Serotonin transporter polymorphism related to amygdala excitability and symptom severity in patients with social phobia

Neuroscience Letters 362 (2004) 189–192 www.elsevier.com/locate/neulet

Serotonin transporter polymorphism related to amygdala excitability and symptom severity in patients with social phobia Tomas Furmarka,*, Maria Tillforsa,b, Ha˚kan Garpenstrandc, Ina Marteinsdottird, Bengt La˚ngstro¨me, Lars Orelandc, Mats Fredriksona a

b

Department of Psychology, Uppsala University, Uppsala, Sweden ¨ rebro University, O ¨ rebro, Sweden Department of Behavioral, Social and Legal Sciences, O c Department of Neuroscience, Pharmacology, Uppsala University, Uppsala, Sweden d Department of Neuroscience, Psychiatry, Uppsala University, Uppsala, Sweden e Uppsala Imanet AB, University Hospital, Uppsala, Sweden

Received 10 December 2003; received in revised form 22 January 2004; accepted 26 February 2004

Abstract A functional polymorphism in the promoter region of the human serotonin transporter (5-HTT) gene has been related to negative affect and amygdala activity. We studied amygdala activation during social anxiety provocation in relation to affective ratings and 5-HTT genetic variation. [H2 15O]positron emission tomography was used to estimate amygdala blood flow during private and public speaking (baseline and anxiety conditions) in 17 patients with social phobia. Genotyping identified patients with long and short alleles in the promoter region of the 5-HTT. Individuals with one or two copies of the short allele exhibited significantly increased levels of anxiety-related traits, state anxiety, and enhanced right amygdala responding to anxiety provocation, compared with subjects homozygous for the long allele. Thus, 5-HTT genetic variation was associated with symptom severity and amygdala excitability in social phobia. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Anxiety; Emotion; Genotyping; Amygdala; Positron emission tomography; Serotonin transporter; Social phobia

There is abundant evidence that mood and anxiety are at least partly modulated by serotonergic neurotransmission [8]. Disturbances in the serotonergic system are likely to be involved in the pathogenesis of disorders of the affective spectrum. The reuptake of serotonin in the synapse is mediated by the serotonin transporter (5-HTT). In the untranslated regulatory region upstream of the gene encoding the 5-HTT, there is a 44 base pair insertion/ deletion gene-linked polymorphic region divided into alleles inducing either low or high transcriptional activity, i.e. short or long alleles [12]. The presence of two long alleles, rather than one or two copies of the short allele, is associated with an increased serotonin reuptake [14]. This difference appears to have behavioral consequences, in particular for anxiety- or depression-related personality traits and other characteristics related to negative affect. * Corresponding author. Department of Psychology, Uppsala University, Box 1225, SE-751 42 Uppsala, Sweden. Tel.: þ 46-18-471-21-53; fax: þ 46-18-471-21-23. E-mail address: [email protected] (T. Furmark).

Initial studies suggested that the presence of the short allele was associated with higher levels of neuroticism [9,14], harm-avoidance [16] and other anxiety-related scales [9,14, 16]. The short allelic variant has also been related to fear conditionability [7] and affective illness [3,17]. Allelic variation in functional expression of the 5-HTT may thus be a susceptibility factor for developing anxiety and/or mood disorders. On the other hand, several attempts to replicate findings on 5-HTT promoter polymorphism and psychopathology have been negative, particularly regarding the association between the short allele and neuroticism (cf. Ref. [6]). Studies of psychiatric anxiety conditions have not demonstrated a link between the 5-HTT gene and panic disorder [10] or social phobia [19]. Evidence is inconclusive also with regard to depression [15]. It is possible that variations in the 5-HTT gene are not directly associated with anxiety or mood disorders, although the 5-HTT gene may interact with environmental conditions thereby affecting the individual’s vulnerability for developing psychopathological

