Placebo effects on human �-opioid activityduring painTor D. Wager*†, David J. Scott‡, and Jon-Kar Zubieta‡§

*Department of Psychology, Columbia University, 1190 Amsterdam Avenue, New York, NY 10027; and Departments of §Radiology and ‡Psychiatry andMolecular and Behavioral Neuroscience Institute, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48109-0720

Communicated by Edward E. Smith, Columbia University, New York, NY, March 15, 2007 (received for review October 26, 2006)

Placebo-induced expectancies have been shown to decrease pain ina manner reversible by opioid antagonists, but little is knownabout the central brain mechanisms of opioid release duringplacebo treatment. This study examined placebo effects in pain byusing positron-emission tomography with [11C]carfentanil, whichmeasures regional �-opioid receptor availability in vivo. Noxiousthermal stimulation was applied at the same temperature forplacebo and control conditions. Placebo treatment affected endog-enous opioid activity in a number of predicted �-opioid receptor-rich regions that play central roles in pain and affect, includingperiaqueductal gray and nearby dorsal raphe and nucleus cunei-formis, amygdala, orbitofrontal cortex, insula, rostral anteriorcingulate, and lateral prefrontal cortex. These regions appeared tobe subdivided into two sets, one showing placebo-induced opioidactivation specific to noxious heat and the other showing placebo-induced opioid reduction during warm stimulation in anticipationof pain. These findings suggest that a mechanism of placeboanalgesia is the potentiation of endogenous opioid responses tonoxious stimuli. Opioid activity in many of these regions wascorrelated with placebo effects in reported pain. Connectivityanalyses on individual differences in endogenous opioid systemactivity revealed that placebo treatment increased functional con-nectivity between the periaqueductal gray and rostral anteriorcingulate, as hypothesized a priori, and also increased connectivityamong a number of limbic and prefrontal regions, suggestingincreased functional integration of opioid responses. Overall, theresults suggest that endogenous opioid release in core affectivebrain regions is an integral part of the mechanism wherebyexpectancies regulate affective and nociceptive circuits.

neuroimaging � periaqueductal gray � expectancy � affective neuroscience

P lacebo effects are treatment effects caused not by thephysical properties of a treatment but by the meaning

ascribed to it. They are particularly strong in experimental andclinical studies of pain. However, much remains to be learnedabout the neural and cognitive mechanisms by which placebotreatments have their effects. Placebo analgesic treatments elicitexpectations of pain relief, which are thought to change theaffective and motivational context in which nociceptive signalsare interpreted (1–6). Although ample evidence exists thatplacebo expectancies reduce reported pain, the neurobiology ofhow expectancies interact with nociceptive brain processes isrelatively unexplored.

Early pharmacological studies showed that placebo analgesia isreduced by the opioid antagonist naloxone (7), suggesting thatendogenous opioids play a crucial role. Follow-up work pointed toboth opioid and nonopioid mechanisms of placebo (8). An emerg-ing idea is that expectancy-based placebo effects are opioid-mediated, but conditioned placebo effects may depend on othermechanisms (9–13). Although these studies support a strong rolefor opioids, little is known about the brain mechanisms that linkexpectancy with opioid release and pain relief in humans. Opioid-mediated placebo effects are thought to involve the periaqueductalgray (PAG), which contains many of the brain’s opioid-containingneurons and has been linked in a large animal literature with pain

relief (14–21). Opioid activity in PAG mediates the analgesic effectsof frontal stimulation in rats, and PAG is a target for humantherapeutic treatments for chronic pain (22–25).

Previously, we found that placebo induced increases in functionalMRI (fMRI) activity in lateral prefrontal cortex (PFC) and or-bitofrontal cortex (OFC). The magnitude of these activations wascorrelated with increases in the midbrain around the PAG, sup-porting the hypothesis that PAG and opioids play a role inexpectancy-based placebo (26). However, the only previous directstudy of regional placebo-induced opioid activity found effects inregions related to motivation and reward value, including thenucleus accumbens (NAC) and anterior insula (aINS), but not thePAG (27). Thus, involvement of PAG opioid activity in placeboanalgesia has not yet been directly demonstrated, and the relation-ships between endogenous opioid activity in PAG and other brainregions, such as PFC and rostral anterior cingulate (rACC), areunknown.

