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educed Serotonin-1A Receptor Binding in Social nxiety Disorder

upert R. Lanzenberger, Markus Mitterhauser, Christoph Spindelegger, Wolfgang Wadsak, ikolas Klein, Leonhard-Key Mien, Alexander Holik, Trawat Attarbaschi, Nilufar Mossaheb, Julia Sacher, homas Geiss-Granadia, Kurt Kletter, Siegfried Kasper, and Johannes Tauscher

ackground: Results from studies in serotonin-1A (5-HT1A) knockout mice and previous positron emission tomography (PET) studies in umans imply a role for 5-HT1A receptors in normal state anxiety as well as in certain anxiety disorders. The objective of this study was to

nvestigate 5-HT1A receptor binding potential (BP) in social anxiety disorder (SAD).

ethods: Using PET and [carbonyl-11C]WAY-100635, we compared a homogeneous group of 12 unmedicated, male SAD patients with 18 ealthy control subjects (HC). A multivariate ANOVA with all regional BP values as dependent variables, age and four radiochemical variables s covariates was performed.

esults: We found a significantly lower 5-HT1A BP in several limbic and paralimbic areas but not in the hippocampus (p � .234) of SAD atients. The difference in 5-HT1A binding was most significant in the amygdala (�21.4%; p � .003). There was also a more than 20% lower -HT1A BP of SAD patients in the anterior cingulate cortex (p � .004), insula (p � .003), and dorsal raphe nuclei (p � .030).

onclusions: The lower 5-HT1A binding in the amygdala and mesiofrontal areas of SAD patients is consistent with 1) preclinical findings of levated anxiety in 5-HT1A knockout mice, 2) a previous PET study in healthy volunteers showing an inverse correlation between 5-HT1A BP nd state anxiety, and 3) another human PET study in patients with panic disorder showing reduced 5-HT1A binding, thus corroborating the

otential validity of 5-HT1A receptors as targets in the treatment of human anxiety disorders.

ey Words: Affective disorders, anxiety, PET, serotonin, social hobia, STAI, 5-HT1A

he neurotransmitter serotonin (5-HT) is involved in the regulation of mood and affective states. Drugs modulating the serotonergic system are widely used in the treatment

f anxiety disorders and depression, and agonists of the seroto- in-1A (5-HT1A) receptor have modest anxiolytic properties Blier and de Montigny 1999). Recent studies in humans Neumeister et al. 2004; Tauscher et al. 2001a), nonhuman rimates (Bethea et al. 2005), and rodents (Gross and Hen 2004; ross et al. 2002) suggest a central role for the 5-HT1A receptor

n the modulation of anxiety and fear. Neumeister et al. (2004) howed a significant reduction of 5-HT1A receptor binding in the ingulate cortex and midbrain raphe nuclei in patients with panic isorder, indicating both lower presynaptic and postsynaptic -HT1A receptor densities. The same research group reported no ignificant change in patients with posttraumatic stress disorder ompared with healthy subjects (Bonne et al. 2005). Sullivan et l. (2005) reported an association between lower 5-HT1A binding otential (BP) in patients suffering from major depression and omorbid panic disorder. In patients with chronic fatigue syn- rome and elevated Spielberger State-Trait Anxiety Inventory STAI) scores, a widespread reduction in 5-HT1A receptor bind- ng has been observed (Bailer et al. 2005).

rom the Departments of General Psychiatry (RRL, CS, NK, AH, TA, NM, JS, TG-G, SK, JT) and Nuclear Medicine (MM, WW, L-KM, KK), Medical Univer- sity of Vienna, Vienna, Austria; the Department of Pharmaceutical Tech- nology and Biopharmaceutics (MM, L-KM), University of Vienna, Vienna, Austria; and Eli Lilly and Co. (JT), Lilly Research Laboratories, Indianapolis, Indiana.

ddress reprint requests to Rupert Lanzenberger, M.D., Department of Gen- eral Psychiatry, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria; E-mail: [email protected].

eceived March 29, 2006; revised May 27, 2006; accepted May 31, 2006.

006-3223/07/$32.00 oi:10.1016/j.biopsych.2006.05.022

In a previous study, we demonstrated a negative correlation between 5-HT1A receptor BP and trait anxiety in healthy subjects (Tauscher et al. 2001a). This is in accordance with studies in 5-HT1A receptor knockout mice demonstrating increased anxi- ety-like behavior in several tests (Gross et al. 2000; Parks et al. 1998; Ramboz et al. 1998). To differentiate between state and trait effects of 5-HT1A receptor expression on the establishment of adult anxiety-like behavior, Gross et al. (2002) and Gross and Hen (2004) developed a transgenic mice model using time- dependent and tissue-specific regulation of 5-HT1A receptor expression. They revealed critical developmental periods for the establishment of adult anxiety behavior. These data suggest that early postnatal 5-HT1A receptor levels, especially in the frontal cortex and hippocampus, are important factors in the develop- ment of adult anxiety thresholds.

Using positron emission tomography (PET) and the highly specific and selective 5-HT1A receptor radioligand [carbonyl- 11C]WAY-100635, we have investigated the regional 5-HT1A BP in symptomatic patients suffering from social anxiety disorder (SAD) compared with a group of matched healthy control subjects for the first time. This study was focused on the limbic and paralimbic system including the amygdala, hippocampus, insula, cingulate, and orbitofrontal cortices, which have been shown to be hyperactive or hypoactive in anxiety disorders (Schneider et al. 1999; Talbot 2004; Tillfors et al. 2001) and anxious states (Benkelfat et al. 1995; Chua et al. 1999; Liotti et al. 2000). Given the stimulus-specific hyperreactivity of the amyg- dala in social phobia (Stein et al 2002; Tillfors et al. 2001; Veit et al. 2002; Wright et al. 2003) and the critical role of the amygdala in social fear conditioning (Morris et al. 2001; Pine et al. 2001; Robinson et al. 2005), we differentiated the hippocampal region from the amygdala in the mesiotemporal region. The anterior cingulate and orbitofrontal cortices were included in the pre- defined region of interest analysis because of the dense inter- connections between it and the amygdala, the central role of the

mesial frontal cortex in fear extinction (Amargos-Bosch et al.

