Effects of cognitive-behavioral therapy on brain activation in specific phobia

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1 NeuroImage 29 (2006) Effects of cognitive-behavioral therapy on brain activation in specific phobia Thomas Straube, a, * Madlen Glauer, a Stefan Dilger, a Hans-Joachim Mentzel, b and Wolfgang H.R. Miltner a a Department of Biological and Clinical Psychology, Friedrich-Schiller-University, Am Steiger 3,1, D Jena, Germany b Institute of Diagnostic and Interventional Radiology, Friedrich-Schiller-University, Jena, Germany Received 9 February 2005; revised 3 June 2005; accepted 5 July 2005 Available online 8 August 2005 Little is known about the effects of successful psychotherapy on brain function in subjects with anxiety disorders. The present study aimed to identify changes in brain activation following cognitive-behavioral therapy (CBT) in subjects suffering from specific phobia. Using functional magnetic resonance imaging (fmri), brain activation to spider videos was measured in 28 spider phobic and 14 healthy control subjects. Phobics were randomly assigned to a therapy-group (TG) and a waiting-list control group (WG). Both groups of phobics were scanned twice. Between scanning sessions, CBT was given to the TG. Before therapy, brain activation did not differ between both groups of phobics. As compared to control subjects, phobics showed greater responses to spider vs. control videos in the insula and anterior cingulate cortex (ACC). CBT strongly reduced phobic symptoms in the TG while the WG remained behaviorally unchanged. In the second scanning session, a significant reduction of hyperactivity in the insula and ACC was found in the TG compared to the WG. These results propose that increased activation in the insula and ACC is associated with specific phobia, whereas an attenuation of these brain responses correlates with successful therapeutic intervention. D 2005 Elsevier Inc. All rights reserved. Keywords: ACC; Insula; Phobia; Therapy; Amygdala; Threat Introduction The most common forms of specific phobia, an anxiety disorder with a high prevalence of approximately 10% in the general population, are related to small animals such as spiders, snakes, or rodents (Fyer, 1998). The functional neuroanatomy associated with symptoms of animal or other kinds of specific phobia is not yet clear. Neuroimaging studies investigating neuronal correlates of the processing of threat in specific phobia have provided mixed results. * Corresponding author. Fax: address: straube@biopsy.uni-jena.de (T. Straube). Available online on ScienceDirect ( Several previous positron-emission tomography (PET) studies found increased regional cerebral blood flow (rcbf) in extrastriate visual cortex but not in other brain regions during visual phobogenic stimulation in animal phobics (Fredrikson et al., 1993, 1995; Wik et al., 1993). A PET-study with animal phobics by Rauch et al. (1995) demonstrated activation of the anterior cingulate cortex (ACC), somatosensory cortex, thalamus, and a fronto-temporal region including the anterior insula during symptom provocation induced by tactile imagery of the feared stimulus. Similar results were also reported by other PET studies, which showed increased responses in anterior insula, ACC/ medial frontal cortex, and in the thalamus or midbrain to real feared animals or pictures of these animals (Carlsson et al., 2004; Reiman, 1997). Furthermore, recent functional resonance magnetic imaging (fmri) studies from our group found significant activation of the insula, ACC, and prefrontal cortex in response to visually presented phobia-related stimuli in animal phobics (Dilger et al., 2003; Straube et al., 2004b, in press). Especially, the findings regarding activation of the insula and ACC are in accordance with brain activation patterns observed during threat processing and symptom provocation in other anxiety disorders. For example, insula activation was shown in social phobia (Straube et al., 2004a, but see Stein et al., 2002; Tillfors et al., 2002), panic disorder (Reiman, 1997), or posttraumatic stress disorder (Osuch et al., 2001; Rauch et al., 1997). Significantly increased activation of the insula associated with provocation of clinically relevant anxiety symptoms was also reported by Rauch et al. (1997), who used pooled data across different anxiety disorders (obsessive compulsive disorder, specific phobia, posttraumatic stress disorder). In all studies included in the analysis of Rauch et al., activation of the ACC was observed as well. ACC responses during clinically relevant anxiety were also described by other authors (Aouizerate et al., 2004; Boshuisen et al., 2002; Osuch et al., 2001; Pissiota et al., 2002). Furthermore, an involvement of ACC and insula was found during the provocation of anxiety and during evaluation of visual threat signals such as fear conditioned fear stimuli and fearful facial expressions in healthy subjects (for /$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi: /j.neuroimage