In this study, we aimed to address these gaps in knowledge bytesting placebo effects on �-opioid activity in PAG and otheropioid-rich regions of interest (ROIs). We delivered identicalthermal stimuli to patches of skin treated with identical, phar-macologically inert placebo and control creams. The conditionsdiffered only in the instructions: Participants were told that theplacebo treatment was a ‘‘highly effective pain reliever,’’ and thatthe control treatment would ‘‘have no effect on pain.’’ Weexamined placebo-induced changes in �-opioid receptor bindingpotential (BP) as measured with 11C-radiolabeled carfentanil (a�-opioid receptor-selective agonist) and positron-emission to-mography (PET). With this method, endogenous opioids inter-fere with the binding of [11C]carfentanil to the receptors,resulting in reductions in BP (28). Thus, the reduction in BP forplacebo vs. control treatment is a measure of placebo-inducedopioid system activation (27).

With this design, we can address another important unresolvedissue. In Zubieta et al. (27), placebo and control conditions weredesigned to produce equal subjective pain by using an adaptiveprocedure. Because noxious input differed for placebo and controlconditions, it is possible that (placebo � control) opioid effectscould be caused by differences in input or even minimized because

Author contributions: T.D.W. and J.-K.Z. designed research; T.D.W. and D.J.S. performedresearch; T.D.W. and J.-K.Z. contributed new reagents/analytic tools; T.D.W. and D.J.S.analyzed data; and T.D.W., D.J.S., and J.-K.Z. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Abbreviations: aINS, anterior insula; BP, binding potential; CH, control with painful heat;CW, control with nonpainful warmth; DLPFC, dorsolateral PFC; DRN, dorsal raphe nucleus;DVR, distribution volume ratio; fMRI, functional MRI; lOFC, lateral OFC; mOFC, medial OFC;NAC, nucleus accumbens; NCF, nucleus cuneiformis; OFC, orbitofrontal cortex; PAG, peri-aqueductal gray; PET, positron-emission tomography; PFC, prefrontal cortex; PH, placebowith painful heat; pgACC, pregenual anterior cingulate; PW, placebo with nonpainfulwarmth; rACC, rostral anterior cingulate; ROI, region of interest; SVC, small volumecorrected.

†To whom correspondence should be addressed. E-mail: tor@psych.columbia.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0702413104/DC1.

© 2007 by The National Academy of Sciences of the USA

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of the psychological equivalence of the conditions. Here, weexamine whether there are placebo effects on opioid activity whennoxious input is equivalent.

Placebo expectancy may affect opioid activity in several ways,three of which are shown in Fig. 1A. Placebo may enhanceendogenous opioid release caused by noxious stimulation (mech-anism no. 1), in which case placebo-induced �-opioid activityshould be higher during hot than warm stimulation; thus, a tem-perature (hot vs. warm) � placebo (placebo vs. control) interactionis predicted. This effect is consistent with studies showing largerplacebo effects with more intense pain (29). Alternatively, appli-cation of the placebo cream may itself trigger endogenous opioidactivity (mechanism no. 2), which should result in evidence forplacebo-induced opioid increases with both hot and warm stimu-lation. Finally, a function of opioid analgesia may be to prepare anorganism to deal with an imminent threat, in which case painanticipation may result in opioid release (mechanism no. 3; Fig. 1ARight). In this case, placebo may reduce threat (30, 31) and thusreduce �-opioid release during the anticipatory warm stimulationperiod. Different brain regions may independently show evidencefor one or more of these predicted patterns. Our hypothesis was thatplacebo treatment would potentiate pain-related opioid release (no.2) in opioid-rich regions such as PAG, OFC, and aINS.

In addition to regional opioid activity, we sought to test forplacebo effects on the functional integration of opioid responses infrontal, limbic, and brainstem regions, which relate to differentcomponents of the endogenous analgesic response (28). We ex-pected frontal regions, which may maintain expectancies for painrelief, to be more correlated with limbic regions and PAG underplacebo conditions. Connectivity between frontal cortex and brain-stem appears to play an important role in the modulation of painby attention (32, 33) and placebo (26), as predicted from animalstudies (14, 34). Recent evidence suggests that correlations betweenrACC and PAG activity are stronger with placebo (35) and hypnoticanalgesia (36). In the present study, multivariate analyses, whichcombined nonmetric multidimensional scaling (NMDS) and per-mutation tests with hierarchical clustering, allowed us to identifyseveral functional opioid subsystems and to test for the modulationof connectivity within and between subsystems by placebo.