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004; Milad and Quirk 2002; Milad et al. 2004), and the integra- ion of affective values of reinforcers (Kringelbach 2005).

ethods and Materials

ubjects Fourteen medication-free male outpatients with social anxiety

isorder and 18 healthy male control subjects were scanned with ET. Two patients were excluded from analysis: one because of rug consumption prior to the scan, discovered afterwards, and he second because of missing structural magnetic resonance maging (MRI). Details of subject characteristics including psy- hometric assessment results are given in Table 1. All healthy ubjects and most of the patients were recruited from the ommunity via advertisements, with four patients being recruited rom the outpatient clinic. Only male subjects were included in he study because of the influence of sex steroid hormones on he 5-HT1A receptor expression (Bethea et al. 2002; Bouali et al. 003; Parsey et al. 2002) and the large between-subject 5-HT1A eceptor variability that has been found in comprehensive inves- igations (Gunn et al. 1998; Rabiner et al. 2002). Consensus iagnoses according to DSM-IV-TR criteria were established by wo psychiatrists performing a routine clinical interview, the ini-International Neuropsychiatric Interview (MINI) (Sheehan

t al. 1998), and the Spielberger State-Trait Anxiety Inventory STAI) (Spielberger and Vagg 1984) on the day of the screening isit. The STAI was repeated on the day of the PET scan. All ubjects underwent a medical examination including general hysical and neurologic status, electrocardiogram, routine labo- atory screening, and medical history at the screening visit to xclude subjects with relevant abnormalities, psychiatric comor- idities, and drug abuse. Six (50%) of 12 patients also fulfilled the riteria for agoraphobia. None of the patients had major depres- ion, because comorbid depression was an exclusion criterion, iven the reduced 5-HT1A receptor BP reported in several studies Drevets et al. 1999; Parsey et al. 2006; Sullivan et al. 2005). To xclude treatment effects (Bhagwagar et al. 2004; Rabiner et al. 000, 2002, 2004; Riad et al. 2004), it was ensured that patients ere naïve to psychotropic drug treatment targeting the seroto- ergic system (except one who had ceased selective serotonin euptake inhibitor [SSRI] medication 3 months prior to the PET

able 1. Demographic and Clinical Characteristics, and Radiochemical ariables. Values Represent Mean (�SD) in Healthy Control Subjects and ocial Anxiety Disorder (SAD) Patients

Control Subjects (n � 18)

SAD Patients (n � 12)

ge range (years) 19–44 23–44

mean � SD mean � SD ge (years) 27.0 � 5.8 30.3 � 6.4 ody mass index (kg/m2), BMI 23.4 � 2.5 25.0 � 4.1 pielberger State Anxiety Score, STAIa 32.4 � 4.5 46.8 � 7.9 pielberger State Anxiety Score, STAIb 32.3 � 5,0 43.8 � 7,8 pielberger Trait Anxiety Score, STAIa 34.3 � 6.6 51.3 � 9.2 pielberger Trait Anxiety Score, STAIb 33.3 � 6,8 48.5 � 9,4 ctivity Injected, ID (MBq) 421.3 � 15.7 398.7 � 35.2 adiochemical Purity, RCP (%) 97.8 � 1.4 97.9 � 1.1 pecific Activity (GBq/�mol) 29.7 � 21.7 23.4 � 13.9 eight of WAY 100634 (�g) 7.2 � 3.6 5.1 � 2.9 eight of unlabeled WAY100635 (�g) 10.7 � 10.1 11.2 � 10.3

a,b Spielberger State and Trait Anxiety Score, STAI (max � 80); ascreening

isit, bPET day.

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scan). Both the patients and the healthy volunteers gave written informed consent after the procedures had been fully explained at the screening visit. The study was approved by the Ethics Committee of the Medical University of Vienna and the General Hospital of Vienna. All subjects received reimbursement for participation. No significant differences of demographic and radiochemical variables were found between the groups (un- paired t test, p � .05, corrected).

PET Scanning Protocol and Preprocessing Positron emission tomography scans were performed on a GE

Advance PET scanner (General Electric Medical Systems, Mil- waukee, Wisconsin) at the Department of Nuclear Medicine, Medical University of Vienna, Austria. The heads of subjects were positioned in the scanner parallel to the orbitomeatal line using a laser beam system to ensure the covering of the cerebellum in the field of view (FOV). Head movements were minimized with a polyurethane molded cushion and straps about the forehead and chin, and subjects were instructed not to move or speak during the measurement. A 5-minute transmission scan was performed in two-dimensional mode for correction of tissue attenuation using a retractable 68Ge ring source. Dynamic PET scans were acquired in three-dimensional mode. Measurements started simultaneously with intravenous bolus injection of [car- bonyl-11C]WAY-100635 in phosphate-buffered saline using an average dose of 5.37 � .86 MBq per kilogram body weight. A series of 30 successive time frames (15 � 1 minute, 15 � 5 minutes) were collected, resulting in a total acquisition time of 90 minutes. The emission data were scatter and attenuation cor- rected using the transmission data. Thirty-five contiguous slices (matrix 128 � 128) with a slice thickness of 4.25 mm were reconstructed using an iterative filtered back-projection algo- rithm (FORE-ITER). The spatial resolution of the final recon- structed volume was 4.36 mm full-width at half maximum (FWHM) at the center of the FOV. No realignment for head movement or partial volume correction was applied. The 30 frames of dynamic PET imaging were summed (PETADD) for MRI-PET co-registration.

Radiochemistry [Carbonyl-11C]WAY-100635 was prepared at the Cyclotron

Unit of the PET center according to methods described previ- ously (Matarrese et al. 2002; McCarron et al. 1996), with slight modifications, in a fully automated PET synthesizer (GE Health- care, Uppsala, Sweden). Details of radiochemical variables are shown in Table 1. Injected activity, specific activity, radiochem- ical purity, weight of WAY-100634 (precursor), and weight of unlabeled WAY-100635 were used as covariates in the multivar- iate analysis of variance (ANOVA). The specific activity is defined as activity (GBq) per amount of substance (WAY 100635, �mol) determined by high-performance liquid chromatography (HPLC). The values were corrected for the time of tracer administration.

Magnetic Resonance Imaging High-resolution T1-weighted structural magnetic resonance

(MR) images (magnetization–prepared rapid gradient–echo [MPRAGE] sequence, 256 � 256 matrix, .78 �.86 mm voxel size, slice thickness 1.56 mm, 128 slices) were acquired in each subject using a 3 Tesla Medspec whole-body MR scanner (Bruker BioSpin, Ettlingen, Germany).

Region of Interest Analysis The structural MRI images were co-registered to the PET

ADD

images and resliced using Statistical Parametric Mapping version

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002 (SPM2; The Wellcome Department of Imaging Neuro- cience, University College London; www.fil.ion.ucl.ac.uk/spm/) Meyer et al. 1999). Regions of interest (ROIs) were defined a riori to enable comparison to other 5-HT1A receptor studies Bonne et al. 2005; Neumeister et al. 2004; Tauscher et al. 2001a) nd based on the functional MRI and PET literature in anxiety isorders (Adolphs 2002; Liotti et al. 2000; Pine et al. 2001; chneider et al. 1999; Tillfors et al. 2001) in areas expressing high evels of the 5-HT1A receptor (Rabiner et al. 2002; Varnas et al. 004). Five ROIs in the anterior cingulate cortex, medial orbito- rontal cortex, insula, amygdala, hippocampus, and one refer- nce region in the cerebellum were drawn bilaterally on co- egistered MR images using the anatomical criteria established by remner et al. (1998). Due to the absence of MRI criteria for aphe boundaries, the raphe region in the midbrain was directly raced onto the PETADD image. To delineate the dorsal raphe ucleus, we identified the slices showing the interpeduncular istern on the triplanar view of co-registered MR images and laced a fixed-size cubic volume of interest (VOI) (747mm3) over he area with highest signal on the corresponding PETADD slices Abrams et al. 2004).