2 126 T. Straube et al. / NeuroImage 29 (2006) reviews see Critchley, 2003; Phan et al., 2002; Reiman, 1997). It has been suggested that areas such as the ACC and anterior insula, but also the thalamus and prefrontal cortex might be part of a core anxiety system relevant for sustained evaluation of (potential) danger and the subjective experience of fear (Critchley, 2003; Rauch et al., 1997; Reiman, 1997). Remarkably, numerous functional imaging studies with patients suffering from specific phobia failed to show amygdala activation when subjects were exposed to phobia-relevant stimulation (Fredrikson et al., 1993, 1995; Mountz et al., 1989; Paquette et al., 2003; Pissiota et al., 2003; Rauch et al., 1995; Reiman, 1997; Wik et al., 1993, 1997). This is in contrast to the widely demonstrated role of the amygdala in the processing of fear-related stimuli and the mediation of fear responses in healthy subjects (e.g., Anderson et al., 2003; Breiter et al., 1996; Büchel et al., 1998; Critchley et al., 2002; Morris et al., 1998) and patients with other anxiety disorders (e.g., Birbaumer et al., 1998; Gilboa et al., 2004; Liberzon et al., 1999; Rauch et al., 2000; Shin et al., 1997a,b, 2004; Stein et al., 2002; Straube et al., 2004a; Tillfors et al., 2001, 2002). Previously, we suggested (Dilger et al., 2003; Straube et al., in press) that the absence of amygdala activation in several studies on specific phobia might be due to sustained periods of symptom provocation, leading to an attenuation of rapid amygdalar responses (see also Breiter et al., 1996; Büchel et al., 1998; Keightley et al., 2003; Taylor et al., 2003; Veltman et al., 2004; Wright et al., 2001). By means of event-related fmri, we were able to show increased amygdala activation in spider phobics during the processing of briefly presented phobia-related pictures (Dilger et al., 2003; Straube et al., in press; see also Carlsson et al., 2004). These findings strongly suggest a crucial function of the amygdala in the initial processing of phobia-related threat and in the induction of fear, while sustained processing of threat-related stimuli, such as confrontation with real, imagined, or filmed feared objects, does not seem to be based on amygdalar activity (see also Rauch et al., 1997; Walker et al., 2003). Although the most conclusive indices regarding the functional neuroanatomy of specific phobia may arise from studies investigating the neuronal effects of successful therapeutic intervention, only one such study has been performed so far (Paquette et al., 2003). In this fmri study, spider phobics showed a therapyinduced attenuation of hyperactivity, which had been observed pretreatment in the parahippocampal gyrus and dorsolateral prefrontal cortex (DLPFC) in response to spider vs. control videos. Results were interpreted to reflect a reduction in coping strategies (DLPFC) and in stimulus-related mnemonic processes (parahippocampal gyrus). Remarkably, no phobia-related activation of the insula, ACC, or other areas was detected. This outcome was attributed to the fact that threatening visual stimuli might not be suited to induce activation in these areas. However, this interpretation is not justified by results of several functional imaging studies (see above), which showed that the (anxious) evaluation of visual fear-relevant stimuli is associated with activation in the insula and ACC in phobics and healthy subjects. Although of high impact for the research regarding neurobiological correlates of psychotherapy, the study of Paquette and colleagues had the important limitation that no untreated phobic control group was included. Since repeated scanning sessions may be associated with confounding variables such as habituation, anticipation, and novelty effects (e.g., Johansen-Berg et al., 2002; McGonigle et al., 2000; Stark et al., 2004), a phobic waiting-list control group offers the possibility to properly control for these influences. Furthermore, the videos, which were used in the pre- and posttreatment scanning sessions, were also presented extensively between scanning sessions as part of the therapy. Therefore, no clear conclusion was possible whether the modification of brain activation was due to effects of the psychotherapy applied, or to other factors such as stimuli-specific habituation. The present study aimed at the investigation of brain activation to phobogenic videos in spider phobics and the effects of successful therapeutic intervention on these brain responses. The study included a healthy control group as well as a phobic waitinglist group. As therapeutic intervention, we administered CBT, which has been consistently shown to be highly effective in the reduction of phobic symptoms (e.g., Öst, 1989, 1996; Paquette et al., 2003). Data analysis was focused on those brain regions that have been suggested to play a critical role in the processing of visual threatening stimuli in specific phobia and/or in the mediation of phobic fear (DLPFC, anterior insula, ACC, thalamus, amygdala, parahippocampal gyrus, extrastriate cortex). Methods Subjects Twenty-eight female spider phobic subjects and 14 healthy female control subjects (mean age: 22.07, SD: 1.98) participated in the study. The participants were recruited by public advertisements. Subjects were diagnosed as spider phobics prior to the experiment according to the criteria of the diagnostic and statistic manual of mental disorders for spider phobia (DSM-IV, American Psychiatric Association, 1994) as assessed by a structured clinical interview (Wittchen et al., 1997). In addition, spider phobics had to show high scores on a spider phobia questionnaire [SPQ (Klorman et al., 1974); TG (finally analyzed subjects): mean = 22.69, SD = 2.95; WG (finally analyzed subjects): mean = 22.33, SD = 2.84]. Control subjects had to be free of any