Genes, Brain and Behavior (2007) 6: 672–676
# 2006 The Authors Journal compilation # 2006 Blackwell Publishing Ltd
Serotonergic genes modulate amygdala activity in major depression U. Dannlowski*,†,‡, P. Ohrmann†, J. Bauer†, H. Kugel§, B. T. Baune†,¶, C. Hohoff†, A. Kersting†, V. Arolt†, W. Heindel§, J. Deckert†,|| and T. Suslow† †
Department of Psychiatry, University of Mu¨nster, Mu¨nster, Germany ‡ IZKF-Research Group 4, IZKF Mu¨nster, University of Mu¨nster, Mu¨nster, Germany §Department of Clinical Radiology, University of Mu¨nster, Mu¨nster, Germany ¶ Department of Psychiatry, James Cook University, Townsville, Australia ||Department of Psychiatry, University of Wu¨rzburg, Wu¨rzburg, Germany *Corresponding author: U. Dannlowski, Department of Psychiatry, University of Mu¨nster, Albert-Schweitzer-Str. 11, 48149 Mu¨nster, Germany. E-mail:
[email protected]
Serotonergic genes have been implicated in the pathogenesis of depression probably via their influence on neural activity during emotion processing. This study used an imaging genomics approach to investigate amygdala activity in major depression as a function of common functional polymorphisms in the serotonin transporter gene (5-HTTLPR) and the serotonin receptor 1A gene (5HT1A-1019C/G). In 27 medicated patients with major depression, amygdala responses to happy, sad and angry faces were assessed using functional magnetic resonance imaging at 3 Tesla. Patients were genotyped for the 5-HT1A-1019C/G and the 5-HTTLPR polymorphism, including the newly described 5-HTT-rs25531 single nucleotide polymorphism. Risk allele carriers for either gene showed significantly increased bilateral amygdala activation in response to emotional stimuli, implicating an additive effect of both genotypes. Our data suggest that the genetic susceptibility for major depression might be transported via dysfunctional neural activity in brain regions critical for emotion processing. Keywords: 5-HT1A-1019C/G, 5-HTTLPR, amygdala, depression, emotion, fMRI, rs25531, serotonin Received 12 August 2006, revised 17 November 2006, accepted for publication 17 November 2006
Converging evidence suggests that the serotonergic system plays a crucial role in the etiology of affective disorders (Wong & Licinio 2001). In particular, the serotonin receptor 1A (5HT1A) and the serotonin transporter (5-HTT) are believed to be involved in the pathophysiology of major depression.
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In the 5-HT1A gene, the G-allele of the 1019C/G promoter polymorphism was reported to derepress 5-HT1A autoreceptor expression by disrupting an inhibitory transcription factorbinding site and thereby reducing serotonergic neurotransmission, which was further reported to be associated with elevated risk for major depression and suicide (Lemonde et al. 2003) and with depression-related personality traits (Strobel et al. 2003). In the promoter region of the 5-HTT gene, a functional variable repeat sequence polymorphism (5HTTLPR) resulting in a short (S) and a long (L) variant has been identified. Carriers for the S-allele are more vulnerable for the depressogenic effect of stressful life events than individuals homozygous for the L-allele (Caspi et al. 2003). For both the genes, risk allele carriers were reported to show poor treatment response to antidepressant treatment (e.g. Lemonde et al. 2004; Smits et al. 2004). However, other studies failed to find associations of serotonergic genes with depression (e.g. Willis-Owen et al. 2005). Inconsistencies in association findings with depression could be in part because of the heterogeneity and complexity of the clinically defined phenotype. It was suggested that the examination of regional brain activation that is critical for emotion processing represents a more promising approach to link neural dysfunction to genes that are probably involved in the pathogenesis of depression with effect sizes 10–20 times larger than in classical association studies (Hariri & Weinberger 2003). The amygdala is a central structure in a limbic emotionprocessing circuit. Amygdala hyperactivity has been shown in acutely depressed patients compared with controls at rest (Drevets et al. 1992), in expectation of negative pictures (Abler et al., in press), in response to verbal stimuli (Siegle et al. 2002) and emotional faces (e.g. Sheline et al. 2001), which was shown to resolve after antidepressant therapy (Fu et al. 2004). Therefore, amygdala hyperactivity has been implicated in the pathogenesis of major depression, probably by causing negatively biased emotion processing (Whalen et al. 2002; Phillips et al. 2003) and consequently, the amygdala was selected for a region of interest analysis in the present study. Furthermore, it was repeatedly shown that amygdala activity is elevated in healthy carriers of at least one S-allele of the 5HTTLPR (Hariri et al. 2002, 2005; Heinz et al. 2005; Pezawas et al. 2005). Hence, it was speculated that the genetic susceptibility for depression could be mediated via alterations of amygdala activity during emotion processing (Hariri et al. 2002), but no data of depressed patients have been reported hitherto. Recently, our group has reported the effect of the 5HT1A-1019C/G polymorphism on neural activity in panic disorder patients (Domschke et al. 2006). In the present study, we have investigated amygdala activation in response to emotional faces in relation to variations in the 5-HT1A and 5-HTT doi: 10.1111/j.1601-183X.2006.00297.x
Serotonergic genes in depression
genes in patients with major depression. It was hypothesized that risk allele carriers for both the genes show stronger amygdala activity during the processing of emotional stimuli.
