Brain, Behavior, and Immunity 19 (2005) 500–509

www.elsevier.com/locate/ybrbi

Named Series: Brain Mechanisms of Placebo

Expectation enhances autonomic responses to stimulation of the human subthalamic limbic region �

Michele Lanotte, Leonardo Lopiano, Elena Torre, Bruno Bergamasco, Luana Colloca, Fabrizio Benedetti ¤

Department of Neuroscience, University of Turin Medical School, Turin, Italy

Received 14 April 2005; received in revised form 8 June 2005; accepted 13 June 2005Available online 1 August 2005

Abstract

Recent studies show that the placebo component of a treatment can be investigated by administering therapies either overtly orcovertly, without the administration of any placebo. Here, we analyze the eVects of open (i.e., expected) versus hidden (i.e., unex-pected) stimulations of the human subthalamic region on autonomic responses in Parkinson patients. To do this, we mapped thewhole subthalamic region, from the dorsal to the ventral part, and recorded both heart rate and sympathetic responses by usingspectral analysis of heart rate variability. We found that open stimulations were more eVective than hidden ones only in the ventralsubthalamic region, whereas no diVerence between the two conditions was found in the dorsal aspect. By analyzing the stimulus–response curves in the dorsal, middle, and ventral subthalamic regions, we found that the autonomic response threshold was higherin the hidden than open condition for both heart rate and sympathetic responses only in the ventral part. As this ventralmost portionof the subthalamic region is involved in associative-limbic functions, these data suggest that expectation enhances autonomicresponses only if these are elicited in the limbic system. These results extend previous Wndings on the open–hidden paradigm in deepbrain stimulation [Benedetti, F., Colloca, L., Lanotte, M., Bergamasco, B., Torre, E., Lopiano, L., 2004a. Autonomic and emotionalresponses to open and hidden stimulations of the human subthalamic region. Brain Res. Bull. 63, 203–211.], and indicate that expec-tation plays a major role in the therapeutic outcome. In light of the interactions between the sympathetic adrenergic system and theimmune system, the open–hidden diVerence in autonomic responses might be relevant to the understanding of how expectationsmight aVect the immune system. 2005 Elsevier Inc. All rights reserved.

Keywords: Expectation; Placebo; Deep brain stimulation; Subthalamic region; Parkinson’s disease; Autonomic nervous system

1. Introduction

Expectation of an outcome plays a major role in theplacebo response (Amanzio and Benedetti, 1999; Bened-etti et al., 1999, 2003b; Colloca and Benedetti, 2005). Therecent introduction in biomedical research of the open–hidden paradigm allows the investigation of bothexpected and unexpected therapeutic outcomes and the

� Series Editors: Tor D. Wager and Jack B. Nitschke.* Corresponding author. Fax: +39 011 670 7708.

E-mail address: fabrizio.benedetti@unito.it (F. Benedetti).

0889-1591/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.bbi.2005.06.004

identiWcation of the placebo eVect without the adminis-tration of any placebo (Colloca et al., 2004). An opentreatment is performed in full view of the patient whoknows what is going on and expects an outcome. By con-trast, a hidden treatment is performed covertly, with thepatient completely unaware that a therapy is beingadministered, so that he/she does not expect anything.The diVerence between the open and the hidden treat-ments represents the placebo, or psychological, compo-nent that comes from the patient’s expectations andperception of receiving a therapy. It has been shown thatopen administrations are more eVective than hidden

M. Lanotte et al. / Brain, Behavior, and Immunity 19 (2005) 500–509 501

administrations in many conditions, which indicates thatthe knowledge about a treatment aVects the therapeuticoutcome (for a review, see Colloca et al., 2004).