kind of phobia and to show low scores in the SPQ (mean = 1.57, SD = 1.60). Furthermore, according to the outcome of the structured clinical interview for DSM-IV, all subjects were free from additional psychopathological and neurological disorders. Subjects with spider phobia were randomly assigned to a therapy-group (TG) and a waiting-list control group (WG). The groups did not differ in phobia severity (based on SPQ scores), age, or level of education (all participants were university students). One subject of the TG did not participate in the second scanning session leaving a sample of 13 subjects in this group (mean age: 21.92, SD: 2.02). In the WG, two subjects had to be excluded due to a missing second scanning session and a panic attack during scanning, leaving a sample of 12 subjects for this group (mean age: 21.33, SD: 2.46). Subjects of the healthy control group (CG) received 6 Euro per hour for participation. The study was approved by the ethics committee of the University of Jena and written informed consent was obtained from each participant prior to the experiment. Stimuli and tasks Subjects were exposed to video clips depicting a moving spider on a grey background or as baseline condition a moving black small synthetic cylinder of comparable size. To assure high ecological relevance of spider stimuli, a German house spider (species Tegenaria atrica) most commonly occur-

3 T. Straube et al. / NeuroImage 29 (2006) ring in houses and cellars was used. The baseline control object had a metallic bottom and was moved by a magnet on the same grey background the spider had previously been filmed on. Both objects were filmed from a distance of 1 m. The range and speed of movements of the cylinder were matched as far as possible to the movements of the spider. In the scanner, the stimuli were presented via a back-projection screen on an overhead mirror (screen size: cm). Moving radius of both objects did not exceed 2 cm. Subjects saw a continuous stream of 5 different baseline clip sequences alternating with 4 different spider clip sequences. Each sequence lasted 24 s. The videos showed always the same spider or control object. Each sequence, however, displayed different unpredictable movement patterns. During the second scanning session (after therapy or waiting time), phobics were also presented with a stream of 5 control and 4 spider videos displaying the same spider and control object as during the first scanning session, but movement of both objects were different as compared to the presentations during the first scanning session. All subjects were tested before the experiment proper whether they were able to pay continuous attention to the videos (using an example of the videos), which was confirmed for all subjects After the scanning sessions, participants rated the fear-induction, valence, and arousal during the presentation of the spider and baseline clips using a 9 point Likert scale (fear: 1 = no fear to 9 = strongest fear; valence: 1 = most pleasant to 9 = most unpleasant; arousal: 1 = not arousing to 9 = most arousing). From one subject of the waiting-list group, ratings after the second scanning session could not be obtained due to technical problems. Behavioral data were analyzed by means of repeated measures analysis of variance (ANOVA) using SPSS (Version 10; SPSS, INC., Chicago). For post-hoc comparisons, Bonferroni correction was applied. A probability level of P < 0.05 was considered statistically significant. All data are expressed as means T SEM (standard error of mean). Therapy and therapy outcome measurements The cognitive-behavioral therapy was based mainly on the rapid gradual exposure to the feared animals according to Öst (1989, 1996) during two sessions with a duration of 4 5 h each. In the therapy group, the two sessions were conducted on succeeding days to the first scanning session, while the waiting group received therapy after the second scanning session. The CBT started with a problem analysis and general education of the types and role of misbeliefs about the dangerousness and properties of spiders and the major psychological reasons for the maintenance of fear reactions. Thereafter, the following therapeutic aims were stipulated: (1) to hold a living tarantula for about 10 min; (2) to catch moving and non-moving spiders at least 10 times with a glass at different locations within the therapy room; (3) to catch any species of spiders at least 3 times in the basement of the institute; (4) to touch a rapid moving house spider. Furthermore, all four treatment goals had to be fulfilled by subjects without strong feelings of anxiety. At the end of each exposure, anxiety ratings had to be below 50 on a scale that ranged from 0 (no fear at all) to 100 (strongest fear). Therapy was given in groups of 2 to 3 subjects by advanced students of psychotherapy under supervision of experienced psychologists (TS., SD., WHRM.). Gradual exposure started with the presentation of spider pictures. Then, subjects were exposed to the skin of a tarantula, followed by exposure to a real tarantula, which, at the end, had to be touched and to be taken into the hand. Thereafter, subjects were exposed to rapid moving spiders whose size increased across succeeding exposures. This procedure was continued until all therapeutic aims were reached. Participants were encouraged to catch as many spiders as possible outside the therapeutic setting and to