ResultsPain-Specific Placebo Effects. The four conditions in the study wereas follows: placebo with painful heat (PH), control with painful heat(CH), placebo with nonpainful warmth (PW), and control withnonpainful warmth (CW). Placebo treatment led to significantreported analgesia during noxious heat [(PH � CH) in pain reports,5.58 for CH vs. 5.07 for PH, t (13) � 1.87, P � 0.042, one-tailed test],shown in supporting information (SI) Fig. 6C. In the brain, we first

tested for opioid activation differences in the temperature �placebo interaction contrast, (CH � PH) � (CW � PW), in whichpositive values indicate larger placebo-induced opioid activation inheat relative to warm stimulation (mechanism no. 1 in Fig. 1A).

Of the 11 key ROIs, 8 showed significant positive values, indi-cating placebo-induced �-opioid system activation [(P � 0.05, smallvolume corrected (SVC)], and none showed decreases. Across all27 ROIs, 14 regions showed significant placebo-induced increasesand none showed decreases. At the set level, activation of three keyROIs or five ROIs in the complete set were required for the numberof significant ROIs to reach the P � 0.05 corrected level. Thus,findings of 8 of 11 key ROIs and 14 of 27 in the full set of ROIs werehighly significant (P � 0.001 for both tests), demonstrating thatmany more regions showed placebo increases in opioid activity thanwould be expected under chance.

ROIs are shown in green in Fig. 2 and SI Fig. 6B, and voxels thatreached corrected significance within ROIs (P � 0.05, SVC) areshown in yellow. Significant regions include PAG, rACC, pregenualanterior cingulate (pgACC), multiple loci within OFC, aINS,thalamus, dorsolateral PFC (DLPFC), and amygdala. The activa-tion extent is shown in Fig. 2 and subsequent figures at P � 0.005,0.01, and 0.05 (two-tailed) in hot colors. Activation volumes andcenter-of-mass coordinates in Montreal Neurological Institute(MNI) space are presented in SI Table 1. Placebo effect magnitudesranged from 5.0% of baseline BP (left DLPFC) to 16.4% (leftamygdala), large enough to be of practical significance and com-parable to previous work (27).

Pain-Specific Placebo Effects Predict Reported Placebo Analgesia. Weexpected correlations between temperature � placebo interactionsin significant ROIs and reported placebo analgesia, demonstratingBP differences between relative placebo ‘‘responders’’ and ‘‘non-responders.’’ We first tested for correlations in the exact voxels thatshowed the significant group effects above. Opioid activity effectmagnitudes were significantly negatively correlated with reportedplacebo analgesia in four of these regions: PAG, right lateral OFC(lOFC), pgACC, and rACC (partial r’s � �0.47 to �0.77; SI Table1), indicating, contrary to our initial hypotheses, greater placeboopioid activation in low responders. We return to a discussion ofthese effects below. Two other regions, left anterior OFC andlOFC, showed negative trends (r’s � �0.57 to �0.60). (For moreinformation on robust partial correlations, see SI Methods.) Only

Nociception Opioidrelease

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Fig. 1. Study hypotheses and procedures. (A) Mechanisms by which placebomay affect opioid binding. See text for details. �, positive effect; �, inhibitoryeffect. (B) Study procedures.

Fig. 2. ROIs showing significant pain-specific opioid activation, [(CH � PH) �(CW � PW)]. ROI extent is shown in green, and significant voxels in orcontiguous with ROIs are shown in red/yellow (positive effects) or lavender/blue (negative effects). Amy, amygdala; aOFC, anterior OFC; lOFC, lateralOFC/inferior frontal border; mOFC, medial OFC; thal, thalamus.

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one region showed a positive correlation, left DLPFC (r � 0.79).Correlation scatter plots are shown in SI Fig. 7.

Tests within key ROIs revealed three additional negativelycorrelated regions: PAG, right lOFC, and right NAC (P � 0.05,SVC, for each; set-level P � 0.04). Two regions in the extended ROIset showed positive correlations, both within left DLPFC (set-levelP � 0.12). Overall, opioid temperature � placebo interactions wereboth significant in the group and predicted the magnitude ofreported analgesia, supporting mechanism no. 1 (Fig. 1).

Placebo Effects in Heat Vs. Anticipation. If placebo potentiates opioidrelease during pain (mechanism no. 1), then the effects reportedabove should be caused by placebo-related increases in opioidsystem activity during heat (CH � PH). Alternatively, the observedtemperature � placebo interactions could be caused by placebo-induced opioid decreases during pain anticipation (PW � CW;mechanism no. 3). Therefore, we assessed simple effects of placeboin heat (CH � PH) and warmth (CW � PW) separately.