0304-3940/03/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.02.070

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T. Furmark et al. / Neuroscience Letters 362 (2004) 189–192

reactions. Caspi and colleagues [3] noted that individuals having the short allele of the 5-HTT promoter polymorphism were more prone to develop depression following stressful life events as compared to individuals homozygous for the long promoter variant. Thus, it is possible that the effect of the gene is uncovered mainly when related to stressful experiences. Functional neuroimaging techniques enable studies of 5HTT genetic influences on brain activity during stressful emotional conditions in humans. The amygdala has long been implicated in the regulation of fear and anxiety [13] and would be a prime region of interest in such imaging studies. Recently, Hariri and co-workers [11] were able to demonstrate greater amygdala neuronal responding to fearful and angry facial stimuli in short allele individuals compared with individuals homozygous for the long allele. Thus, amygdala excitability might contribute to the 5-HTT genetic influence on fear and anxiety. The aim of the present study was to relate genetic variation at the 5-HTT locus to amygdala activity and symptom severity in individuals with social phobia (also known as social anxiety disorder). This is a disabling psychiatric condition characterized by a marked fear of being scrutinized in social interaction or performance situations such as public speaking [1]. We related 5-HTT allelic variation to regional cerebral blood flow (rCBF) in the amygdala during an anxiogenic public speaking task, compared with baseline measurements, and also to state and trait anxiety, depression and neuroticism ratings. The present report presents refined analyses of a previously published positron emission tomography (PET) study on social phobia in which the method is described in detail [20]. Briefly, 18 patients (ten men/eight women) diagnosed with DSM-IV [1] social phobia were scanned using PET and oxygen-15 labeled water, while they were performing a stressful speaking task in front of an audience or alone, i.e. public and private speaking corresponding to anxiety provocation and baseline [20]. Approvals were obtained from the local ethics and radiation safety committees. Written informed consent was obtained from all participants. A PET scanner (GEMS PC2048-15B) with a 100 mm axial field of view and 6 mm of axial/transaxial resolution was used. A venous catheter was inserted and subjects were positioned in the scanner. A 10 min transmission scan was completed while subjects prepared the speech. The 15Owater tracer, approximately 15 MBq/kg, was thereafter injected. Subjects started speaking immediately after the injection and continued until they received instructions to stop 2.5 min later [20]. Data obtained during the first 70 s after bolus arrival to the brain were summed and individual images were reconstructed. Images were automatically aligned to a standard stereotactic space and corrected for head movements using the Computerized Brain Atlas (CBA; Applied Medical Imaging, Uppsala, Sweden). Statistical rCBF analyses were focused on the amygdala.