avoid avoidance behavior. All subjects of the therapy group responded successfully to the therapy and reached the therapeutic aims. Additional outcome measurements included ratings of the video presentations (see Results) and posttherapy scores of the SPQ. For SPQ scores, the ANOVA showed a significant main effects of scanning session [ F(1,23) = 106.8, P < ] and group [ F(1,23) = 27.7, P < ], and an interaction of group by session [ F(1,23) = 109.2, P < ]. Posthoc tests revealed that posttherapy scores of the TG (mean = 7.69, SD = 5.38) were significantly reduced compared to pretherapy scores (t = 19.52, P < ) and to postwaiting time scores of the WG (mean = 22.42, SD = 3.6; t = 14.36, P < ). fmri In the 1.5 T magnetic resonance scanner ( Magnetom Vision plus, Siemens, Medical Systems), one run of 65 volumes was acquired per session using a T2*-weighted echo-planar sequence (TE = 50 ms, flip angle = 90-, matrix = 64 64, FOV = 192 mm, TR = 3.9 s). Phobics underwent two sessions separated by an interval of 2 weeks, except of one TG subject, who had to be measured with a 3 week interval due to illness. Control subjects were examined only once. Each volume comprised 38 axial slices (thickness = 2 mm, no gap, in plane resolution = 3 3 mm), which were acquired using a tilted angel to reduce susceptibility artifacts in inferior brain areas (Deichmann et al., 2003). The slices covered the whole brain except for the most superior part of the parietal cortex. Additionally, a high-resolution T1-weighted anatomical volume was recorded. Preprocessing and analysis of the functional data were performed using the software Brain Voyager 2000 (Version 4.9; Brain Innovation, Maastricht, The Netherlands). The first four volumes of each run were discarded from the analysis to ensure that steady state tissue magnetization was reached. The volumes were realigned to the first volume in order to minimize the effects of head movements on data analysis. Succeeding data preprocessing comprised spatial (8 mm fullwidth half-maximum isotropic Gaussian kernel) as well as temporal (high pass filter: 3 cycles per run) smoothing. Anatomical and functional images were coregistered and Table 1 Ratings: descriptive data Variable CG TG WG First First Second First Second Valence Spider 3.64 (0.39) 8.15 (0.25) 5.46 (0.24) 8.17 (0.27) 8.10 (0.25) Baseline 3.86 (0.40) 3.93 (0.37) 4.31 (0.38) 4.0 (0.48) 4.10 (0.55) Arousal Spider 2.14 (0.39) 7.70 (0.33) 3.15 (0.50) 7.42 (0.36) 7.45 (0.28) Baseline 1.64 (0.20) 2.08 (0.31) 1.54 (0.31) 2.67 (0.47) 3.10 (0.62) Fear Spider 1.07 (0.07) 6.30 (0.65) 2.77 (0.51) 6.25 (0.65) 6.27 (0.62) Baseline 1.00 (0.00) 1.38 (0.24) 1.15 (0.10) 1.58 (0.26) 2.27 (0.68) Data are given as mean (standard error of the mean).

4 128 T. Straube et al. / NeuroImage 29 (2006) normalized to the Talairach space (Talairach and Tournoux, 1988). Statistical analysis was performed by multiple linear regression of the signal time course at each voxel. The expected blood oxygen level-dependent (BOLD) signal change for the spider videos (=predictor) relative to the baseline videos was modeled by a canonical hemodynamic response function (modified gamma function; delta = 2.5, tau = 1.25). Withinand between-group statistical comparisons were conducted using a mixed effect analysis, which considers inter-subject variance and permits population-level inferences. In the first step, voxelwise statistical maps were generated and the relevant, planned contrasts of predictor estimates (beta-weights) were computed for each individual. In the second step, a random effect group analysis of these individual contrasts was performed. The results of the analysis were considered statistically significant for t values with P < within the a priori defined ROIs (see Straube et al., 2004a,b). A cluster threshold of 5 contiguously activated voxels was used to minimize false positive results. Only activated voxels (according to the statistical and cluster thresholds) within the ROIs were used for further analysis. The following ROIs were defined a priori: DLPFC, anterior insula, ACC, amygdala, parahippocampal gyrus, thalamus, and as extrastriate region the fusiform gyrus (Dilger et al., 2003; Straube et al., in press). The ROIs were defined using Talairach daemon software ( edu/projects/talairachdaemon.html). For exploratory analysis outside of the ROIs (without cerebellum), thresholds were set at P < (of at least 5 contiguous voxels). Results Behavioral data First scanning session Descriptive data for the ratings of valence, arousal, and fear associated with the stimuli are given in Table 1. Valence and arousal ratings of the videos showed a significant main effect of Table 2 Significant activation to spider vs. control videos in all groups during the first scanning session Region WG TG Controls x y z t value x y z t value x y z t value ROI DLPFC R L ACC Insula R L Amygdala R L Parahippocampal g R L Fusiform gyrus R L Thalamus R L Exploratory DLFC R L VLPFC R L Precentral gyrus R L Postcentral gyrus R Temporal gyri R L Parietal gyri R L Cuneus R L Precuneus R L Lingual gyrus R L Occipital gyri R L Basal ganglia R L DLFC, dorsolateral frontal cortex; DLPFC, dorsolateral prefrontal cortex; g, gyrus; L, left; R, right; VLPFC, ventrolateral prefrontal cortex; (x,y,z), Talairach coordinates of maximally activated voxel [Activation threshold: P < 0.005, uncorr. (ROI), P < , uncorr. (Exploratory), cluster 135 mm 3 ].