racer Kinetic Modeling of [carbonyl-11C]WAY-100635 mission Data

For quantification of the 5-HT1A receptor BP, we used the inetic modeling tool of the biomedical image quantification oftware PMOD 2.7 (PMOD Technologies Ltd., Zurich, Switzer- and) (Mikolajczyk et al. 1998). The simplified reference tissue odel (SRTM), based on a two-tissue compartmental model

Gunn et al. 1998; Lammertsma and Hume 1996), was applied sing the cerebellum as a reference region because of its low -HT1A receptor density (Burnet et al. 1997; Hall et al. 1997). ecay-corrected time activity curves (TACs) were obtained using

he 30 frames of the dynamic PET data and the three-dimensional OIs. We calculated the regional BP and the regional relative elivery of the radioligand normalized to the cerebellum (R1). Right nd left ROIs were combined (except for the raphe region) to mprove signal-to-noise ratio. The SRTM is sensitive to group ifferences in the radioligand kinetics of the reference region. e therefore examined the cerebellar TACs and found no

ignificant group difference (two-tailed t test, p � .163) between reas under the curve between cerebellar TACs of SAD patients nd healthy control subjects.

tatistical Analysis Statistical analyses were performed using SPSS 12.0.1 (SPSS

nc., Chicago, Illinois). The Kolmogorov-Smirnov test (p � .05) or normal distribution and the Levene’s test for equality of ariance (p � .05) were used prior to parametric statistical nalyses. All parametric tests were two-tailed. Significance was et at p � .05. Pearson product moment correlation coefficients ere calculated between regional BP values and demographic

age, body mass index) and radiochemical variables. To exclude ny possible bias by summation effects, we included all radio- hemical variables (see Table 1) as covariates in the multivariate NOVA. Age-dependent reduction of 5-HT1A BP is a matter of ebate (Rabiner et al. 2002; Tauscher et al. 2001b) and there may e age-dependent regional effects on perfusion and tracer deliv- ry, so age was controlled for. A multivariate ANOVA with all egional BP values as dependent variables; group (patients ersus control subjects) as a fixed factor; and age, injected ctivity, specific activity, weight of WAY-100634, and radiochem-

cal purity as covariates was performed. To exclude the influence

on regional BP of tracer delivery differences between groups, we performed an additional multivariate ANOVA with all regional R1 values as dependent variables, group as a fixed factor, and age and the four radiochemical variables as covariates. When signif- icant group-by-region differences for BP values were obtained with ANOVA, post hoc t tests (unpaired, two-tailed, alpha level p � .05) in eight ROIs were calculated. Significance levels were corrected for the multiple comparisons involved in employing eight ROIs. The Bonferroni adjusted p value was p � .00625. A Pearson product moment correlation and partial correlations controlling for age and radiochemical variables between anxiety scores (STAI) and regional 5-HT1A BP were performed (two- tailed, p � .05).

Results

Comparison Between SAD Patients and Healthy Control Subjects

The multivariate ANOVA revealed a significant reduction of 5-HT1A BP in SAD patients compared with healthy control subjects (p � .041; Pillai’s Trace). Including age and radiochem- ical variables as covariates in the multivariate ANOVA, we found a highly significant reduction in 5-HT1A binding in the amygdala (p � .003; �21.4%, uncorrected for multiple comparison, post hoc t test), in the anterior cingulate cortex (p � .004; �23.8%), and in the insula (p � .003; �28.0%), and a significant reduction in the raphe region (p � .030; �36.4%) and the medial orbito- frontal cortex (p � .032; �18.4). Applying a Bonferroni correc- tion for multiple comparisons, the amygdala region (p � .024), the insula (p � .024), and the anterior cingulate cortex (p � .032) remained significant. Details are given in Table 2. Excluding age as a covariate in the multivariate ANOVA, we found a highly significant group difference in the anterior cingulate cortex (p � .003), insula (p � .002), and amygdala (p � .004), and a significant difference in the raphe nuclei (p � .025) and the medial orbitofrontal cortex (p � .028), indicating a minor effect of age. To exclude possible bias introduced by ROI drawing, we additionally used a fixed size VOI in the amygdala and calculated a multivariate ANOVA, which further improved the significance level (p � .001; �23.8% group difference) of the post hoc t test. There was no relevant improvement applying a fixed size VOI in the hippocampal head (p � .234; �9.7% to p � .210; �11.5%). Parametric 5-HT1A receptor binding potential maps superim- posed on co-registered T1-weighted MR images are shown in Figure 1. Figure 2 shows scatter histograms of the 5-HT1A receptor BP values comparing patients with healthy control subjects.

An additional ANOVA with all regional R1 values as depen- dent variables and age as a covariate showed no significant group effect (p � .504). Including the radiochemical variables additionally as covariates showed no significant effect (p � .361). Both ANOVA results indicate no bias due to tracer delivery differences between groups. No significant correlations (p � .05, corrected) between 5-HT1A BP or R1 and demographic as well as radiochemical variables were found. There were no significant group differences between the SAD patients and the healthy control subjects concerning age, body mass index, and radio- chemical variables.

To investigate the effect of comorbid agoraphobia on 5-HT1A BP, we performed calculations on subgroups. Excluding the six SAD patients with comorbid agoraphobia reduced the signifi- cance levels of the multivariate ANOVA only slightly (p � .075,

n � 24; controlled for age and radiochemical variables). Exclud-

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ng the six SAD patients without comorbid agoraphobia dramat- cally reduced the significance level of the multivariate ANOVA p � .387, n � 24).

elationships Between Anxiety Scores (STAI) and Regional -HT1A Receptor Binding Potential

Spielberger State-Trait Anxiety Inventory scores obtained at he screening visit and on the day of the PET scan were ignificantly lower in the patient than the healthy control group p � .001). Details are given in Table 1. State and trait scores on oth days correlated significantly (p � .001). A Pearson correla- ion analysis revealed no significant correlation between state or rait anxiety scores and regional 5-HT1A BP in both groups. A artial correlation controlling for age and radiochemical vari- bles also showed no significant relationship between 5-HT1A inding and anxiety scores. As shown in Figure 3, the values in he correlation plot are clustered into two groups, differentiating learly between social anxiety disorder patients and healthy ontrol subjects.