Results revealed separate sets of regions that showed effects inheat and anticipation (P � 0.05, SVC). Significant results for (CH �PH) are shown in Fig. 3A and SI Table 2. During heat, opioidactivity increases were found primarily in OFC areas [anterior OFC,medial OFC (mOFC), and lOFC bilaterally] and right amygdala,and at lower thresholds, thalamus (P � 0.005), rACC (P � 0.005),and NAC (P � 0.01, all two-tailed). In these regions, there wasvirtually no effect of placebo during warm stimulation (Fig. 3B). Ofthese regions, significant negative correlations between average(CH � PH) and reported placebo analgesia were found in rightamygdala (r � �0.61), mOFC (r � �0.78), and lOFC (r � �0.74).Voxel-wise searches within ROIs yielded the same set of regions(P � 0.05, SVC). Scatter plots and coordinates are shown in SI Fig.8. Subsequent analyses suggested that negative correlations couldbe caused by greater nonspecific release in control conditions inplacebo responders (SI Fig. 9 and SI Text), which was observed inareas including medial PFC, aINS, mOFC, and lOFC.

A different set of regions showed evidence of placebo-inducedanticipatory decreases (PW � CW; mechanism no. 3) (Fig. 3C).These regions include left amygdala, left ventral aIns, pgACC,dorsal PAG, caudate, and right PFC [DLPFC, superior frontalsulcus (SFS), and inferior frontal junction, (IFJ)]. Notable is thedissociation between rACC, reported in Wager et al. (26), andpgACC, reported in Zubieta et al. (27): rACC responded specifi-cally during pain, whereas pgACC responded specifically duringanticipatory warm stimulation. Right vs. left amygdala showed asimilar distinction. Among these regions, negative correlationsbetween (CH � PH) and reported analgesia were found in rightDLPFC (r � �0.65; P � 0.05, SVC) and positive correlations in leftventral aINS (r � 0.67; SI Fig. 8).

Detailed Analysis of Midbrain. Results of exploratory analysis (P �0.05, two-tailed) in midbrain nuclei are shown in SI Fig. 10.Anticipatory placebo decreases in opioid activity (PW � CW) werefound in the dorsal PAG and overlying superior colliculus. Placeboincreases during heat (CH � PH) were found in midventral regionsof the PAG and most strongly in the nucleus cuneiformis (NCF)and dorsal raphe nucleus (DRN) (inferior to the red nucleus),extending into the ventral tegmental area (VTA). The tempera-ture � placebo interaction (Fig. 2 Inset and SI Fig. 10) wassignificant and positive in both dorsal and midventral PAG, ex-tending into DRN as well. These results are consistent withmechanisms nos. 1 and 3.

Multivariate Connectivity Analysis. Portions of ROIs that showedplacebo effects in one or more contrasts, 32 contiguous regions inall were selected for network analysis. We analyzed average opioidBP across conditions for each participant, computing Spearman’s �values between pairs of regions. These interregion correlations canreveal whether individual differences in opioid binding are consis-tent across the brain or whether there are functional subsystemsin opioid responses that show different patterns of individualdifferences.

To test for functional subsystems, the matrix of �’s for all pairs wasdecomposed with nonmetric multidimensional scaling (NMDS)(eight-dimensional solution; SI Fig. 11 A and B and SI Text), andhierarchical cluster analysis with average linkage was used oncomponent scores to group regions into sets (‘‘subsystems’’). Non-parametric tests were used to determine the number of subsystemsand to establish significance. We found that the best solution wasa seven-subsystem solution, and this was reliably better than the nullhypothesis of no functional subsystems (P � 0.0001, Z � 4.0) (fordetails, see SI Fig. 11 C and D and SI Text).

Fig. 4A shows locations of the 32 regions in the brain, and Fig. 4Bshows the relationships between regions (marked with circles) in thespace of the first two components. Each circle represents a uniqueregion, although some regions share the same name (e.g., DLPFC).Regions closer together on the graph were on average more highlycorrelated, and the marker color indicates subsystem membership.Lines show pairs of regions with significant bivariate �’s [gray, P �0.05; black, false discovery rate (FDR) (37) corrected P � 0.05].The size of each region indicates its centrality in the overallnetwork; regions with larger circles are more centrally connectedhubs.

Fig. 4 shows that OFC regions (yellow) have high connectivitywithin-subsystem and low connectivity with other subsystems, as dotwo separable prefrontal subsystems (red and magenta). PAG–rACC (green) and amydgala–pgACC subsystems (light blue) alsoshow patterns of binding distinguishable from the other subsystems.