Normalized rCBF values were extracted from the CBA central voxel of the right and left amygdala, located at x23, y 2 4, z 2 17 and x 2 22, y 2 6, z 2 17 in the Talairach space (see Ref. [5]). State anxiety, associated with the speaking tasks, was evaluated after each scan using Spielberger’s State-Trait Anxiety Inventory (STAI-S; range 20 – 80) [18] and subjective ratings of fear and distress (range 0– 100) [20]. Trait anxiety (STAI-T) [18], neuroticism according to the NEO-PI [4] and Beck’s Depression Inventory (BDI) scores [2] were assessed prior to PET scanning. Details on the 5-HTT genotyping procedure have been described in another protocol [7]. Briefly, genomic DNA was extracted from blood samples by use of a Qiampw DNA extraction kit (Qiagen GmbH, Hilden, Germany). The genotype of the 5-HTT promoter was analyzed by polymerase chain reaction (PCR) in a 20 ml reaction volume in a RoboCyclerw 96 (Stratagene GmbH, Heidelberg, Germany). The specific oligonucleotides were 50 -CAA CCT CCC AGC AAC TCC CTG TA-30 (forward primer) and 50 -GAG GGA CTG AGC TGG ACA ACC AC-30 (reverse primer). The reaction mixture included 1 £ Taq DNA polymerase buffer, 400 mM dNTP, 1 mM of each primer, 5% dimethyl sulfoxide (DMSO), 100 ng genomic DNA, and 2 units Taq DNA polymerase. The PCR protocol employed consisted of initial denaturing at 94 8C for 1 min followed by 36 cycles of denaturing at 94 8C for 1 min, annealing at 63 8C for 1 min, and extension at 72 8C for 1 min. Group differences (presence of short vs. long alleles only) were analyzed by use of Student’s t-tests and analyses of variance (ANOVAs) if applicable. Effect sizes (ES) corresponding to Cohen’s d were also calculated. Results showed that seven individuals were homozygous for the long allele of the 5-HTT gene, whereas the short form was present in ten subjects. Two subjects were homozygous for the short allele. Due to technical failure genetic information is missing from one male subject. The short allele was present in six of eight (75.0%) individuals diagnosed as having the generalized social phobia subtype and among four of nine (44.4%) subjects with nongeneralized social phobia. The distribution of allelic long/short forms did not differ significantly across subtypes (x2 ð1Þ ¼ 1:6, n.s.) or across men and women (x2 ð1Þ ¼ 0:5, n.s.). Descriptive and comparative statistics for the behavioral outcome variables are presented in Table 1. Trait anxiety and BDI depression scores were significantly elevated in individuals with short as compared to long alleles. Neuroticism scores followed the same pattern although the two-tailed significance value was marginal at P ¼ 0:052. ESs were large for all these scales. The ANOVAs showed significant effects of group (long/short) and condition (anxiety provocation/baseline) on all state anxiety measures [Fð1; 15Þ ¼ 13:8 2 57:4, P # 0:002]. Follow-up analyses revealed that short allele individuals exhibited significantly

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Table 1 Behavioral outcome variables: descriptive (mean, SD) and comparative (t-value, effect size) statistics for individuals with social phobia homozygous for the long allele of the serotonin transporter promoter gene as compared with those having a short allele present Variable Questionnaire data STAI-T Neuroticism BDI State-measures: anxiety provocation (public speaking) STAI-S Fear Distress State-measures: baseline (speaking alone) STAI-S Fear Distress

Long alleles (n ¼ 7)

Short alleles (n ¼ 10)

t-value (corresponding d:f: ¼ 15)

ES

41.0 (9.8) 79.0 (25.0) 4.7 (4.8)

51.1 (8.1) 103.1 (22.0) 11.3 (5.9)

2.3* 2.1# 2.4*

1.12 1.02 1.23

48.9 (9.8) 21.4 (19.6) 39.3 (27.6)

62.0 (5.3) 52.8 (13.8) 63.0 (16.2)

3.6*** 3.9*** 2.2*

1.66 1.85 1.04

36.4 (9.1) 1.8 (2.8) 6.1 (6.9)

48.3 (6.9) 21.5 (13.8) 30.2 (9.2)

3.1** 3.7*** 5.9****

1.47 1.98 2.96

ES, effect size; STAI, Spielberger State (STAI-S) and Trait (STAI-T) Anxiety Inventory; BDI, Beck’s Depression Inventory; *P , 0:05, **P , 0:01, ***P , 0:005, ****P , 0:0001, #P ¼ 0:052.

higher (P , 0:005) overall ratings, and reactions were overall higher (P , 0:0001) during public than private speaking, on all state anxiety scales. The STAI-S, fear, and distress ratings were in each case significantly higher in short allele individuals both during public speaking and baseline, and ESs were large (see Table 1). Significant group £ condition interactions were not obtained. Amygdala rCBF did not differ between groups at baseline (tð15Þ , 1, n.s.). The ANOVA yielded a significant group £ laterality £ condition interaction (Fð1; 15Þ ¼ 5:34, P ¼ 0:036). Follow-up tests demonstrated greater rCBF increase from baseline to provocation in the right (tð15Þ ¼ 2:67, P ¼ 0:018) but not left amygdala (tð15Þ ¼ 1:78, n.s.) in subjects with short as compared to long alleles only (see Fig. 1). Thus, the presence of a short 5-HTT promoter allele was associated with elevated trait anxiety and depression scores, with a tendency also for neuroticism, in subjects with social