Although most of the studies with the open–hiddenapproach have used overt and covert infusions of drugs(Amanzio et al., 2001; Gracely et al., 1983; Levine andGordon, 1984; Levine et al., 1981), recently intraopera-tive overt and covert stimulations in awake humanshave been investigated (Benedetti et al., 2004a). Forexample, it has been shown that the open stimulationof the limbic portion of the subthalamic region is moreeVective in eliciting both autonomic and emotionalresponses than the hidden stimulation. In addition, byanalyzing the thresholds for inducing autonomicresponses, an increase in threshold in the hidden condi-tion was found, suggesting that overt stimulation has afacilitating eVect on the autonomic responses, probablythrough the involvement of expectation and anticipa-tion mechanisms (Benedetti et al., 2004a). In previousstudies, it has also been shown that the eVects of sub-thalamic nucleus (STN) stimulation can be modulatedby complex psychological factors. In fact, context-related cues, like verbal suggestions, can produce diVer-ent outcomes following STN stimulation (Benedettiet al., 2003a,b; Pollo et al., 2002). Context-related cuesand placebo administration have also been shown toinduce a release of dopamine in the striatum (de laFuente-Fernandez et al., 2001) and a change of neuro-nal activity of the STN of Parkinsonian patients(Benedetti et al., 2004b), thus suggesting that the entirebasal ganglia circuitry can be aVected by psychologicalmanipulation.

On the basis of these considerations and of our previ-ous study on the open–hidden stimulation of the subtha-lamic region (Benedetti et al., 2004a), in the present workwe performed a detailed analysis of the autonomicresponses (heart rate and sympathetic activity) to stimu-lation of the subthalamic region, to better understandwhen and where a signiWcant diVerence between overtand covert stimulations occurs. In light of the close inter-action among psychosocial context, autonomic nervoussystem, and immune system (Bierhaus et al., 2003), theopen–hidden diVerence in autonomic responses might berelevant to the understanding of how expectations mightaVect the immune system.

2. Methods

2.1. Subjects

Twenty patients participated in the study after theysigned a written informed consent for both surgery andmapping procedures. All the experimental procedureswere carried out in accordance with the Declaration ofHelsinki. The patients were diagnosed with idiopathicParkinson’s disease. Clinical evaluation was performed bymeans of the UniWed Parkinson’s Disease Rating Scale(UPDRS) (Fahn et al., 1987) and the Hoehn and Yahr’s(1967) Wve stages of the disease. Table 1 shows the charac-teristics of each patient, the UPDRS scores before the sur-gical implantation of the electrodes, and the duration ofthe disease, as well as the drug therapy before surgery. Alldrugs were interrupted the day before surgery.

Table 1Characteristics of the subjects

ama, amantadine; ami, amitriptiline; apo, apomorphine; b, bromazepam; ca, cabergoline; ci, citalopram; cl, clozapine; d, diazepam; m, madopar; pe,pergolide; pr, pramipexol; q, quetiapine; re, reboxetine; ro, ropinirol; s, sinemet; v, venlafaxine.

Patient Age (years)

Sex Duration of Parkinson’s disease (years)

UPDRS before surgery

Therapy before surgery

1 57 f 14 60.5 s, pe2 70 f 20 50.5 m, s, ca, apo, ama3 53 f 15 39 m, cl, b4 54 f 14 58 m, ami, ama5 65 m 22 68 m, ro, v6 53 m 11 44 s, q7 65 f 20 65.5 m, pr, d, ci, re, ama8 66 f 14 70 m, pe, ama9 68 m 10 58.5 m, pe, b,

10 67 f 16 45.5 s, pr, q11 50 m 12 56.5 s, pe12 69 m 24 59 m, s, ca, apo13 58 f 19 48.5 m, cl, b14 64 f 10 58 m, ama, cl15 55 f 17 71.5 m, pe, ama16 56 m 15 47.5 s, q17 69 f 17 65 m, pr, ci, re, ama18 70 m 14 63 m, pe, ama19 59 f 11 51.5 m, pe, b20 67 f 20 48 s, q

502 M. Lanotte et al. / Brain, Behavior, and Immunity 19 (2005) 500–509

2.2. Localization of STN

All the patients performed a brain 3-D MRI scanbefore surgery. At surgery, after positioning of a Cos-man–Roberts–Wells stereotactic frame (CRW Radion-ics, Burlington, MA, USA), a stereotactic CT scan wasperformed. Then, the MRI and CT slices were fused bythe Stereoplan system (Radionics, Burlington, MA,USA), to obtain in the same images the spatial precisionof CT and the better tissue deWnition of MRI. Then, weidentiWed the anterior and posterior commissurae coor-dinates and the length of the intercommissural line. TheSTN was anatomically localized 2.5 mm posterior and4 mm inferior with respect to the mid-commissural pointand 12 mm from the midline. The electrode track wasplanned using a 58–63 anterior–posterior angle and14–20 lateral angle, as shown in Fig. 1A. A 14-mm pre-coronal burr was performed under local anesthesia andthe electrode lowered into the brain.