iscussion

We have demonstrated a significantly lower 5-HT1A receptor inding potential in male patients suffering from SAD than in an ge-matched healthy control group. Significant 5-HT1A BP reduc- ions (corrected for multiple tests) were found in the amygdala �21.4%), the anterior cingulate cortex (�23.8%), and insula �28.0%), indicating alterations in limbic and paralimbic areas of AD patients. The reduction was most prominent in the raphe uclei (�36.4%) but did not survive correction for multiple esting in this area. Given the inclusion criteria of our subjects, e can exclude the possibility that the reduction in 5-HT1A BP as caused by exposure to psychotropic drugs or comorbid epression (Bailer et al. 2005; Drevets et al. 1999; Sargent et al. 000; Sullivan et al. 2005). Our results are partly concordant with recently published study in panic disorder patients (Neumeister t al. 2004). This group showed a reduction in 5-HT1A receptor inding of 27.6% in the anterior cingulate cortex, comparable ith our result of 23.8%. In contrast to our study, they found no ifference in the anterior insula and mesiotemporal cortex ncluding both the hippocampus and amygdala. We differenti- ted the amygdala from the hippocampus region a priori because f comprehensive literature emphasizing the central role of the

able 2. Regional 5-HT1A Receptor Binding Potential Values in SAD Patient

egion of Interest

Binding Potential Values

Controls SAD Subjects (n � 18) (n � 12)

Mean SD Mean SD

ippocampus (head) 6.2 � 1.9 5.6 � 2.2 ippocampus (fixed VOI)c 6.1 � 1.9 5.4 � 2.2

nsula 5.0 � 1.0 3.6 � 1.2 nterior Cingulate Cortex 4.2 � .8 3.2 � 1.0 mygdala 4.2 � 1.1 3.3 � 1.2 mygdala (fixed VOI)c 4.2 � 1.2 3.2 � 1.2 edial Orbitofrontal Cortex 3.8 � .8 3.1 � 1.1

aphe Nuclei (fixed VOI)c 2.2 � .7 1.4 � .6

aCalculation of mean BP reduction: (100*BPpatients/BPcontrols) � 100. bSignificance levels corrected for age and four radiochemical covariates cfixed VOI, fixed size for volume of interest. dp values surviving the Bonferroni correction for multiple comparison im

mygdala in neural processing of fear and anxiety. Investigating

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six regions of interest, we found the most significant group difference in the amygdala, suggesting an area-specific effect on serotonergic transmission in SAD. Given the high percentage (50.0%) of comorbid agoraphobia in the patient sample, our results could be thought to reflect a main effect of agoraphobia on reduced 5-HT1A BP. The subgroup analysis detailed in Results did not support the view that our results could reflect effects attributable solely to agoraphobia, although the small sample size might limit its validity.

Modulation of Amygdala Activity by Mesiofrontal Areas Given the central role of the amygdala in fear conditioning

and processing (Adolphs et al. 2005; Morris et al. 2001), we expected and found significant differences in 5-HT1A binding of the amygdala region between SAD patients and control subjects. The amygdala is strongly modulated by mesiofrontal areas. The anterior cingulate cortex and neighboring prefrontal areas con- trol the attention to threat-related stimuli and inhibit amygdala activity by top-down regulation (Bishop et al. 2004). In rats, destruction of ventral prefrontal cortex blocks recall of fear extinction, indicating the storage of long-term extinction memory in paralimbic areas and not in the amygdala (Milad and Quirk 2002). Studies in nonhuman primates have confirmed the central role of the anterior cingulate region (Kalin et al. 2005) and the orbitofrontal cortex (Kalin and Shelton 2003) in the regulation of amygdala activity. Increased amygdala activity is associated with diminished activation of the mesiofrontal cortex in anxiety disorders (Shin et al. 2005). The exaggerated amygdala respon- siveness frequently shown in functional magnetic resonance imaging (fMRI) studies investigating SAD patients (Birbaumer et al 1998; Lorberbaum et al. 2004; Schneider et al. 1999; Tillfors et al. 2002) might be associated with alterations of the seroto- nergic system in mesiofrontal areas. Several functional studies have reported increased or altered amygdala activation to facial stimuli in social phobia, confirming the specific association of faces with anxiety in this disorder. A recently published PET study of panic disorder patients demonstrated a lack of appro- priate top-down regulation of the orbitofrontal region on amyg- dala activity (Kent et al. 2005). These results in limbic and paralimbic regions are consistent with our data, which demon- strate area-specific 5-HT1A alterations in anxiety circuitry includ-

Healthy Controls

Volume of Interest, VOI [cm3]

Comparison of Means(n � 30)

Mean SD Percentage Reductiona pb

1.2 � .2 �9.7% .234 1.2 � .1 �11.5% .210 5.9 � 1.3 �28.0% .003d

2.1 � .6 �23.8% .004d

1.5 � .3 �21.4% .003d

1.3 � .1 �23.8% .001d

9.3 � 2.2 �18.4% .032 .7 � .0 �36.4% .030

dicated in the statistical section (post hoc t tests, ANOVA).

g eight ROIs (p � 0.00625; Bonferroni adjusted p value at 8 ROIs).

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he Function of the Insula Region in the Anxiety Circuitry In SAD patients, we found a significantly lower 5-HT1A

eceptor binding in the insula region, including the anterior and osterior parts, in the region of interest analysis. Neumeister et l. (2004), investigating the anterior insula, did not find lower -HT1A receptor binding in patients with panic disorder in this egion. The same group reported no significant reduction in -HT1A binding in the anterior insula when investigating patients ith posttraumatic stress disorder (Bonne et al. 2005). Given the

mportant role of the polymodal insula region in the anxiety ircuitry, we would expect changes of serotonergic transmission n this region. Patients suffering from social phobia had increased ctivity in the insula, amygdala, pons, and temporal pole, and ecreased activity in the anterior cingulate and prefrontal cortex n an fMRI paradigm using anticipatory anxiety (Lorberbaum et l. 2004). Chua et al. (1999), investigating healthy subjects with

igure 1. Parametric 5-HT1A receptor binding potential (BP) maps superim A) compared with a representative patient suffering from social anxiety dis he corresponding area in the mesial orbitofrontal cortex. The color table i inding potential; MR, magnetic resonance; SAD, social anxiety disorder.

MRI, revealed an area-specific activiation pattern restricted to

the insula, orbitofrontal region, anterior cingulate, and temporal cortices in anticipatory anxiety. Anxiety in healthy subjects was associated with specific activations in the ventral insula, orbito- frontal cortex, and anterior temporal region (Liotti et al. 2000). To summarize, functional data suggest the specific and strong involvement of the insula and mesiofrontal region in processing of anxiety consistent with our finding of reduced 5-HT1A receptor binding.