5 T. Straube et al. / NeuroImage 29 (2006) Table 3 Significant differences between control subjects and phobics a in brain activation to spider vs. control videos during the first scanning session Region of interest Controls > phobics Phobics > controls x y z t value x y z t value ROI ACC Insula R L Amygdala R L Parahippocampal g R L Exploratory Precentral gyrus R Postcentral gyrus R Lingual gyrus L DLFC, dorsolateral frontal cortex; DLPFC, dorsolateral prefrontal cortex; g, gyrus; L, left; R, right; VLPFC, ventrolateral prefrontal cortex; (x, y, z), Talairach coordinates of maximally activated voxel [Activation threshold: P < 0.005, uncorr. (ROI), P < , uncorr. (Exploratory), cluster 135 mm 3 ]. a There was no significant difference in activation between the TG and WG. group [valence: F(2,36) = 19.37, P < ; arousal: F(2,36) = 46.73, P < ] and object [valence: F(1,36) = , P < ; arousal: F(1,36) = , P < ] and a significant interaction of group by object [valence: F(2,36) = 41.24, P < ; arousal: F(2,36) = 50.15, P < ]. Post-hoc analysis revealed that both groups of phobics rated spider videos as significantly more unpleasant and arousing than control subjects (TG vs. CG: valence: t = 9.85, P < ; arousal: t = 12.64, P < ; WG vs. CG: valence: t = 9.59, P < ; arousal: t = 11.50, P < ), while no significant differences between groups were found for the control videos. There was also no significant difference in the valence and arousal ratings of spider videos between both groups of phobics (valence: t = 0.035, P > 0.5; arousal: t = 0.57, P > 0.5). For fear ratings, only phobics were included in the ANOVA, because controls did not show any variance or only marginal variance in their fear ratings of the control objects and spiders, which were rated as not fear-inducing at all (see Table 1). In phobics, the ANOVA showed a significant main effect of object [ F(1,23) = 125.0, P < ], which was based on increased ratings for spider videos as compared to control videos (t = 11.40, P < ). Second scanning session Descriptive data for the ratings of valence, arousal, and fear in response to the stimuli are given in Table 1. Ratings of the clips indicated a significant main effect of group [valence: F(1,22) = 19.10, P < ; arousal: F(1,22) = 34.67, P < ; fear: F(1,22) = 16.32, P < 0.005], object [valence: F(1,22) = 33.58, P < ; arousal: F(1,22) = 58.84, P < ; fear: F(1,22) = 42.58, P < ], and a significant interaction of group by object [valence: F(1,22) = 10.24, P < 0.004; arousal: F(1,22) = 12.43, P < 0.005; fear: F(1,22) = 7.68, P < 0.05]. Post-hoc analysis revealed that the WG compared to the TG rated spiders as significantly more unpleasant (t = 7.49, P < ), arousing (t = 7.23, P < ), and fear-inducing (t = 4.41, P < ), while no significant group difference was found for the control object (valence: t = 0.33, P > 0.5; arousal: t = 2.23, P > 0.05; fear: t = 1.63, P > 0.1). fmri data First scanning session The ROI analysis for the contrast spider > baseline revealed increased brain responses in several areas in control subjects and phobics (see Table 2). However, only phobics showed activation in the insula and ACC, while amygdala activation was restricted to control subjects (see Table 2). For all other ROIs, increased activation to spider videos was found in all Fig. 1. Increased activation of ACC (A, x,y,z = 2,8,41) and insula (B, x,y,z = 40,11,3) to spider vs. control videos in phobic subjects (PS) compared to control subjects (CS) during the first scanning session. Statistical parametric maps ( P < 0.005) are overlaid on a T1 scan (radiological convention: left = right). The plots show the contrasts of parameter estimates (spider vs. baseline; mean T SEM for maximally activated voxel in the ROI).

6 130 T. Straube et al. / NeuroImage 29 (2006) groups (see Table 2). Between-group comparisons did not reveal any significant differences in activation of the ROIs between both groups of phobic subjects. When activation in a combined group of phobics was compared with the non-phobic control subjects, greater responses in the left and right anterior insula (Table 3; Fig. 1), and in the ACC (Table 3; Fig. 1) were detected in phobics, while controls showed increased activation in the left amygdala and bilaterally in the parahippocampal gyrus (Table 3). Exploratory analysis revealed activation in several areas in all groups, above all in primary and secondary visual cortex (Table 2). Between-group comparisons did not reveal significant differences between both groups of phobic subjects in these regions. Phobics as compared to non-phobic control subjects exhibited greater responses in the left extrastriate visual cortex (lingual gyrus; see Table 3), while control subjects showed increased activation in the pre- and postcentral gyri (see Table 3). Second scanning session In contrast to the first scanning session, where no differences between the TG and WG were detected in the ROI analysis, pronounced differences in brain activation emerged between both groups at the second scanning time point. The TG showed an absence of activation to spider videos in the ACC and only a small cluster of activation in the ventral anterior insula, while the WG exhibited pronounced responses bilaterally in the insula and in the ACC (Table 4; Fig. 2). Between-group comparisons demonstrated stronger activation bilaterally in the insula, thalamus, and in the ACC in the WG compared to the TG (Table 2; Fig. 2), while the TG did not exhibit higher activation than the WG in any ROI. In exploratory analysis, increased activation in the WG compared to the TG was detected in the left dorsomedial prefrontal cortex (DMPFC, see Table 4) and left precuneus (Table 4). The TG as compared to the WG showed greater responses in the right cuneus (see Table 4). Table 4 Significant activation to spider vs. control videos in phobics during the second scanning session Region WG TG WG > TG a x y z t value x y z t value x y z t value ROI DLPFC R L ACC Insula R L Parahippocampal g R L Fusiform gyrus R L Thalamus R L Exploratory DMPFC R L DLFC R L VLPFC R L Precentral gyrus R L Postcentral gyrus R L Temporal gyri R L Parietal gyri R L Cuneus R L Precuneus R L Lingual gyrus R L Occipital gyri R L Basal ganglia R L DLFC, dorsolateral frontal cortex; DLPFC, dorsolateral prefrontal cortex; g, gyrus; L, left; R, right; VLPFC, ventrolateral prefrontal cortex; (x,y,z), Talairach coordinates of maximally activated voxel [Activation threshold: P < 0.005, uncorr. (ROI), P < , uncorr. (Exploratory), cluster 135 mm 3 ]. a There was no significant result for the contrast TG > WG, except for the cuneus (x,y,z = 29, 80, 30; t = 5.07).