After this anatomical localization, neurophysiologicalidentiWcation of the STN was carried out. In fact, electri-cal activity microrecording was performed starting from10 mm above the anatomical target (MicrotargetingElectrodes Type BP, FHC, Bowdoinham, USA). Theelectrical signals were acquired by means of the Neuro-trek system (NeuroTrek, Alpha Omega, Nazareth,Israel). After a low background activity correspondingto a region encompassing the zona incerta (ZI), the STNwas identiWed by a background noise with a sustainedand irregular pattern of discharge at a frequency of 25–45 Hz (Hutchinson et al., 1998). Moreover, single neu-rons responsive to contralateral proprioceptive stimuliwere identiWed and, in some cases, “tremor neurons”were recorded with an oscillatory discharge of 4–6 Hz,corresponding to the typical Parkinsonian tremor. Whenthe microelectrode exited the STN, a low backgroundnoise was followed by a regular and high frequency (HF)discharge of units belonging to the substantia nigra parsreticulata (SNr). After the deWnition of the extension ofthe STN recording area, with its dorsal and ventral bor-ders, and the identiWcation of the other subthalamicstructures, according to the neuronal Wring pattern, themicrostimulation procedures were started (Lanotte et al.,2002; Limousin et al., 1998; Lopiano et al., 2001).

2.3. Microstimulation

ConWrmation of good positioning of the electrode tipin the STN was also obtained by means of microstimula-tion for the assessment of both clinical eVects (reductionof rigidity and tremor) and side eVects (muscle contrac-tions, dyskinesia, and tingling sensations). In fact, someside eVects are sometimes a good index of correct posi-tioning. The stimulus width was 60�s with a frequencyof 130 Hz. During the microstimulation session, the sub-thalamic region was stimulated both dorsally and in the

middle part and ventrally. Each of these sites was stimu-lated at 1, 3, and 5 V to determine the stimulus–responsecurve and the autonomic response threshold. In addi-tion, this stimulation was performed in two diVerentconditions: hidden stimulation and open stimulation. Inthe hidden condition, the three stimulus intensities weredelivered with the patient completely unaware that thestimulation was started. To do this, the buzz that is asso-

Fig. 1. (A) Magnetic resonance image showing the electrode track withits tip in the right subthalamic region. (B) During stimulation of thesubthalamic region, the ECG was continuously recorded. Data analy-sis consisted in measuring the R–R intervals to assess heart rate.(C) The R–R intervals were plotted versus time (number of heartbeats) to obtain a tachocardiogram, which represents heart rate vari-ability. (D) Spectral analysis of heart rate variability showing the PSDof the LF sympathetic and HF parasympathetic components at rest(broken line) and during stimulation (bold line) of a site in the subtha-lamic region.

M. Lanotte et al. / Brain, Behavior, and Immunity 19 (2005) 500–509 503

ciated with the stimulus was turned oV and the patientdid not know if and when the stimulus was going to bedelivered. We paid particular attention to the possibleunblinding of the patients, due to stimulation-inducedside eVects or straightforward motor improvement. Todo this, the patients were asked to report any sensationthey perceived. In no case did the patients realize that thestimulator had been turned on. Conversely, in the opencondition the buzz was turned on to make the patientaware that stimulation was being delivered. The patientwas also told when the stimulator was switched on.Microstimulation was performed at the level of the mostsuperWcial (dorsal) subthalamic region, the middle por-tion, and the deepest (ventral) region. The intervalsbetween diVerent stimuli and diVerent experimental con-ditions (hidden and open) ranged from 1.5 to 3.5 min.The sequence of the diVerent conditions was random.