The 5-HT1A Receptor in the Hippocampus In the hippocampus, we found no group difference in 5-HT1A

receptor binding between patients and healthy control subjects. This is consistent with functional neuroimaging studies in healthy subjects (Benkelfat et al. 1995; Chua et al. 1999; Liotti et al. 2000), patients suffering from anxiety disorders (Bonne et al. 2005; Kent et al. 2005; Tillfors et al. 2001; Wright et al. 2003), and studies in

d on the co-registered T1-weighted MR images of a single healthy subject (SAD) (B). White cross in the triplanar view (coronal, sagittal, axial) indicates tes the 5-HT1A receptor binding potential values. 5-HT1A, serotonin-1A; BP,

pose order

nonhuman primates (Kalin et al. 2004, 2005). These studies have

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hown that the hippocampus is not a central part of anxiety ircuitries. To exclude possible effects of hippocampal and mygdala size differences between the groups, we additionally alculated the binding potential using fixed volumes of interest 1.2 cm3 for the hippocampus, 1.3 cm3 for the amygdala). The ignificance levels were slightly higher in the amygdala (p � .003 o p � .001) and the hippocampus (p � .234 to p � .210), ndicating that no bias was introduced in the manual tracing of OIs on co-registered MR images. Given the long PET measure- ent time of 90 minutes, a relevant underestimation of BP by late inding equilibrium in receptor-rich regions such as the hip- ocampus or insula is unlikely (Parsey et al. 2000; Tauscher et al. 002).

nxiety Scores and Regional 5-HT1A Binding Investigating 30 subjects, we found no significant relationship

etween either trait or state anxiety scores (STAI) and regional -HT1A BP in male SAD patients and healthy control subjects. hese results are consistent with the comprehensive PET study ublished by Rabiner et al. (2002) performing the STAI in 66 healthy ale subjects. In addition, Neumeister et al. (2004) compared atients suffering from panic disorder to healthy control subjects 67.7% female subjects) using the Panic Disorder Severity Scale PDSS) and did not report a correlation between PDSS scores and egional 5-HT1A binding. Investigating patients with chronic atigue syndrome and healthy control subjects, Cleare et al. 2005) also found no significant correlation between STAI scores nd regional 5-HT1A receptor binding in 21 ROIs. We reported an nverse relationship between 5-HT receptor binding and anx-

igure 2. Scatter histograms of the 5-HT1A receptor BP values for healthy c isorder (SAD �‘, red, n � 12). Mean (� SD) bars indicate the significant di

003*), anterior cingulate cortex (p � .004), and raphe nucleus (p � .030). omparison (Bonferroni correction) implying six ROIs. 5-HT1A, serotonin-1A OI, region of interest.

1A

ety scores in a group of 19 healthy subjects (42% female

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subjects) using the Revised NEO Personality Inventory (Tauscher et al. 2001a). However, none of the correlations in the four ROIs delineated (prefrontal, parietal, occipital, and anterior cingulate cortices) survived a correction for multiple comparisons (Taus- cher et al. 2001a). In addition, Sullivan et al. (2005) recently reported a positive correlation between 5-HT1A binding and anxiety scores in 28 patients with major depressive disorder (MDD) including comorbid anxiety disorders (43%) and found lower 5-HT1A BP in MDD patients with comorbid anxiety disor- der than in those with MDD only. It is possible that our finding of no significant relationship between regional 5-HT1A BP and patients’ anxiety scores arises because of insufficient statistical power, with a sample size of only 12. Given the high intersubject variability of 5-HT1A BP (Gunn et al. 1998; Rabiner et al. 2002) and the strong effects of steroid hormones on 5-HT1A receptor expression (Bethea et al. 2002; Parsey et al. 2002), the results in our homogeneous group of 18 male control subjects do not support a significant relationship between trait or state anxiety and 5-HT1A BP in the amygdala, hippocampus, insula, anterior cingulate, and orbitofrontal cortices in healthy male subjects.

The Relationship Between Postsynaptic 5-HT1A Levels and Serotonergic Neurotransmission

The 5-HT1A receptor is the major inhibitory serotonergic receptor on glutaminergic and gamma-aminobutyric acid (GABA)ergic neurons in the frontal cortex (Amargos-Bosch et al. 2004; Puig et al. 2005; Santana et al. 2004). In addition, 5-HT1A receptors are involved in the control of serotonergic neurons in the dorsal raphe nuclei by the medial prefrontal cortex (Celada et

l subjects (HC � ●, blue, n � 18) and patients suffering from social anxiety ces between SAD and control group in the insula (p � .003*), amygdala (p � risk (*) indicates significant values (p � .05) after adjustment for multiple inding potential; HC, healthy control subjects; SAD, social anxiety disorder;

ontro fferen

Aste ; BP, b

al. 2001, 2002), suggesting an important feedback mechanism

b a t e n p r a 5 g c s l i s l a ( F a D c o c 5 1 i a d e i i h

F s ( s vento

R.R. Lanzenberger et al. BIOL PSYCHIATRY 2007;61:1081–1089 1087

etween serotonergic firing in the raphe region and prefrontal ctivity. Therefore, the balance of inhibitory 5-HT1A and excita- ory serotonin-2A (5-HT2A) receptors strongly influences the ffects of serotonergic firing on glutaminergic and GABAergic eurons in the frontal cortex but also in the amygdala, hippocam- us, and insula (Martin-Ruiz et al. 2001). Pharmacological studies in odents have shown the anxiolytic effect of both 5-HT1A agonism nd 5-HT2A antagonism (Delgado et al. 2005). Lower postsynaptic -HT1A receptor levels reduce the inhibitory influence of serotoner- ic neurons on glutaminergic and GABAergic neurons in the frontal ortex (Amargos-Bosch et al. 2004; Santana et al. 2004). Increased erotonergic phasic firing by reduced presynaptic 5-HT1A receptor evels in the raphe nuclei and increased serotonergic excitatory nfluence on glutaminergic and GABAergic neurons might shift the erotonergic influence on frontal neurons. Lower 5-HT1A receptor evels might be induced by a downregulation mechanism in the dult subject, e.g., by hormonal influence on receptor expression Andrade et al. 2005; Bethea et al. 2002, 2005; Bouali et al. 2003; ernandes et al. 1997; Meijer and de Kloet 1998) or congenital nd genetic effects (Gross and Hen 2004; Gross et al. 2002). ownregulation of 5-HT1A receptors in the anterior cingulate ortex, insula, and amygdala suggests an area-specific regulation f 5-HT1A receptor expression in these areas involved in pro- essing of anxiety-associated stimulation. Given the lower -HT1A BP in depression (Bhagwagar et al. 2004; Drevets et al. 999; Sargent et al. 2000), reduced 5-HT1A receptor levels might ncrease the vulnerability for affective disorders, including both nxiety disorders and depression. Our data are not sufficient to ifferentiate between neuroplastic processes on 5-HT1A receptor xpression in glutaminergic and GABAergic neurons and primar- ly reduced 5-HT1A receptor levels changing the serotonergic nfluence on frontal and limbic neurons. In addition, steroid

igure 3. Relationship between 5-HT1A receptor BP values in the amygdala a cores. Values are clustered into two groups, and grouping differentiates bet ●, blue). There was no significant correlation between STAI state or trait scor erotonin-1A; BP, binding potential; STAI, Spielberger State-Trait Anxiety In

ormones such as glucocorticoids regulate the 5-HT1A receptor

expression by transcriptional repression of the receptor promoter sequence (Meijer et al. 2000). Dysregulations of the hypothalam- ic-pituitary-adrenal (HPA) axis have been frequently shown in anxiety disorders and depression. Therefore, reduced 5-HT1A receptor levels and changes in the serotonin system across different types of anxiety disorders and depression might be caused by HPA axis dysregulation.