Fig. 3. Placebo effects in heat vs. anticipation. (A)Regions showing placebo-induced opioid increasesduring heat (CH � PH). Color coding is as in Fig. 1. (B)Bar graphs showing placebo-induced opioid activity inwarm (black bars) and heat (gray bars) averaged acrossregions (x axis). R, right; L, left. Other labels are as inFig. 2. (C) Regions showing placebo-induced anticipa-tory opioid decreases (PW � CW). (D) Bar graphs foreffects in C. Amy, amygdala; aOFC, anterior OFC; Cau,caudate; IFJ, inferior frontal junction; lOFC, lateralOFC/inferior frontal border; mOFC, medial OFC; thal,thalamus

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A large, interconnected subsystem of limbic regions (dark blue)includes NAC, insula, lateral OFC, thalamus, and some frontal andcingulate regions.

Averaging �-opioid BP within subsystems, we tested whetherconnectivity between networks was stronger with painful than withwarm stimulation and stronger with placebo than with controlduring heat (ref. 38; see SI Fig. 12 and SI Text). This analysis cantest whether individual differences are more coherent across brainsubsystems with heat (analysis 1) and with placebo (analysis 2).Increased connectivity with placebo would indicate that placeboinduces coherent opioid activity across multiple regions that varieson an individual basis and would support the idea that there are trueindividual differences in placebo responses. We found that heatincreased integration of OFC with DLPFC and the limbic sub-system (statistics in SI Table 3). Correlations between the PAG–rACC and the rACC–caudate networks were stronger with placebothan with control treatment (�placebo � 0.67 vs. �control � 0.09, Z �2.25, P � 0.02, two-tailed). Placebo treatment also increasedconnectivity between the PAG and the rACC themselves,(�placebo � 0.71 vs. �control � 0.06, Z � 2.31, P � 0.02). Scatter plotsare shown in Fig. 5.

Finally, we tested for placebo-induced increases in functionalintegration among regions as a whole. We compared interregioncorrelations under PH with those under CH, and counted thenumber of increased vs. decreased correlations (SI Fig. 12). Wefound 36 increased pairwise correlations under placebo comparedwith 5 decreased (difference � 26, P � 0.0001 by using a permu-tation test). All significant placebo effects on pairwise correlationsare listed in SI Table 4. Notable is the increased connectivity amonglimbic regions (scatter plots for left NAC and left aINS are in SI Fig.12 Inset 2) and between limbic regions and DLPFC (scatter plots forleft DLPFC–amygdala are in SI Fig. 12 Inset 3).

DiscussionUnderstanding expectancy effects on endogenous opioid neuro-transmission is an important step in explaining how cognitive

processes modulate perception and affect, with implications forhealthy endogenous regulation and clinical pain syndromes (39).Consistent with behavioral studies (7, 11, 40), the results indicatethat placebo treatment induces increases in endogenous opioidactivity in �-opioid-rich limbic and paralimbic regions, includingPAG, NCF, DRN, OFC, amygdala, two dissociable regions of theanterior cingulate (rACC and pgACC), ventral aINS, and thalamus.All of these regions have shown a response to placebo or expectancyeffects in pain or pharmacological administration of opioids. NCFand DRN are and have been reported in recent fMRI studies ofcentral pain modulation (41–43).

The results suggest that placebo may affect opioid responsesin multiple ways. Placebo treatment worked in a subset of regions[OFC, rACC, NAC, NCF, DRN, ventral tegmental area (VTA),right amygdala, and thalamus] by potentiating the opioid releasecaused by noxious stimulation (mechanism no. 1 in Fig. 1 A). Inanother set of regions (lateral prefrontal cortex, aIns, pgACC,and left amygdala), placebo treatment appears to reduce antic-ipatory opioid activity, perhaps by reducing anticipatory threatresponses (mechanism no. 3 in Fig. 1 A). These regions may beinvolved in preparing for upcoming pain. Connectivity analysessupported this distinction, as the regions showing each pattern ofresponses belonged to different, separable subsystems. Althoughthese results suggest a prominent role for endogenous opioids inplacebo analgesia, placebo analgesia does not appear to beopioid-mediated in all conditions; for example, after condition-ing with a nonopioid analgesic (11) and in a recent study ofirritable bowel syndrome (44).