7 T. Straube et al. / NeuroImage 29 (2006) Fig. 2. Increased activation of ACC (A, x,y,z = 0,13,40) and insula (B, x,y,z = 40,8, 4) to spider vs. control videos in the waiting-list group (WG) compared to the therapy group (TG) during the second but not the first scanning session. Statistical parametric maps ( P < 0.005) for the second scanning session (WG > TG) are overlaid on a T1 scan (radiological convention: left = right). The plots show the contrasts of parameter estimates for both scanning sessions (spider vs. baseline; mean T SEM for maximally activated voxel in the ROI). For those ROIs where decreased activation was found in the TG compared to the WG, we compared the second with the first scanning session in the TG using one-tailed t tests. These comparisons confirmed significant reductions of activation in the ACC [Talairach coordinates (x,y,z) and statistical values of peak activation: 3, 8, 34, t = 4.20, P < 0.005], left insula [Talairach coordinates (x,y,z) and statistical values of peak activation: 39, 22, 7, t = 3.31, P < 0.005], and left thalamus [Talairach coordinates (x,y,z) and statistical values of peak activation: 9, 17, 11, t = 3.90, P < 0.005]. For the ROIs with differential activation between the TG and WG, we also contrasted BOLD responses of each phobic group with BOLD responses of the control subjects from the first scanning session. These comparisons showed no significant differences between control subjects and the TG, while the WG exhibited increased activation in the right insula [Talairach coordinates (x,y,z) and statistical values of peak activation: 38, 4, 0, t = 3.59, P < 0.005] and the ACC [Talairach coordinates (x,y,z) and statistical values of peak activation: 15, 23, 31, t = 4.13, P < 0.005], suggesting a normalization of brain activation in the TG but not in the WG. Discussion The present study revealed that the processing of phobogenic threat is associated with increased activation in the insula and ACC in subjects suffering from specific phobia. Most importantly, successful cognitive-behavioral therapy led to a reduction of hyperactivity within these brain regions. Thus, in comparison to subjects of the untreated WG, subjects of the treated TG showed a marked attenuation of phobic symptomatology and of responses in the insula and ACC during the second scanning session. The activation pattern observed in the insula fits theoretical accounts and increasing empirical evidence that this brain region is important for pathological but also non-pathological emotional experiences (for reviews see Critchley, 2003; Phan et al., 2002; Reiman, 1997). For example, involvement of the insula has been described in spider phobics in response to real or imagined feared animals (Rauch et al., 1995; Reiman, 1997), spider pictures (Dilger et al., 2003; Carlsson et al., 2004; Straube et al., in press), and spider-related words (Straube et al., 2004b). Significant insula activation was also demonstrated in social phobics and healthy subjects during the processing of angry faces (Straube et al., 2004a), and during symptom provocation in other anxiety disorders (Osuch et al., 2001; Rauch et al., 1997; Reiman, 1997). Significantly increased responses of the insula associated with provocation of clinically relevant anxiety symptoms were reported by Rauch et al. (1997) when using pooled data across different anxiety disorders. Furthermore, in healthy subjects, the insular cortex has been shown to be involved in the recognition and experience of aversive states such as disgust, fear, or pain (e.g., Critchley et al., 2002; Peyron et al., 2002; Phillips et al., 1997; Reiman, 1997). The insular cortex is also activated by interoceptive stimulation and correlates with autonomic activity (Aziz et al., 2002; Critchley et al., 2003). Above all, the work of Critchley et al. (2001, 2002) provided strong evidence that the insula might support the interaction of perceived threat signals and bodily states of arousal leading to subjective emotional experiences such as the feeling of fear. However, although a growing number of studies strongly implicate a critical involvement of the insula in the processing of phobia-related threat, several studies failed to find support for this assumption (e.g., Fredrikson et al., 1993, 1995; Tillfors et al., 2002; Wik et al., 1993, 1997). Such discrepancies between studies require further research, in which experimental conditions should be systematically varied. As suggested elsewhere (Straube et al., 2004a, in press), an important aspect might be the amount of subjects attentional resources while threatening stimuli are present. For example, attentional distraction by concurring cognitive tasks reduces insula responses to threat (e.g., Anderson et al., 2003; Critchley et al., 2002; Gorno-Tempini et al., 2001; Straube et al., 2004a). Recently, we showed that activation of the insula and ACC was even blocked when spider phobics were requested to solve a demanding cognitive task that was displayed