Conclusion The main finding of this PET study is a significantly lower

5-HT1A receptor binding in patients suffering from social anxiety disorder compared with healthy control subjects, indicating an altered serotonergic neurotransmission in SAD and specifically implicating 5-HT1A receptors as a potential target of interest for novel therapeutics to treat this condition. These results further emphasize the central role of the amygdala, the mesiofrontal cortex, and the polymodal insula in the modulation of anxious states.

This research was supported by Grants from the Austrian Science Fund (P16549) and the National Alliance for Research on Schizophrenia and Depression (NARSAD) to JT and a Grant from the Austrian National Bank (OENB P11468) to RRL.

We are grateful to M. Willeit, D. Hussey, and U. Bailer for discussions. We thank A. Konstantinidis, A. Schosser, J. Kindler, and H. Nassan-Agha for blood sampling and subject screening assessments. We are especially indebted to C. Poetzi and C. Novotny for clinical support and M. Paul, G. Dobrozemsky, G. Wagner, B. Reiterits, I. Leitinger, and R. Bartosch for technical support during the positron emission tomography (PET) scans.

Parts of this work were presented at the American College of Neuropsychopharmacology (ACNP), December 2005, Hilton

Spielberger State-Trait Anxiety Inventory state (left) and trait (right) anxiety social anxiety disorder (SAD) patients (‘, red) and healthy control subjects 5-HT1A binding in patients (SAD) and healthy control subjects (HC). 5-HT1A,

ry; SAD, social anxiety disorder; HC, healthy control subjects.

nd the ween es and

Waikoloa Village, Waikoloa, Hawaii.

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brams JK, Johnson PL, Hollis JH, Lowry CA (2004): Anatomic and functional topography of the dorsal raphe nucleus. Ann N Y Acad Sci 1018:46 –57.

dolphs R (2002): Neural systems for recognizing emotion. Curr Opin Neuro- biol 12:169 –177.

dolphs R, Gosselin F, Buchanan TW, Tranel D, Schyns P, Damasio AR (2005): A mechanism for impaired fear recognition after amygdala damage. Nature 433:68 –72.

margos-Bosch M, Bortolozzi A, Puig MV, Serrats J, Adell A, Celada P, et al. (2004): Co-expression and in vivo interaction of serotonin1A and serotonin2A receptors in pyramidal neurons of prefrontal cortex. Cereb Cortex 14:281–299.

ndrade TG, Nakamuta JS, Avanzi V, Graeff FG (2005): Anxiolytic effect of estradiol in the median raphe nucleus mediated by 5-HT1A receptors. Behav Brain Res 163:18 –25.

ailer UF, Frank GK, Henry SE, Price JC, Meltzer CC, Weissfeld L, et al. (2005): Altered brain serotonin 5-HT1A receptor binding after recovery from anorexia nervosa measured by positron emission tomography and [carbonyl11C]WAY-100635. Arch Gen Psychiatry 62:1032–1041.

enkelfat C, Bradwejn J, Meyer E, Ellenbogen M, Milot S, Gjedde A, et al. (1995): Functional neuroanatomy of CCK4-induced anxiety in normal healthy volunteers. Am J Psychiatry 152:1180 –1184.

ethea CL, Lu NZ, Gundlah C, Streicher JM (2002): Diverse actions of ovarian steroids in the serotonin neural system. Front Neuroendocrinol 23:41– 100.

ethea CL, Pau FK, Fox S, Hess DL, Berga SL, Cameron JL (2005): Sensitivity to stress-induced reproductive dysfunction linked to activity of the seroto- nin system. Fertil Steril 83:148 –155.

hagwagar Z, Rabiner EA, Sargent PA, Grasby PM, Cowen PJ (2004): Persis- tent reduction in brain serotonin1A receptor binding in recovered de- pressed men measured by positron emission tomography with [11C]WAY-100635. Mol Psychiatry 9:386 –392.

irbaumer N, Grodd W, Diedrich O, Klose U, Erb M, Lotze M, et al. (1998): fMRI reveals amygdala activation to human faces in social phobics. Neurore- port 9:1223–1226.

ishop S, Duncan J, Brett M, Lawrence AD (2004): Prefrontal cortical function and anxiety: Controlling attention to threat-related stimuli. Nat Neurosci 7:184 –188.

lier P, de Montigny C (1999): Serotonin and drug-induced therapeutic responses in major depression, obsessive-compulsive and panic disor- ders. Neuropsychopharmacology 21(suppl 2):91S–98S.

onne O, Bain E, Neumeister A, Nugent AC, Vythilingam M, Carson RE, et al. (2005): No change in serotonin type 1A receptor binding in patients with posttraumatic stress disorder. Am J Psychiatry 162:383–385.

ouali S, Evrard A, Chastanet M, Lesch KP, Hamon M, Adrien J (2003): Sex hormone-dependent desensitization of 5-HT1A autoreceptors in knock- out mice deficient in the 5-HT transporter. Eur J Neurosci 18:2203–2212.

remner JD, Bronen RA, De Erasquin G, Vermetten E, Staib LH, Ng CK, et al. (1998): Development reliability of a method for using magnetic reso- nance imaging for the definition of regions of interest for position emis- sion tomography. Clin Positron Imaging 1:145–159.

urnet PW, Eastwood SL, Harrison PJ (1997): [3H]WAY-100635 for 5-HT1A receptor autoradiography in human brain: A comparison with [3H]8-OH- DPAT and demonstration of increased binding in the frontal cortex in schizophrenia. Neurochem Int 30:565–574.

elada P, Puig MV, Casanovas JM, Guillazo G, Artigas F (2001): Control of dorsal raphe serotonergic neurons by the medial prefrontal cortex: In- volvement of serotonin-1A, GABA(A), and glutamate receptors. J Neuro- sci 21:9917–9929.

elada P, Puig MV, Martin-Ruiz R, Casanovas JM, Artigas F (2002): Control of the serotonergic system by the medial prefrontal cortex: Potential role in the etiology of PTSD and depressive disorders. Neurotox Res 4:409 – 419.

hua P, Krams M, Toni I, Passingham R, Dolan R (1999): A functional anatomy of anticipatory anxiety. Neuroimage 9:563–571.

leare AJ, Messa C, Rabiner EA, Grasby PM (2005): Brain 5-HT1A receptor binding in chronic fatigue syndrome measured using positron emission tomography and [11C]WAY-100635. Biol Psychiatry 57:239 –246.

elgado M, Caicoya AG, Greciano V, Benhamu B, Lopez-Rodriguez ML, Fernandez-Alfonso MS, et al. (2005): Anxiolytic-like effect of a serotoner- gic ligand with high affinity for 5-HT1A, 5-HT2A and 5-HT3 receptors. Eur J Pharmacol 511:9 –19.

revets WC, Frank E, Price JC, Kupfer DJ, Holt D, Greer PJ, et al. (1999): PET imaging of serotonin 1A receptor binding in depression. Biol Psychiatry

46:1375–1387.

ww.sobp.org/journal

Fernandes C, McKittrick CR, File SE, McEwen BS (1997): Decreased 5-HT1A and increased 5-HT2A receptor binding after chronic corticosterone associated with a behavioural indication of depression but not anxiety. Psychoneuroendocrinology 22:477– 491.