Placebo Effects in the Midbrain. The finding of PAG opioid increasesin placebo is important because it is perhaps the key region linkingplacebo expectancies with analgesia at both the spinal and supraspi-nal levels. In addition to placebo modulation of �-opioid activity, wefound that correlations between the PAG and rACC during noxiousstimulation were strengthened with placebo, corroborating findingsof Bingel et al. (35) on placebo-dependent connectivity betweenrACC and PAG in fMRI. Interestingly, however, the strongestmidbrain placebo effects during noxious heat were found in andaround the NCF and DRN (SI Fig. 10), two key nuclei in descendingantinociceptive pathways (16, 45) that have been reported in recentimaging studies to show expectancy-related effects (41–43).

Functional Integration. We assessed interregional connectivityacross participants (rather than on dynamic measurements overtime), as has much of the previous work on connectivity in pain (32,33, 35, 36). Such analyses are informative about the pattern ofindividual differences in brain activity. Connectivity analyses sug-gested that there are multiple subsystems in the human �-opioid

Fig. 4. Connectivity analysis of opioid binding poten-tial. (A) 3D rendering of connectivity among regions thatshow placebo opioid responses. (B) Nonmetric multidi-mensional scaling (NMDS) connectivity graph of the re-gions in A. See text for explanation. L, left; R, right; amy,amygdala; aofc, anterior OFC; cau, caudate; ifj, inferiorfrontal junction; lofc, lateral OFC/inferior frontal border;mofc, medial OFC; thal, thalamus.

Fig. 5. Placebo-modulated connectivity between PAG and rACC.

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system, as evidenced by the findings that opioid BP levels were notcorrelated across ROIs. Rather, opioid BP levels were correlatedwithin subsets of anatomically distributed but functionally coherentregions (identified by cluster analysis). For example, our findingssuggest that one subsystem is composed of left and right lateral andmidlateral OFC. Opioid BP levels among these regions was corre-lated (although the regions are distinct and each is at least 2 cm fromeach other one) and correlations with other regions are lower.Other subsystems included dissociable superior and mid-dorsolateral subsystems, a limbic subsystem, a midline (rACC andPAG) subsystem, and perilimbic subsystems (comprised of pgACC,amygdala, caudate, and one prefrontal region). These are shown inFig. 4. These findings argue against the notion of a unitary ‘‘opioidsystem.’’

In addition to increasing the PAG–rACC connectivity hypoth-esized a priori, placebo treatment increased functional connectivityamong the distinct subsystems (SI Fig. 12), particularly amongsubcortical regions, including NAC, thalamus, amygdala, and in-sula, and between these regions and DLPFC and OFC regions inother subsystems. The most straightforward interpretation of in-creased opioid–BP connectivity with placebo is that individualsdiffer in the magnitude of their opioid response to placebo and thatan individual’s response magnitude is consistent across ‘‘con-nected’’ regions. That is, the ‘‘placebo responders’’ in one region arethe same as those in other regions, implying a central commonmechanism for placebo-induced opioid release that varies acrossindividuals. These results suggest that it may be meaningful tocharacterize individuals on the basis of brain activity, i.e., as ‘‘opioidplacebo responders,’’ in future studies.

Correlations Between Reported Placebo and Opioid Activation. Opi-oid activity increases in a substantial proportion of ROIs wascorrelated with reported analgesia, in many cases in the same voxelsthat showed group effects. However, the correlations were negativein most regions, suggesting that greater reported reduction in painis associated with more modest opioid increases. One reason maybe that placebo responders show nonspecific opioid activity acrossboth placebo and control conditions and thus smaller (CH � PH)differences. There was evidence for this effect in a number ofregions; a representative example in the right lateral OFC is shownin SI Fig. 9 A–C. Strong placebo responders showed evidence ofhigher opioid activation (lower BP values) averaged across taskconditions (r � 0.56, P � 0.03; SI Fig. 9B) as well as evidence ofhigher opioid activation (lower BP) in the CH condition (r � 0.72,P � 0.003). SI Fig. 9D shows whole-brain maps of correlationsbetween reported placebo and BP in the CH and CW conditions.The widespread negative correlations reinforce the notion thatplacebo responders show relatively lower BP in many opioid-richregions. This could occur for two reasons: Placebo responders mayrelease more endogenous opioids in response to the experimentalcontext or to the manipulation phase preceding PET, or placeboresponders may have higher receptor-binding affinity, which wouldlead to lower overall BP levels for these participants in the contextof an existing opioid tone. In the former case, inclusion of theexpectancy manipulation may be an important experimental dif-ference from previous work (27), because it may elicit opioidrelease by itself. In the later case, affinity differences may helpexplain observed correlations between opiate- and placebo-inducedanalgesic responses (11). More detail and discussion of additionalalternatives is provided in SI Text.