8 132 T. Straube et al. / NeuroImage 29 (2006) in the foreground of phobia-relevant pictures (Straube et al., in press). Similarly, high processing demand associated with the coordination of ongoing active behavior (e.g., Tillfors et al., 2002) could also attenuate or suppress activation of the insula. Finally, attentional distraction that is self-induced as a kind of avoidance or coping behavior might lead to an attenuation of insula responses (see also Paquette et al., 2003). Even more clearly as for the insula, responses in the ACC were normalized in the TG at the second scanning time point, whereas the waiting-list group did not show such an effect. Activation of the ACC during threat processing has been shown in specific phobia and in other anxiety disorders (Rauch et al., 1995, 1997; Osuch et al., 2001; Boshuisen et al., 2002; Pissiota et al., 2002; Aouizerate et al., 2004; Carlsson et al., 2004). Remarkably, direct stimulation of the ACC evokes anxiety in humans (Laitinen, 1979) suggesting a functional relevance of ACC activity in anxiety provocation studies. In healthy subjects, involvement of the ACC, especially of its rostral part, has been repeatedly described during the emotional evaluation of threatening and other salient emotional stimuli (for reviews see Bush et al., 2000; Phan et al., 2002). Several studies reported also emotion-related activation of dorsal ACC (for review see Phan et al., 2002), which has been implicated in cognitive operations such as attentional control or response selection (for review see Bush et al., 2000). Recently, Carlsson et al. (2004) demonstrated augmented rcbf in dorsal ACC to phobogenic vs. other fear-related pictures in spider phobics during passive viewing, suggesting a stimuli-specific, phobia-related increase of activation in this area. In the present study, ACC activation was also found rather in the dorsal part of the ACC. Furthermore, the dorsal ACC has been implicated in the elicitation and control of sympathetic autonomic arousal (Critchley et al., 2003). Thus, the ACC activation observed in the present study might be associated with the sympathetic hyperarousal that has been repeatedly described to be a main physiological feature during the processing of phobogenic stimuli in animal phobics (e.g., Hamm et al., 1997; Globisch et al., 1999; Cuthbert et al., 2003). Successful therapy strongly reduces these responses (e.g., Antony et al., 2001; Bracht et al., 1999; Gutberlet and Miltner, 1999, 2001; Hellstrom and Öst, 1995, 1996; Öst, 1996; Öst et al., 2001), which suggests that the therapy-induced elimination of ACC activation might be coupled to attenuated fear-associated arousal. Besides for the insula and ACC, we found stronger activation in extrastriate visual cortex in phobics than in controls during the first scanning session. This finding is in accordance with several reports showing that the processing of threatening visual stimuli is associated with increased activation in extrastriate visual areas in phobics (Dilger et al., 2003; Fredrikson et al., 1993, 1995; Straube et al., 2004a; Wik et al., 1993; but see Paquette et al., 2003) and healthy controls (e.g., Keightley et al., 2003; Vuilleumier et al., 2001). However, we found no clear therapy-effect in visual areas. This suggests that increased activation in these regions is not necessarily coupled to the fear-relevance of the visual stimulus during sustained stimulus presentation. Thus, the role of other factors, such as novelty, attention, and general salience of stimuli, or an interaction of fear with these factors has to be taken into account (see also Stark et al., 2004; Ishai et al., 2004). In the DMPFC and thalamus, the WG showed stronger activation than the TG at the second scanning time point, whereas no differences of activation were observed in the first scanning session. Increased responses in DMPFC has been shown in phobics during the evaluation of threat-relevant stimulation (Carlsson et al., 2004; Rauch et al., 1995; Stein et al., 2002; Straube et al., 2004a). This region has been implicated in anxiety and the general assessment of the emotional significance of stimuli (Phan et al., 2002; Simpson et al., 2001). Evidence for a significantly increased activation of the thalamus during threat is less consistent (for review see Phan et al., 2002) and seems to be more strongly associated with the internal induction of emotion during recall or imagery (e.g., Rauch et al., 1997). The assumed role of the DMPFC and thalamus in threat processing is partially supported by the therapy effects found in these areas in the present study. However, the data for the first scanning session do not confirm this hypothesis. At least, the observed differences between treated and untreated phobics indicate that augmented responses of the DMPFC and thalamus correlate with the maintenance of anxiety during a repeated confrontation with phobia-related stimuli (see also Furmark et al., 2002). In contrast to the study by Paquette et al. (2003), we did not find activation in DLPFC or parahippocampal gyrus in phobics as compared with healthy subjects. In parahippocampal gyrus, rather less activation was detected in phobics. Furthermore, we found no evidence of a therapy effect in these areas. Activation of the DLPFC during anxiety provocation paradigms was proposed to reflect coping strategies and inhibitory control but also anticipatory anxiety or worrying (e.g., Johanson et al., 1998; Paquette et al., 2003; Reiman, 1997). Interestingly, a study by Johanson et al. (1998) suggested that rcbf in prefrontal areas in phobics might depend on the amount of panic during phobogenic stimulation. In this study, an increase of prefrontal rcbf was associated with a higher level of control of anxiety responses, while a decrease was observed when subjects experienced panic. Therefore, it seems reasonable that studies with inhomogeneous groups of phobic subjects