Gross C, Hen R (2004): The developmental origins of anxiety. Nat Rev Neuro- sci 5:545–552.

Gross C, Santarelli L, Brunner D, Zhuang X, Hen R (2000): Altered fear circuits in 5-HT(1A) receptor KO mice. Biol Psychiatry 48:1157–1163.

Gross C, Zhuang X, Stark K, Ramboz S, Oosting R, Kirby L, et al. (2002): Serotonin1A receptor acts during development to establish normal anx- iety-like behaviour in the adult. Nature 416:396 – 400.

Gunn RN, Sargent PA, Bench CJ, Rabiner EA, Osman S, Pike VW, et al. (1998): Tracer kinetic modeling of the 5-HT1A receptor ligand [carbonyl- 11C]WAY-100635 for PET. Neuroimage 8:426 – 440.

Hall H, Lundkvist C, Halldin C, Farde L, Pike VW, McCarron JA, et al. (1997): Autoradiographic localization of 5-HT1A receptors in the post-mortem human brain using [3H]WAY-100635 and [11C]way-100635. Brain Res 745:96 –108.

Kalin NH, Shelton SE (2003): Nonhuman primate models to study anxiety, emo- tion regulation, and psychopathology. Ann N Y Acad Sci 1008:189–200.

Kalin NH, Shelton SE, Davidson RJ (2004): The role of the central nucleus of the amygdala in mediating fear and anxiety in the primate. J Neurosci 24:5506 –5515.

Kalin NH, Shelton SE, Fox AS, Oakes TR, Davidson RJ (2005): Brain regions associated with the expression and contextual regulation of anxiety in primates. Biol Psychiatry 58:796 – 804.

Kent JM, Coplan JD, Mawlawi O, Martinez JM, Browne ST, Slifstein M, et al. (2005): Prediction of panic response to a respiratory stimulant by re- duced orbitofrontal cerebral blood flow in panic disorder. Am J Psychia- try 162:1379 –1381.

Kringelbach ML (2005): The human orbitofrontal cortex: Linking reward to hedonic experience. Nat Rev Neurosci 6:691–702.

Lammertsma AA, Hume SP (1996): Simplified reference tissue model for PET receptor studies. Neuroimage 4:153–158.

Liotti M, Mayberg HS, Brannan SK, McGinnis S, Jerabek P, Fox PT (2000): Differential limbic– cortical correlates of sadness and anxiety in healthy subjects: Implications for affective disorders. Biol Psychiatry 48:30 – 42.

Lorberbaum JP, Kose S, Johnson MR, Arana GW, Sullivan LK, Hamner MB, et al. (2004): Neural correlates of speech anticipatory anxiety in gener- alized social phobia. Neuroreport 15:2701–2705.

Martin-Ruiz R, Puig MV, Celada P, Shapiro DA, Roth BL, Mengod G, et al. (2001): Control of serotonergic function in medial prefrontal cortex by serotonin-2A receptors through a glutamate-dependent mechanism. J Neurosci 21:9856 –9866.

Matarrese M, Sudati F, Soloviev D, Todde S, Turolla EA, Kienle MG, et al. (2002): Automation of [11C]acyl chloride syntheses using commercially available 11C-modules. Appl Radiat Isot 57:675– 679.

McCarron JA, Turton DR, Pike VW, Poole KG (1996): Remotely-controlled production of the 5-HT1A receptor radioligand, [carbonyl-11C]-WAY- 100635, via 11Ccarboxylation of an immobilized Grignard reagent. J Labelled Comp Radiopharm 38:941–953.

Meijer OC, de Kloet ER (1998): Corticosterone and serotonergic neurotrans- mission in the hippocampus: Functional implications of central cortico- steroid receptor diversity. Crit Rev Neurobiol 12:1–20.

Meijer OC, Williamson A, Dallman MF, Pearce D (2000): Transcriptional re- pression of the 5-HT1A receptor promoter by corticosterone via miner- alocorticoid receptors depends on the cellular context. J Neuroendocri- nol 12:245–254.

Meyer JH, Gunn RN, Myers R, Grasby PM, et al. (1999): Assessment of spatial normalization of PET ligand images using ligand-specific templates. Neuroimage 9:545–553.

Mikolajczyk K, Szabatin M, Rudnicki P, Grodzki M, Burger C (1998): A JAVA environment for medical image data analysis: initial application for brain PET quatitation. Med Inform (Lond) 23:207–214.

Milad MR, Quirk GJ (2002): Neurons in medial prefrontal cortex signal mem- ory for fear extinction. Nature 420:70 –74.

Milad MR, Vidal-Gonzalez I, Quirk GJ (2004): Electrical stimulation of medial prefrontal cortex reduces conditioned fear in a temporally specific man- ner. Behav Neurosci 118:389 –394.

Morris JS, Büchel C, Dolan RJ (2001): Parallel neural responses in amygdala subregions and sensory cortex during implicit fear conditioning. Neuro-

image 13:1044 –1052.

N

P

P

P

P

P

P

R

R

R

R

R

R

S

S

R.R. Lanzenberger et al. BIOL PSYCHIATRY 2007;61:1081–1089 1089

eumeister A, Bain E, Nugent AC, Carson RE, Bonne O, Luckenbaugh DA, et al. (2004): Reduced serotonin type 1A receptor binding in panic disorder. J Neurosci 24:589 –591.

arks CL, Robinson PS, Sibille E, Shenk T, Toth M (1998): Increased anxiety of mice lacking the serotonin1A receptor. Proc Natl Acad Sci U S A 95: 10734 –10739.

arsey RV, Oquendo MA, Ogden RT, Olvet DM, Simpson N, Huang YY, et al. (2006): Altered serotonin 1A binding in major depression: A [carbon- yl-C-11]WAY100635 positron emission tomography study. Biol Psychia- try 59(2):106 –113.

arsey RV, Oquendo MA, Simpson NR, Ogden RT, Van Heertum R, Arango V, et al. (2002): Effects of sex, age, and aggressive traits in man on brain serotonin 5-HT1A receptor binding potential measured by PET using [C-11]WAY-100635. Brain Res 954:173–182.