Due to both ethical constraints and the patient’sintraoperative conditions, most of the stimulationslasted less than 3 min, and were stopped when at least200 heart beats were acquired for subsequent analysis(see below). In addition, to reduce the duration of theexperimental session, only the subthalamic region of oneside was tested, and the side was randomized (12 patientswere tested on the left, 8 on the right). Therefore, eachpatient was tested in three sites (dorsal, middle, and ven-tral STN of one side) and the total number of microsti-mulation sites we studied was 60 (3 sites £ 20 subjects).Since each site was tested 6 times (3 stimulusintensities £ 2 experimental conditions), a total of 360stimulations, either open (N D 180) or hidden (N D 180),were delivered in all the subjects.

2.4. IdentiWcation of sympathetic and parasympathetic responses

The electrocardiogram (ECG) was recorded by usingconventional techniques with two electrodes on thechest. Heart rate was analyzed by measuring the R–Rintervals of the ECG (Fig. 1B) and then transformingthem into frequency (1/R–R). The heart rate baselinewas represented by the mean of 10 R–R intervals justbefore the stimulus, whereas the maximum post-stimulusresponse was represented by the shortest R–R interval.The ECG beat-to-beat series (R–R intervals) werechecked for ectopic beats. All the patients of this studydid not show any ectopic beat.

The sympathetic and parasympathetic components ofthe ECG were analyzed by means of the spectral analysisof heart rate variability. To do this, we transformed theECG into a tachocardiogram (R–R intervals over time),which represents heart rate variability (Fig. 1C), and car-ried out a spectral analysis of the tachocardiogram. Byusing this procedure, it is possible to identify a low fre-quency (LF) component (0.04–0.15 Hz), which corre-sponds to sympathetic activity, and a HF component

(0.15–0.4 Hz), which corresponds to parasympatheticactivity (Kamath and Fallen, 1993; Malliani et al., 1991;Task Force, 1996) (Fig. 1D). Power spectral analysis wasapplied to the R–R interval sequences at rest (200 beatsbefore stimulation) and during stimulation. We used theautoregressive method with an Esaote System(EBNeuro, Florence, Italy), in which the spectral compo-nents were automatically obtained by the spectraldecomposition method that measured the area undereach spectral peak. Our spectral analysis detected a LFcomponent in the range of 0.04–0.15 Hz and a HF in therange of 0.15–0.4 Hz. The power spectrum densities(PSD) were calculated from these frequency ranges.However, due to the high variability of the parasympa-thetic component in the HF domain, we analyzed onlyLF. In fact, due to the vagal parasympathetic inXuenceon the respiratory rate, HF responses are better detect-able if breathing is kept constant (the subject has to syn-chronize his/her breathing with a metronome) (Kamathand Fallen, 1993; Malliani et al., 1991; Task Force,1996). Due to ethical constraints, we did not ask ourpatients to control their breathing and decided not toanalyze parasympathetic HF activity.

2.5. Statistical analysis

The three stimulus intensities at each site and in eachcondition (open and hidden) were used to create a stimu-lus–response curve and to determine the threshold for anautonomic response. Statistical analysis was performedby means of a paired t test. In addition, reconstruction ofthe anatomical localization of the stimulation site wasbased on depth coordinates. To do this, we calculatedthe progression of the electrode tip (in mm) startingfrom 10 mm above the supposed anatomical target (i.e.,the STN), as assessed by means of the stereotactic coor-dinates. This was the site from where we started therecording session. Correlation between depth andresponses was performed by means of linear regressionanalysis. Data are presented as mean § SD, and the levelof signiWcance is p < .05.

3. Results

Autonomic responses were induced by the stimula-tion of the entire subthalamic region. At low intensity ofstimulation (1 V), we found heart rate increase in 13/20sites (65%) in the dorsal region, 2/20 (10%) in the middle,and 11/20 (55%) in the ventral region. Likewise, wefound sympathetic LF increase in 8/20 sites (40%), 0/20(0%) in the middle, and 6/20 (30%) in the ventral region.By increasing the intensity of stimulation up to 5 V, alarger number of responsive loci was obtained. In fact,5 V stimulation induced an increase of heart rate in 18/20loci (90%) in the dorsal part, 5/20 (25%) in the middle,

504 M. Lanotte et al. / Brain, Behavior, and Immunity 19 (2005) 500–509

and 17/20 (85%) in the ventral part. Similarly, 5 V stimu-lation elicited sympathetic LF responses in 12/20 loci(60%) in the dorsal portion of the subthalamic region, in2/20 (10%) in the middle, and in 10/20 (50%) in the ven-tral portion.

