data, additional resolution limi-tations developed from normalizing images of all subjects to a com-mon space. By convention, normalized fMRI data were smoothedwith an 8 mm kernel filter. The principal consequence of the low res-olution is a partial volume effect, whereby signals classified as origi-nating from a single gyri will contain some signal from neighboringregions. Such effects would tend to blur differences, which reinforcesthe findings that the differences here are likely to be robust.

Cerebral blood flow and resting cerebral blood volume vary acrossbrain regions (Kastrup et al., 1999; Rostrup et al., 2000). Biophysicalmodels of the BOLD signal indicate that multiple hemodynamicfactors not related to neuronal activation can influence the BOLD re-sponse, including hematocrit, resting blood volume, and other factors(Ogawa et al., 1993; Boxerman et al., 1995; Bandettini and Wong,1997). However, the differential BOLD responses between left andright gyri and lateralization of insula function are unlikely to be an ar-tifact of these hemodynamic factors, since each gyrus has similar vas-culature on the left and right sides (Ture et al., 1999; Varnavas andGrand, 1999), and we noted significantly different patterns of activa-tion on each side. However, vascular influences on the lateralizationof responses cannot be completely ruled out, given the presence ofdifferences in left and right vascularization.

Another consideration is that gyral functions likely further dividealong inferior and superior directions (Cerf-Ducastel et al., 2001). In-vestigating inferior/superior functional topography would be a logicalnext step from the present study. However, one difficulty is the iden-tification of anatomical boundaries, which are not as clear as the gyraldistinctions; thus, the method here of tracing over the average of allsubjects' anatomical images would likely need to be altered, depend-ing upon what details could be resolved with available scans.

The cold pressor challenge elicited varying degrees of pain acrossthe subjects, and a measurement, such as a pain rating on a visual-analog scale would have been beneficial. Such a rating would likelyhave contributed to the model, as a greater pain experience wouldlikely be associated with greater activation in certain brains regionssuch as the amygdala.

Autonomic functioning changes with aging (O'Brien et al., 1986),and the wide age range in the present sample means that such varia-tion is contained within the data presented here. Sex differences inresponses are also likely present.

5. Conclusions

The insular cortices show functional differentiation between ante-rior and posterior gyri during Valsalva maneuver, hand grip, and coldpressor autonomic challenges, with significant laterality differencesin the Valsalva maneuver. Large onset and offset transient responsesappeared to the cold pressor and Valsalva maneuver challenges, andgyri differed on recovery periods in particular challenges. Posteriorand anterior gyri differed in lateralization patterns during the Valsalvamaneuver, and anterior gyri showed significantly different responsesto the sympathetic-dominant phase of the Valsalva maneuver, and an-terior–posterior differences were noted during the hand grip challenge.The insular gyri are useful anatomical markers of functional boundariesrelated to autonomic processing. Assessing fMRI responses or the im-pact of neural injury within specific gyri may help refine our under-standing of the impaired physiology in certain disease conditions.

Supplementary materials related to this article can be found onlineat doi:10.1016/j.autneu.2012.01.009.

Acknowledgments

We acknowledge the support for PW of the UCLA Howard HughesUndergraduate Research andUCLAUndergraduate Research FellowshipPrograms.

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