ConclusionsPlacebo-induced expectancies of pain relief have been shown todecrease pain in a manner reversible by opioid antagonists, but littleis known about the central brain mechanisms of opioid releaseduring placebo treatment. This study examined placebo effects onendogenous opioid neurotransmission with PET and the �-opioidreceptor-selective radiotracer [11C]carfentanil in relation to several

alternative mechanisms: Placebo treatment may potentiate eitheranticipatory or pain-related endogenous opioid release or elicitopioid release itself. Noxious stimulation under placebo and controlconditions differed only in contextual instructions (temperatureswere equivalent). Increases in endogenous opioid neurotransmis-sion (decreases in �-opioid receptor availability in vivo) were foundin a number of key opioid-rich regions identified in previous studiesof placebo and cognitive regulation, including the PAG, amygdala,OFC, insula, rACC, and lateral PFC. Two distinct patterns werefound: First, placebo-induced increases in opioid activity specific toheat in OFC, right amygdala, and rACC suggests that placebopotentiates pain-related opioid release; opioid activity in many ofthese regions was correlated with reported placebo analgesia.Second, placebo-induced decreases in opioid activity specific toanticipation in pregenual cingulate, insula, and PFC suggests thatanticipatory anxiety may result in opioid release, and placebo mayblock this release (although this is speculative); these anticipatoryopioid responses were not correlated with reported placebo anal-gesia. Finally, an application of nonmetric multidimensional scalingand cluster analysis revealed distinct clusters of regions with similaropioid activity across participants, suggesting that coherent sub-systems within the endogenous opioid system can be identified.Painful heat (vs. warm stimulation) increased the functional inte-gration of opioid activity in OFC with those in other areas. Placebotreatment increased connectivity between the PAG and rACC andincreased functional integration among limbic regions and betweenlimbic regions and PFC. Overall, placebo treatment had widespreadeffects on endogenous opioid activity in cortical and subcorticalregions critical for the determination of affective value and context-based control of pain.

Materials and MethodsExperimental Design. Fifteen healthy volunteers (details in SI Text)underwent two 90-min scans with PET and [11C]carfentanil (Fig.1B). Each scan consisted of 30 warm trials (one per minute) and 30noxious heat trials (one per minute), in that order. We stimulatedthe placebo-treated forearm region during one scan and thecontrol-treated region in the other, with order randomized, coun-terbalanced, and controlled for by regression in all analyses. Carfen-tanil was delivered at subpharmacological doses, 0.026 � 0.01 �g/kgper scan. These doses occupy �0.5% of the available �-opioidreceptors and are devoid of physiological effects.

During each trial, a 1-s warning tone was followed by 4 s ofanticipation. Next, 24.5 s of thermal stimulation (3 s ramp up, 17 sat target, 4.5 s ramp down) was applied using a Medoc TSA 2001(Medoc Ltd., Chapel Hill, NC). Next, a warning tone cued partic-ipants to rate the stimulus intensity on a 0–10 visual analogue scalewith the following verbal anchors: 0 was ‘‘no sensation’’; 1 was‘‘detectable sensation’’; 2 was ‘‘nonpainful warmth’’; 3 was ‘‘justpainful’’; and 10 was ‘‘unbearable.’’ The interval between stimula-tion offset and the next warning cue was 30.5 s to allow physiologicalrecovery and prevent habituation (see SI Text and SI Fig. 6). Beforescanning, warm and noxious temperatures were chosen on anindividualized basis for each subject (3, 26, 46). Warm temperatures(44–46.5°C) were chosen at calibration level 2. Noxious tempera-tures (48–49.5°C) were chosen at level 8. After calibration, placeboand control treatments were applied to two regions of the volarforearm of each volunteer. As in previous work, before scanning,subjects also underwent an expectancy manipulation phase in whichbelief in the placebo was strengthened (3, 26, 46). The manipulationinvolves surreptitiously presenting reduced temperatures duringstimulation on placebo-treated skin (using different skin areas fromthose used in scanning). Stimulation during PET scanning followeda minimum of 20 min after the manipulation phase. See SI Text formore details.