Although the responses in the dorsal–middle regionwere constant over time in the open and hidden condi-tions, there was a large variation in the ventralmost partof the subthalamic area. Figs. 2A and B show that thediVerence between the open and the hidden stimulationsat 1 V (black circles) for heart rate and sympathetic LFchanges, was always around 0 for the most dorsal andmiddle loci of stimulation, while variations up to 50%occurred in the most ventral loci. A linear regressionanalysis, performed on the 26 sites that responded with aHR increase to 1 V stimulation (see above), showed that

Fig. 2. The diVerence between open and hidden stimulations of a givensite in the subthalamic region is plotted against its depth in the brain,both for heart rate responses (A) and sympathetic LF responses (B).The depth is expressed in millimeter from the beginning of recording(see Section 2). Note that, at low intensity of stimulation (1 V), largeopen–hidden diVerences are present only in the deepest loci of stimula-tion (black circles). Conversely, at high intensity of stimulation (5 V),the open–hidden diVerences are small even in the deepest sites of stim-ulation (white circles).

the correlation between open–hidden diVerence anddepth was high (r D .863, t(24) D 8.362, p < .001). InFig. 2A, it can also be seen that high intensity stimula-tions of 5 V reduced the open–hidden diVerence in theventral region compared with 1 V stimulation(t(10) D 5.47, p < .001), although correlation betweenopen–hidden diVerence and depth, performed on the 40sites that responded with a HR increase to 5 Vstimulation (see above), was still signiWcant (r D .735,t(38) D 6.668, p < .002).

Likewise, the correlation between open–hidden diVer-ence and depth, performed on 14 sites that responded witha sympathetic LF increase to 1 V stimulation (see above),was high (rD .903, t(12)D9.212, p < .001). In Fig. 2B, it canalso be seen that high intensity stimulations of 5 V reducedthe open–hidden diVerence in the ventral region comparedwith 1 V stimulation (t(5)D8.845, p < .001), although cor-relation between open–hidden diVerence and depth, per-formed on 24 sites that responded with a LF increase to5 V stimulation (see above), was still signiWcant (r D .816,t(22)D10.128, p <.001). Therefore, hidden stimulations athigh intensities were capable of inducing larger autonomicresponses that were similar to those elicited by open stim-ulations, which suggests a change in threshold. Fig. 2 alsoshows that two loci at 11 and 12 mm (i.e., in the ventral-most region) in two diVerent patients elicited subjectivesensations of pleasure, thus indicating that this part of thesubthalamic region is involved in limbic–emotionalresponses. Interestingly, these pleasant sensations wereevoked only by open stimulations at low intensity (1 V)and by both open and hidden stimulations at highintensity (5 V).

To better appreciate the relationship between thedepth of the stimulated loci and the anatomical struc-tures of the subthalamic region, particularly the STN,Fig. 3 was obtained from the Schaltenbrand and Wahren(1977) atlas at the level of a sagittal slice taken 12 mmfrom the midline. Although there is a large inter-individ-ual variability in both size and shape of diVerent nucleiand tracts, Fig. 3 indicates that the dorsal region stimu-lation is likely to involve the dorsal pole of the STN, theZI and the Forel’s Weld, the middle region, the core ofthe STN, and the ventral region stimulation, the ventralpole of the STN, and the SNr. Interestingly, we found afew, or no response at all, in this middle region. This isparticularly evident in Fig. 2B, in which a clear-cut gapbetween the dorsal and ventral regions can be seen. Inthis gap, no sympathetic LF response was found. There-fore, it can be assumed that structures other than theSTN were stimulated, particularly in dorsalmost andventralmost stimulation loci.