Materials and methods Datasets of 27 right-handed inpatients with acute major depression according to the Diagnostic and Statistical Manual of Mental Disorders criteria (DSM-IV, American Psychiatric Association, 1994), diagnosed with the Structured Clinical Interview for DSM-IV interview (SCID-I, Wittchen et al. 1997), were analyzed (Table 1). Given previously reported effect sizes concerning the 5-HTTLPR effect on amygdala activity (Cohen’s d ¼ 1.0, Hariri et al. 2002; d ¼ 1.17, Heinz et al. 2005), the present sample size provided sufficient statistical power [1-ß ¼ 79.8%, calculated using G-POWER (Erdfelder et al. 1996)]. Exclusion criteria were neurological abnormalities, substance abuse, former electroconvulsive therapy and benzodiazepine treatment. All the patients were under antidepressant treatment, which was coded in terms of dose and treatment duration into medication levels from 1 to 4, according to the suggestions of Sackheim (2001). The study was approved by the Ethics Committee of the University of Mu¨nster. After complete description of the study to the subjects, written informed consent was obtained. Only patients with primary major depression were included (indicated by earlier onset and predominant symptoms). Secondary life-time diagnoses were social phobia (n ¼ 3), agoraphobia (n ¼ 1), panic disorder (n ¼ 7) and obsessive compulsive disorder (OCD) (n ¼ 1). Facial stimuli consisted of sad, angry, happy and neutral expressions (Ekman & Friesen 1976). Patients were presented with alternating 30-s blocks of a facial emotion category or a no-face stimulus (a gray rectangle). Facial stimuli were presented twice per second in a random sequence for 500 ms. The order of blocks was counterbalanced across subjects. Each facial emotion block was preceded by a no-face block and was presented twice, resulting in an overall presentation time of 8 min. Patients were told that they would see human faces and that they should pay attention to them. T2* functional data were acquired at a 3-Tesla scanner (Gyroscan Intera 3.0T, Philips Medical Systems, Best, The Netherlands) using a single shot echoplanar sequence with parameters selected to minimize distortion in the amygdala region, while retaining adequate S/N and T2* sensitivity. Volumes consisting of 25 axial slices were acquired (matrix 128 128, resolution 1.75 1.75 3.5 mm, repetition time (TR) ¼ 3 s, echo time (TE) ¼ 30 ms, flip angle (FA) ¼ 908)
Table 1: Clinical characteristics of the patient sample (n ¼ 27) Age Gender Total education time Duration of illness (months) Age of onset Life-time hospitalization (weeks) Time since first hospitalization (months) Number of episodes Family history HRSD GAF
36.7 (12.5) 20 women, 7 men 14.6 (1.9) 113.1 (115.2) 27.2 (12.8) 6.9 (10.3) 27.1 (52.3) 4.6 (4.5) 16 positive, 11 negative 22.6 (3.6) 55.4 (9.7)
HRSD, Hamilton Rating Scale for Depression (Hamilton, 1960); GAF, Global Assessment of Functioning (American Psychiatric Association, 1994). Values represent mean (SD). Patients in the respective genotype groups did not differ concerning any variable presented in this table, according to t-tests or chi-square tests (all P > 0.1). Genes, Brain and Behavior (2007) 6: 672–676
160 times in block design, 10 times per condition. To optimize the following normalization procedures, the same sequence parameters were used to cover the whole brain with 43 slices. Functional imaging data were motion corrected, spatially normalized to standard MNI space (Montreal Neurological Institute) and smoothed (Gaussian kernel, 6 mm FWHM) using Statistical Parametric Mapping software (SPM2, Wellcome Department of Neurology, London, UK). Statistical analysis was performed by modeling the different conditions (angry, sad, happy, neutral and no-face) as variables within the general linear model (modeled with a standard hemodynamic response function), contrasting emotional faces (angry, sad and happy) with the