in respect to the individual coping or phobic behavior should not obtain consistent results for the DLPFC. The other region for which Paquette et al. found a therapyinduced reduction of hyperactivity was the parahippocampal gyrus. This area has been strongly associated with memory functions (Brewer et al., 1998; Wagner et al., 1998), particularly during processing of emotional stimuli (Kilpatrick and Cahill, 2003). Although there is evidence for increased parahippocampal activation in phobics during threat processing (Paquette et al., 2003; Stein et al., 2002, Straube et al., 2004a), several studies with animal phobics did not report activation or even deactivation in this region (e.g., Dilger et al., 2003; Fredrikson et al., 1995; Rauch et al., 1995; Wik et al., 1993). Eventually, the discrepant findings could be explained by assuming an inverted U-function of activation depending on the amount of stress (see Diamond et al., 1992; Pavlides et al., 1993). Strongest activation would then occur during an optimal level of emotional arousal (see also Straube et al., 2003), while high levels of stress or anxiety would decrease neuronal activity. However, this idea remains to be tested in future studies by using samples with high variance in emotional reactions or by using parametric designs allowing the induction of different levels of anxiety. The absence of activation in animal phobics in the other ROI in the medial temporal lobe, the amygdala, is in line with previous symptom provocation studies that used stimulation paradigms such as imagery (Rauch et al., 1995), video presentation (Mountz et al., 1989; Fredrikson et al., 1993, 1995; Wik et al., 1993, 1997; Paquette et al., 2003), prolonged picture presentation in blocked designs (Pissiota et al., 2003), or in vivo confrontation (Reiman, 1997). These consistent outcomes, therefore, indicate that sus-

9 T. Straube et al. / NeuroImage 29 (2006) tained phobia-related stimulation in specific phobia (in contrast to social phobia, see Furmark et al., 2002; Tillfors et al., 2001, 2002) does not seem to be associated with amygdalar activity. The amygdala, however, has been implicated in the initial processing of threat and the induction of rapid fear responses (Dilger et al., 2003; Carlsson et al., 2004; Öhman and Mineka, 2001). In recent eventrelated fmri studies, we showed activation of the amygdala in spider phobics during brief presentations of spider as compared to phobia-unrelated pictures (Dilger et al., 2003; Straube et al., in press). Most importantly, we also demonstrated that amygdala activation is more pronounced during attentional distraction than during the attended processing of the phobic stimuli (Straube et al., in press). Similar effects of amygdala inhibition during explicit or direct as compared to implicit processing of threat-related stimuli were also described by other studies that investigated anxiety patients and healthy subjects (Keightley et al., 2003; Lange et al., 2003; Straube et al., 2004a). Furthermore, several studies have reported an inverse correlation between activation in ACC/medial prefrontal cortex and amygdala (e.g., Keightley et al., 2003; Ochsner et al., 2004; Shin et al., 2004; Taylor et al., 2003), suggesting that the medial frontal cortex might exert inhibitory control over the amygdala (see also Quirk et al., 2003). Somewhat unexpectedly, amygdala activation was observed in controls in the present study. We interpret this effect as normal response to more interesting/relevant and also perceptually different (especially in respect to the movements of the spider extremities) stimuli in contrast to the baseline videos. These factors together with novelty aspects may lead to amygdala activation in controls (see also Schwartz et al., 2003; Wright et al., 2003). The amygdala of phobics might have been inhibited for example by influences of the ACC during the first scanning (see above). However, if this was the case, there should be amygdala activation due to the absence of ACC activity in the therapy group during the second scanning (at least compared to the WG). On the other hand, this expectation is based on the assumption that the normal activation in healthy controls is stable over time. A limitation of our study is that we cannot answer this question since healthy controls were measured only once. Thus, further studies are needed to reveal the mechanisms by which amygdala activation is modulated during sustained symptom provocation in specific phobia. In conclusion, the observed pattern of phobia-associated brain activation and therapy-related decrease of hyperactivation suggests an important role of the ACC and insula in the effective treatment of specific phobia. Furthermore, successful cognitive-behavioral therapy led also to reduced activation in thalamus and DMPFC in the treated as compared to the untreated phobic subjects, indicating that the reduction of fear is associated with decreased activity in these areas. In accordance with previous studies, we found no evidence of amygdala involvement in sustained processing of phobia-related stimuli. Future studies should investigate whether initial and rather automatic brain responses to phobogenic stimuli, for example in the amygdala, will be also modified by psychotherapy. Acknowledgments The study was supported by the Deutsche-Forschungsgemeinschaft (DFG project number: Mi 265/6-1; Mi 265/6-2). 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