By using three intensities of stimulation (1, 3, and5 V), we were able to obtain a stimulus–response curvefor both heart rate and LF responses, in the dorsal, mid-dle, and ventral subthalamic regions. Figs. 4A and Bshow the stimulus–response relationship in the three

M. Lanotte et al. / Brain, Behavior, and Immunity 19 (2005) 500–509 505

regions for both heart rate and LF responses. It can beseen that a diVerence between the open and hidden con-ditions is present only in the ventral subthalamic region,

Fig. 3. Reconstruction of the subthalamic region in relation to thedepth of our electrode tip, according to the Schaltenbrand andWahren (1977) atlas at the level of a sagittal slice taken 12 mm fromthe midline. The depth is expressed in millimeter from the beginning ofrecording (see Section 2). Th, thalamus; ZI, zona incerta; Hf, Forel’sWeld; STN, subthalamic nucleus; SNr, substantia nigra pars reticulata;CST, corticospinal tract.

whereas no diVerence between the two conditions wasfound in the dorsal and middle portions. These curvesshow that the threshold for evoking autonomicresponses in the ventral subthalamic region was higherin the hidden condition compared to the open one. Infact, the threshold for evoking heart rate responses andsympathetic responses in the dorsal and middle regionsdid not vary signiWcantly in the open and hidden condi-tions, whereas there was a signiWcant diVerence in theventral part for both heart rate (t(16) D 6.18, p < .03) andsympathetic LF activity (t(9) D 9.456, p < .001) (Fig. 5).

Although all drugs were interrupted the day beforesurgery, some psychotropic and autonomic eVects maynot have disappeared in less than 24 h. Therefore, wecompared those patients taking psychotropic drugs, likequetiapine, clozapie, amitriptyline, reboxetine, and ven-lafaxine, with those patients who did not, and found nosigniWcant diVerences between the two groups.

4. Discussion

The present study extends our previous Wndings onthe open and hidden stimulations of the human subtha-lamic region (Benedetti et al., 2004a). In particular, com-pared to our previous study, this work adopted diVerentmapping procedures, whereby three intensities of stimu-lation (they were Wve in Benedetti et al., 2004a) and threestimulation loci (they were two in Benedetti et al., 2004a)

Fig. 4. Stimulus–response curves for heart rate changes (A) and sympathetic LF PSD changes (B) in the dorsal, middle, and ventral subthalamic regionsin the open and hidden stimulation conditions. Note that the stimulus–response relationship, and thus the threshold for eliciting a response, changes onlyin the ventral (limbic) subthalamic region.

506 M. Lanotte et al. / Brain, Behavior, and Immunity 19 (2005) 500–509

were used. This allowed the mapping of the middle por-tion of the subthalamic region as well. We found that theexpected stimulations induced larger autonomicresponses than unexpected ones if, and only if, the stimu-lation involved the ventral portion of the subthalamicregion. In this regard, the subthalamic region is an inter-esting model to be studied, as it is constituted of twoparts, at least from a functional point of view, dorsal andventral, that are both related to autonomic activity andthat are separated by only 3–4 mm from each other. Inthe dorsal portion, no diVerence was found betweenexpected and unexpected stimulations, which indicatesthat the eVects in the ventral subthalamic region arehighly speciWc. This rules out general non-speciWc eVectson autonomic functioning.

Although an attempt to reconstruct the location ofthe responsive sites is shown in Fig. 3, inter-individualdiVerences in both shape and size of the subthalamicstructures does not allow exact localization. Nonethe-less, our Wndings can rely on the depth of the stimulatedsites; in fact, as shown in Fig. 2, the deeper the stimula-tion, the larger the open–hidden diVerence. Therefore,even though the exact localization is diYcult to obtain,the data of the present study clearly indicate that the

Fig. 5. Thresholds for heart rate (A) and sympathetic LF (B) responsesin the open and hidden conditions. Note that the threshold changesonly in the ventral subthalamic region.

eVects we found were conWned to the most ventralaspects of the subthalamic region. As the target of theneurosurgical implantation of the electrodes is the STN,we always refer to this anatomical structure as the stimu-lation locus. However, it would be better, particularly instudies like the present one, to refer to the entire subtha-lamic region.