Before each scan, the control and placebo creams were reappliedto the forearm. During scanning, each block of warm or noxioustrials was preceded by a 10-min rest period, which facilitates the use

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of Logan plots (47) used for BP quantification (see SI Text). Thestudy was designed to maximize the sensitivity of the placebo vs.control comparison with noxious heat, which began 40 min afterinjection in each condition. We were also interested in the placebovs. control comparison during warm stimulation (providing infor-mation relevant for mechanisms no. 2 and no. 3) and the pain(hot–warm) � placebo interaction (relevant for mechanism no. 1).Importantly, however, the design is not suited for a direct compar-ison between hot vs. warm stimulation, because these conditionswere not randomized, and BP calculation bias may differ betweenearly and late scanning periods. The comparison between controland pain states has been the subject of previous work (28, 48) fromwhich we draw inferences about the effects of noxious stimulationon opioid binding.

Image Acquisition and Analysis. [11C]carfentanil PET distributionvolume ratio (DVR) [equal to BP � 1 or (Bmax/Kd) � 1] images foreach condition were acquired, reconstructed, and corrected forattenuation by using a modified Logan plot analysis (27). High-resolution anatomical MRI images were warped into MontrealNeurologic Institute (MNI) standard space, and warping parame-ters were applied to DVR images (details in SI Text). DVR imageswere spatially smoothed with an 8-mm isotropic Gaussian filter. Allopioid effects were localized with reference to the group meanwarped anatomical MRI image, which was used as the anatomicalunderlay in all figures.

Consistent with previous studies (27), we define relative �-opioid‘‘activation’’ as the relative decrease �-opioid receptor BP betweenconditions, i.e., a positive value for (CH � PH) in DVR indicatesthat placebo increases in endogenous opioid activity during heat.Similarly, positive (CW � PW) values indicate that placebo in-creases during anticipatory warm stimulation, and positive inter-action values, [(CH � PH) � (CW � PW)] indicate that placebo-induced opioid increases greater during heat. Contrast values wereentered into a random effects, multiple regression model at thesecond level with mean-centered reported placebo during heat(reported pain in CH � PH) and administration order (contrastcoded) entered as covariates. Robust model estimation was per-formed using iteratively reweighted least squares (IRLS) in Matlabsoftware (Mathworks, Natick, MA), which produces valid P valueswhile minimizing the influence of outliers and violations of nor-mality (49). See SI Text for details.

ROIs. ROIs were identified on the basis of previous imaging studies.Each ROI met all of the following criteria: (i) a priori interest on thebasis of at least one activation in placebo studies of fMRI or�-opioid receptor BP; (ii) high BP in the current sample (� 1.1,reflecting specific binding; see SI Fig. 6); and (iii) activation in atleast two previous studies of either placebo, opiate administration,or emotion regulation, from ref. 50 (SI Fig. 6). In some cases, ROIswere enlarged slightly to encompass high-BP regions in our sample.Eleven ROIs of primary interest included PAG, rACC, aINS,pgACC, medial orbital sulcus (MOS), lateral OFC/inferior frontalgyrus (LOFC), amygdala (Amy), and nucleus accumbens (NAC).Each of these regions has been shown to be important for placeboeffects in fMRI and/or opioid-binding studies. Additional ROIs inthe thalamus (6), DLPFC, medial OFC, and dorsal caudate werealso identified as described above, making a complete set of 27regions. Correction for multiple comparisons across regions wasperformed with a permutation test that compared the number ofsignificant SVC ROIs with the number expected under the nullhypothesis (see SI Text). For ROI sets that show a significantnumber of activated regions, we report SVC-corrected regions(yellow in all figures).

Because of the importance of brainstem pain modulation path-ways, additional exploratory ROIs included the NCF (41, 43),lateral to PAG in the midbrain, the midline DRN (e.g., ref. 42; SIFig. 10), and dopaminergic ventral tegmental area (VTA) due toopioid-dopamine interactions in this region (51, 52). NCF and DRNare heavily interconnected with PAG and the rostral ventralmedulla and play key roles in spinal afferent inhibition and sensi-tization (45). Because too few imaging studies have examined theseregions to define formal ROIs as above, we compared the locationsof midbrain placebo effects with approximate locations of thesenuclei as defined in refs. 41–43 and in the Duvernoy atlas (53).

We thank Drs. Ed Smith and Ken Casey for helpful comments anddiscussion; Raza Zaidi and Alex Sokolik for help with data collection;Matthew Davidson and Brent Hughes for technical assistance; the PETtechnologist at the University of Michigan PET Center for assistance;and the authors of Statistical Parametric Mapping and the MontrealNeurological Institute. Support was provided by the Mind, Brain, Body,and Health Initiative (T.D.W.), the John D. and Catherine T. MacArthurFoundation (T.D.W.), and National Science Foundation Grants 0631637(to T.D.W.) and R01 AT 001415 (to J.K.Z.).

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