Fig. 1. Right amygdala cerebral blood flow alterations, expressed as percentage change in radioactive counts from baseline to anxiety provocation (speaking alone and in public), in patients with social phobia as related to a polymorphism of the serotonin transporter gene. The presence of a short allele (n ¼ 10) as compared to homozygosity for long alleles (n ¼ 7) was associated with an enhanced amygdala excitability. In the short allele group, two subjects were homozygous for the short allele (open squares) whereas the remaining eight had one short and one long allele (filled squares). Lines represent group mean values.

phobia. This is consistent with several previous reports on 5HTT polymorphism and anxiety- or depression-related traits (e.g. Refs. [9,14,16]). However, as previously noted, findings on this topic have been mixed. Subjects having one or two copies of the short allele also gave higher state anxiety ratings, both when speaking in the presence of an audience and alone. Hence, the effect of the 5-HTT gene was observed also when directly related to a stressful experience, consistent with previous findings in mood disordered subjects [3]. Finally, subjects with the short promoter variant exhibited enhanced excitability of the right but not left amygdala in going from the speaking alone baseline to the more pronounced anxious state when speaking in public. Congruently, recent neuroimaging data suggest that short allele individuals show a greater right amygdala response to pictures of fearful and angry faces [11]. The present results extend this finding, suggesting that a genetic serotonergic influence on amygdala excitability is evident, not only during perception of emotional stimuli, but also during production of an aversive emotion. Although enhanced amygdala excitability in short allele individuals could be innate, it could also be related to learning or a gene by environment interaction, because the polymorphism has been associated with enhanced fear conditionability [7]. In contrast to the present findings, a genetic influence on the subjective experience of affect was not observed by Hariri and colleagues [11], suggesting that the brain endophenotype (rCBF) is a more sensitive index of genetic influences than the behavioral phenotype (subjective ratings). In our study, both rCBF and anxiety levels were enhanced in short allele subjects. This may reflect that stronger and more vivid emotions are elicited by speaking tasks in phobic patients than by perception of fearful faces in normal volunteers. Anxiety levels were elevated both during baseline and anxiety provocation in short allele subjects whereas significant group £ condition interactions were not demonstrated. This could, together with the data on STAI-T,

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BDI and neuroticism, support underlying trait-like behavioral differences between the allelic variants. However, amygdala activity differed as a function of the 5-HTT polymorphism only in going from baseline to the provoked condition, supporting that the allelic groups differ in neural responsiveness as the emotional impact of the situation changes. Amygdala rCBF may be a more sensitive index of an emotional response compared with subjective ratings. In a previous report, Stein and colleagues [19] were not able to demonstrate linkage between the 5-HTT gene and generalized social phobia. However, methodological procedures in the present protocol differ from the Stein et al. [19] study in which the 5-HTT allelic forms were not directly compared with regard to symptomatic profiles. The generalizability of the current results is, however, limited because only a relatively small clinical group was studied and subjects with social phobia volunteering for a research project may differ in severity and allelic frequency from phobics typically seen in the clinic. Further comparisons with healthy controls and other psychiatric populations are warranted. In sum, the present results support a genetically determined link between serotonergic functions, anxiety proneness and a brain region central for emotional experience and processing. Amygdala excitability during anxiety provocation may serve as an endophenotype suitable for genetic analyses of neuropsychiatric conditions.

Acknowledgements This study was supported by grants from the Swedish Research Council (M.F., T.F., L.O.), the Bank of Sweden Tercentenary Foundation (M.F.), and the Swedish Brain Foundation (T.F., H.G.).

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