Autonomic responses to the stimulation of the sub-thalamic region have been described in the rat (Spenceret al., 1988; Van del Plas et al., 1995), cat (Angyàn andAngyàn, 1996, 1999), and man (Kaufmann et al., 2002;Priori et al., 2001; Thornton et al., 2002). Both increasesand decreases in heart rate, blood pressure, and respira-tion rate have been described. Both microinjections ofL-glutamate and electrical stimulation of the ZI of therat induced a decrease of blood pressure and heart rate(Spencer et al., 1988; Van del Plas et al., 1995). Con-versely, stimulation of the STN in freely moving catsinduced a conspicuous increase in blood pressure, heartrate, and respiratory rate (Angyàn and Angyàn, 1999).In Parkinson patients, STN stimulation has been foundto induce heart rate increase and variable blood pressureresponses (Kaufmann et al., 2002; Thornton et al., 2002).Additional autonomic responses have been found fol-lowing STN stimulation, such as adrenal secretion ofepinephrine and norepinephrine in the rat (Matsui,1987), and plasma renin increase and sympathetic skinresponse in man (Priori et al., 2001). All these studiesindicate that the stimulation of the subthalamic region,particularly the dorsal part (e.g., the ZI; see Spenceret al., 1988; Van del Plas et al., 1995), involves either neu-rons or passing Wbers that play a role in cardiovascularcontrol.

By contrast, the subthalamic region is also known tobe related to associative and limbic functions (Alexanderet al., 1986, 1990; Parent and Hazrati, 1995). In fact, thestimulation of the human STN has been reported to pro-duce emotional-related responses, such as euphoria andhypomania (Ghika et al., 1999), mirthful laughter(Krack et al., 2001), and pleasant sensations (Benedettiet al., 2004a). Furthermore, stimulation of the SNr hasbeen found to induce acute depression (Bejjani et al.,1999; Kumar et al., 1999). These emotional responses arenot necessarily induced by neuronal populations locatedin the subthalamic region itself, but they can also beevoked by the activation of distant areas. In fact, theSTN is connected to the ventral pallidum, a major limbicoutput region (Turner et al., 2001), and the temporal lobeis an output target of the basal ganglia (Middleton andStrick, 1996). In addition, cingulate cortex activity is mod-ulated by HF STN stimulation (Limousin et al., 1997).Recent studies also indicate that STN stimulation mayalso have important eVects on mood (Berney et al., 2002;Funkiewiez et al., 2003; Schneider et al., 2003).

By taking all these Wndings into consideration, thedeepest sites of stimulation in the present study were likely

M. Lanotte et al. / Brain, Behavior, and Immunity 19 (2005) 500–509 507

to involve the limbic structures of the subthalamic region.This is further supported by the subjective report of plea-sure and well being in two patients when two stimulationswere performed at a depth of 11 and 12 mm from thebeginning of recording (Fig. 2). Most interesting, the overtand covert stimulations of this area produced very diVer-ent outcomes. Sometimes the open–hidden diVerence wasas large as 50%, both for heart rate and sympatheticresponses (Fig. 2). Moreover, the stimulus–responsecurves of both heart rate and sympathetic LF activitywere dramatically reduced in the hidden condition (Figs.4A and B). Thus, these responses in the subthalamic lim-bic region appear to be context dependent, and are inagreement with previous studies which indicate that theparticular responses evoked are not related to speciWcelectrode locations, but rather to the subject’s psychologi-cal traits and concerns. In other words, limbic stimulationappears to produce eVects that are dependent on theongoing context (Benedetti et al., 2004a; Halgren, 1982).

These data suggest that expectation might increase theexcitability of limbic structures, so that unexpected stimu-lations would require an increase in intensity. This notionis supported by another recent study on the expected andunexpected administration of methylphenidate in cocaineabusers (Volkow et al., 2003). In this study, the eVects ofmethylphenidate on brain glucose metabolism, measuredby [18F]deoxyglucose-positron emission tomography, wereanalyzed in two diVerent conditions: when cocaine abus-ers expected to receive the drug and when they did not. Inthe former case, the eVect was greater than in the latter. Inother words, expected methylphenidate induced largermetabolic changes in the brain compared to unexpecteddrug, thus indicating that expectation enhanced its phar-macological eVects. Interestingly, the diVerence in brainglucose metabolism between the expectation and no-expectation conditions was as large as 50%.