arsey RV, Slifstein M, Hwang DR, Abi-Dargham A, Simpson N, Mawlawi O, et al. (2000): Validation and reproducibility of measurement of 5-HT1A receptor parameters with [carbonyl-11C]WAY-100635 in humans: Com- parison of arterial and reference tissue input functions. J Cereb Blood Flow Metab 20:1111–1133.

ine DS, Fyer A, Grun J, Phelps EA, Szeszko PR, Koda V, et al. (2001): Methods for developmental studies of fear conditioning circuitry. Biol Psychiatry 50:225–228.

uig MV, Artigas F, Celada P (2005): Modulation of the activity of pyramidal neurons in rat prefrontal cortex by raphe stimulation in vivo: Involve- ment of serotonin and GABA. Cereb Cortex 15:1–14.

abiner EA, Bhagwagar Z, Gunn RN, Cowen PJ, Grasby PM (2004): Preferen- tial 5-HT1A autoreceptor occupancy by pindolol is attenuated in de- pressed patients: Effect of treatment or an endophenotype of depres- sion? Neuropsychopharmacology 29:1688 –1698.

abiner EA, Gunn RN, Wilkins MR, Sargent PA, Mocaer E, Sedman E, et al. (2000): Drug action at the 5-HT(1A) receptor in vivo: Autoreceptor and postsynaptic receptor occupancy examined with PET and [carbonyl- (11)C]WAY-100635. Nucl Med Biol 27:509 –513.

abiner EA, Messa C, Sargent PA, Husted-Kjaer K, Montgomery A, Lawrence AD, et al. (2002): A database of [(11)C]WAY-100635 binding to 5-HT(1A) receptors in normal male volunteers: Normative data and relationship to methodological, demographic, physiological, and behavioral variables. Neuroimage 15:620 – 632.

amboz S, Oosting R, Amara DA, Kung HF, Blier P, Mendelsohn M, et al. (1998): Serotonin receptor 1A knockout: An animal model of anxiety- related disorder. Proc Natl Acad Sci U S A 95:14476 –14481.

iad M, Zimmer L, Rbah L, Watkins KC, Hamon M, Descarries L (2004): Acute treatment with the antidepressant fluoxetine internalizes 5-HT1A auto- receptors and reduces the in vivo binding of the PET radioligand [18F]MPPF in the nucleus raphe dorsalis of rat. J Neurosci 24:5420 –5426.

obinson S, Hoheisel B, Windischberger C, Habel U, Lanzenberger R (2005): FMRI of the emotions: Towards an improved understanding of amyg- dala function. Current Medical Imaging Reviews 1:115–129.

antana N, Bortolozzi A, Serrats J, Mengod G, Artigas F (2004): Expression of serotonin1A and serotonin2A receptors in pyramidal and GABAergic neurons of the rat prefrontal cortex. Cereb Cortex 14:1100 –1109.

argent PA, Kjaer KH, Bench CJ, Rabiner EA, Messa C, Meyer J, et al. (2000): Brain serotonin1A receptor binding measured by positron emission tomog-

raphy with [11C]WAY-100635: Effects of depression and antidepressant treatment. Arch Gen Psychiatry 57:174–180.

Schneider F, Weiss U, Kessler C, Muller-Gartner HW, Posse S, Salloum JB, et al. (1999): Subcortical correlates of differential classical conditioning of aver- sive emotional reactions in social phobia. Biol Psychiatry 45:863–871.

Sheehan DV, Lecrubier Y, Sheehan KH, Amorim P, Janavs J, Weiller E, et al. (1998): The Mini-International Neuropsychiatric Interview (M.I.N.I.): The development and validation of a structured diagnostic psychiatric inter- view for DSM-IV and ICD-10. J Clin Psychiatry 59 Suppl 20:22–33; quiz 34 –57.

Shin LM, Wright CI, Cannistraro PA, Wedig MM, McMullin K, Martis B, et al. (2005): A functional magnetic resonance imaging study of amygdala and medial prefrontal cortex responses to overtly presented fearful faces in posttraumatic stress disorder. Arch Gen Psychiatry 62:273–281.

Spielberger CD, Vagg PR (1984): Psychometric properties of the STAI: A reply to Ramanaiah, Franzen, and Schill. J Pers Assess 48:95–97.

Stein MB, Goldin PR, Sareen J, Zorrilla LT, Brown GG (2002): Increased amyg- dala activation to angry and contemptuous faces in generalized social phobia. Arch Gen Psychiatry 59:1027–1034.

Sullivan GM, Oquendo MA, Simpson N, Van Heertum RL, John Mann J, Parsey RV (2005): Brain serotonin(1A) receptor binding in major depression is related to psychic and somatic anxiety. Biol Psychiatry 58(12):947–954.

Talbot PS (2004): The molecular neuroimaging of anxiety disorders. Curr Psychiatry Rep 6:274 –279.

Tauscher J, Bagby RM, Javanmard M, Christensen BK, Kasper S, Kapur S (2001a): Inverse relationship between serotonin 5-HT(1A) receptor binding and anxiety: A [(11)C]WAY-100635 PET investigation in healthy volunteers. Am J Psychiatry 158:1326 –1328.

Tauscher J, Kapur S, Verhoeff NP, Hussey DF, Daskalakis ZJ, Tauscher- Wisniewski S, et al. (2002): Brain serotonin 5-HT(1A) receptor binding in schizophrenia measured by positron emission tomography and [11C]WAY-100635. Arch Gen Psychiatry 59:514 –520.

Tauscher J, Verhoeff NP, Christensen BK, Hussey D, Meyer JH, Kecojevic A, et al. (2001b): Serotonin 5-HT1A receptor binding potential declines with age as measured by [11C]WAY-100635 and PET. Neuropsychopharmacol- ogy 24:522–530.

Tillfors M, Furmark T, Marteinsdottir I, Fischer H, Pissiota A, Langstrom B, et al. (2001): Cerebral blood flow in subjects with social phobia during stress- ful speaking tasks: A PET study. Am J Psychiatry 158:1220 –1226.

Tillfors M, Furmark T, Marteinsdottir I, Fredrikson M (2002): Cerebral blood flow during anticipation of public speaking in social phobia: A PET study. Biol Psychiatry 52:1113–1119.

Varnas K, Halldin C, Hall H (2004): Autoradiographic distribution of serotonin transporters and receptor subtypes in human brain. Hum Brain Mapp 22:246 –260.

Veit R, Flor H, Erb M, Hermann C, Lotze M, Grodd W, et al. (2002): Brain circuits involved in emotional learning in antisocial behavior and social phobia in humans. Neurosci Lett 328:233–236.

Wright CI, Martis B, McMullin K, Shin LM, Rauch SL (2003): Amygdala and insular responses to emotionally valenced human faces in small animal

specific phobia. Biol Psychiatry 54:1067–1076.

www.sobp.org/journal