neutral face condition. Voxel values (Tzourio-Mazoyer et al. 2002) of bilateral amygdala were extracted, summarized by mean and tested among the different conditions using the MarsBaR toolbox (Brett et al. 2002). Furthermore, we investigated the effect of genotype on functional connectivity between amygdala and a recently reported region in the subgenual anterior cingulate cortex (sgACC), in which functional connectivity was inversely correlated with anxious temperament in healthy adults (Pezawas et al. 2005). All patients were genotyped for the 5-HT1A-1019C/G, the 5-HTTLPR and the newly described 5-HTT-rs25531 polymorphism according to published protocols (Deckert et al. 1997; Rothe et al. 2004; Wendland et al. 2006). For genotyping, quality control patients were additionally genotyped either by TaqMan SNP Genotyping assay (5-HT1A-1019C/G: all patients; ABI Prism 7900HT Sequence Detection System, Applied Biosystems, Darmstadt, Germany) or by direct automated sequencing (5-HTTLPR/5-HTT-rs25531: patients carrying the LALA or LALG variants; Applied Biosystems 3730 DNA Analyzer), which resulted in concordance rates of 100%. Genotypes were determined blind for phenotypes and independently by two investigators. The genotype distribution of all three polymorphisms was conformed to Hardy– Weinberg equilibrium (all P > 0.1). For the 5-HTTLPR/5-HTT-rs25531 polymorphism, the LG-allele was found to behave similar to the low-expressing S-allele (Hu et al. 2005). Thus, it was treated as ‘risk allele’ for the purpose of grouping. Following previous studies (Hariri et al. 2002; Strobel et al. 2003), patients were grouped into risk allele carriers and non-risk allele carriers for each polymorphism [5-HT1A-1019C/G: GG (n ¼ 8) and CG (n ¼ 13) vs. CC (n ¼ 6); 5-HTTLPR/5-HTT-rs25531: SASA (n ¼ 3), SALG (n ¼ 2), SALA (n ¼ 13) and LALG (n ¼ 3) vs. LALA (n ¼ 6) (SASG, SGSG, SG LA, S GLG and LGL G were not present in our sample)]. The dichotomization of 5-HT1A-1019C/G seemed to be justified because anxiety-like behavior was apparent not only in homozygous 5-HT1A / knockout mice but also in heterozygous mice (þ/), indicating that a partial receptor deficit is sufficient to cause the behavioral phenotype (Toth 2003). For both the genes, subjects in the respective genotype groups were not significantly different concerning age, gender, education, family history, depression severity, trait anxiety, duration of illness, number of episodes, total hospitalization time, medication level, or comorbid diagnoses, according to t-tests or chi-square tests (all P > 0.1). Furthermore, the distribution of allele frequencies of the two polymorphisms was independent, w2 (8) ¼ 4.7, P ¼ 0.79.
Results For both polymorphisms, a 3 (emotion: happy, sad, angry) 2 (hemisphere: left, right) 2 (genotype: risk, non-risk) analysis of variance was conducted on amygdala activity parameters. The main effects of both the polymorphisms were significant, 5-HT1A-1019C/G: F (1,25) ¼ 13.3, P ¼ 0.001, Z2p ¼ 0.35; 5HTTLPR/5-HTT-rs25531: F (1,25) ¼ 6.6, P ¼ 0.017, Z2p ¼ 0.21. Risk allele carriers for either polymorphism had increased amygdala activity elicited by emotional faces regardless of valence, compared with non-risk allele carriers (Fig. 1). No significant effect of emotion or hemisphere was recorded. Adding medication level or current depression severity (Hamilton Rating Scale for Depression score; Hamilton 1960) as covariate did not alter the results. The effect of 5-HTTLPR
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Figure 1: Effect of 5-HTTLPR/5-HTT-rs25531 and 5-HT1A-1019C/G on amygdala activity. Upper panels represent the effect of the 5HTTLPR/5-HTT-rs25531 polymorphism, lower panels the effect of the 5HT1A-1019C/G polymorphism. Left panels reflect left amygdala activation and right panels reflect right amygdala activation. All S-alleles were SA-alleles. Error bars, SEM. fMRI, functional magnetic resonance imaging.