A limitation of the present study is that these eVectsoccurred in Parkinson patients and thus they do not nec-essarily reXect the physiology of healthy subjects.Impairment of the autonomic functions is common inParkinson’s disease (Netten et al., 1995), so that we can-not rule out a defective subthalamic autonomic circuitryin these patients. Nonetheless, it is important to remem-ber that context-dependent responses are also present inother conditions in which the limbic system is stimulated(Halgren, 1982), and similar autonomic responses arealso present in spasmodic torticollis and essential tremor(Thornton et al., 2002), which indicates that Parkinson’sdisease is not the only condition where these eVects canbe found.

The present Wndings highlight the important role ofexpectation in the therapeutic outcome, and underscorehow the placebo component of a treatment may be stud-ied without the administration of any placebo. Indeed,by administering treatments covertly, we eliminateexpectations and thus the placebo, or psychological,

component (Colloca et al., 2004; Price, 2001). We believethat this approach may yield important information onhow expectation aVects diVerent physiological functions,including the immune system. In fact, it is worth notingthat a link among psychosocial context, noradrenaline,and the immune system has been described recently inhumans through the noradrenaline-activated nuclearfactor NF-�s which, in turn, induces mononuclear cellactivation (Bierhaus et al., 2003).

In addition, it has long been known that immuneresponses can be classically conditioned, and this mightrepresent the basis of the placebo eVect in some illnesses.For example, Ader and Cohen (1982) provided experi-mental evidence that immunological placebo responsescan be obtained by pairing a solution of sodium saccha-rin (conditioned stimulus) with the immunosuppressivedrug cyclophosphamide (unconditioned stimulus). Micetreated in this way showed conditioned immunosuppres-sion, that is, immune responses to sodium saccharinalone. Ader et al. (1993) also showed that a conditionedenhancement of antibody production is possible usingan antigen as unconditioned stimulus of the immune sys-tem. In this case, mice were given repeated immuniza-tions with keyhole limpet hemocyanin (KLH) pairedwith a gustatory conditioned stimulus. A classically con-ditioned enhancement of anti-KLH antibodies wasobserved when the mice were re-exposed to the gusta-tory stimulation alone. These studies in animals havebeen repeated in humans. For example, Olness and Ader(1992) presented a clinical case study of a child withlupus erythematosus who received cyclophosphamidepaired with taste and smell stimuli. During the course of12 months, a clinically successful outcome was obtainedby using taste and smell stimuli alone on half themonthly chemotherapy sessions. In another study(Giang et al., 1996), multiple sclerosis patients receivedtreatments with cyclophosphamide (unconditioned stim-ulus) paired with anise-Xavored syrup (conditioned stim-ulus). Eight out of these 10 patients displayed decreasedperipheral leukocyte counts following the syrup alone,an eVect that mimics that of cyclophosphamide. Morerecently, repeated associations between cyclosporin A(unconditioned stimulus) and a Xavored drink (condi-tioned stimulus) have been found to induce conditionedimmunosuppression in humans, in which the Xavoreddrink alone produced a suppression of some immunefunctions, as assessed by means of interleukin-2 (IL-2)and interferon-gamma (IFN-�) mRNA expression,in vitro release of IL-2 and IFN-�, as well as lymphocyteproliferation (Goebel et al., 2002).

Although all these studies suggest a mechanism ofPavlovian conditioning in the placebo responses in theimmune system, we believe that a challenge for futureresearch will be to understand whether or not othermechanisms, like expectancies, are capable of aVectingimmunological placebo responses.

508 M. Lanotte et al. / Brain, Behavior, and Immunity 19 (2005) 500–509

Acknowledgments

This research was supported by grants from the Pro-ject “Neuroscience” of the National Research Council(01.00439.ST97 and 02.00529.ST97), from the Project“Alzheimer’s disease” (PFA/DML/UO6/2001 and PFA/DML/UO6/2001/AA) and “Surgical therapy ofadvanced Parkinson’s disease” of the Italian Ministry ofHealth, and from a grant of the Italian Ministry of Uni-versity and Research-FIRB (RBNE01SZB).

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