without rs25531 was somewhat weaker compared with the combined effect of 5-HTTLPR and rs25531, but still reached significance, F (1,25) ¼ 5.3, P ¼ 0.030, Z2p ¼ 0.17. To explore the interplay of the two observed polymorphisms, two 3 (emotion) 2 (hemisphere) analyses of covariance were conducted with ‘number of risk genes’ (genes with one or two risk alleles, 0–2) and ‘number of risk alleles’ on both genes (0–4) added as covariates. Both covariates produced strong main effects on amygdala activity, F (1,25) ¼ 22.9, P ¼ 0.0001, Z2p ¼ 0.47 and F (1,25) ¼ 16.7, P ¼ 0.0004,
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Zp2 ¼ 0.4, respectively. Amygdala activity increased linear as a function of risk genes and risk alleles, indicating an additive effect of the two variants (Fig. 2). We replicated a positive connectivity between bilateral amygdala and sgACC, t (26) ¼ 3.5, P ¼ 0.002. Functional connectivity predicted Global Assessment of Functioning scores (American Psychiatric Association, 1994) (r ¼ 0.43, P ¼ 0.026) and inversely duration of index episode (r ¼ 0.55, P ¼ 0.003) and life-time hospitalization (r ¼ 0.44, P ¼ 0.023). Non-risk allele carriers for both polymorphisms showed Genes, Brain and Behavior (2007) 6: 672–676
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Figure 2: Amygdala activity as a function of risk alleles. Bilateral amygdala activity in response to angry, sad and happy faces dependant on number of risk alleles (S and LG-allele for 5HTTLPR/5-HTT-rs25531, G-allele for 5-HT1A-1019C/G). Number of risk alleles covaries significantly with amygdala activity, F (1,25) ¼ 16.7, P ¼ 0.0004, indicating an additive effect.
higher amygdala-sgACC connectivity values, although differences did not reach significance (P > 0.1).
Discussion Our data suggest that in patients with major depression, amygdala excitability in response to emotionally relevant stimuli is influenced by the 5HT1A-1019C/G and the 5HTTLPR/5-HTT-rs25531 polymorphisms. Thus, we replicated and extended previous findings concerning the influence of the 5-HTTLPR polymorphism on amygdala activity to a patient sample. Furthermore, it was shown that the 5HT1A-1019C/G polymorphism has an independent and even stronger influence on amygdala excitability in major depression. An additive effect of both genotypes was observed, with highest amygdala activity in individuals carrying a combination of both short variant of 5-HTTLPR and G variant of 5HT1A-1019C/G. Recently, another additive effect of 5-HTTLPR and a tryptophan hydroxylase-2 gene variation on neural activity as measured with event-related potentials has been shown in healthy subjects (Herrmann et al., in press). To the best of our knowledge, the present study is the first to show additive gene effects on neural activity in depressed patients. Genes, Brain and Behavior (2007) 6: 672–676
The finding of genetic variations biasing amygdala activity is probably not specific for major depression because it has been repeatedly shown in healthy subjects. However, genetically based amygdala hyperactivity might mediate the risk for depression in healthy controls and constitute a biological marker for depressed patients. Our findings underscore the notion that the role of the amygdala in major depression is linked to specific genetic risk factors (Hariri et al. 2005). In risk allele carriers, stronger amygdala activity during emotion processing may reflect a tendency for a negatively biased perception of emotionally relevant stimuli. Limitations must be acknowledged. The number of patients is small, albeit in the range of other recently published studies (Hariri et al. 2002; Heinz et al. 2005; Domschke et al. 2006) and the power analysis indicated sufficient statistical power to detect effect sizes estimated from previous reports. Only patients were investigated, but our findings relate to and are consistent with previous ones in healthy subjects (Hariri et al. 2002, 2005; Heinz et al. 2005, Pezawas et al. 2005). Furthermore, stressful life events were not recorded. However, no patient reported having experienced a trauma relevant for post-traumatic stress disorders as indicated by the SCID interview. The correlation analyses of amygdala-sgACC connectivity with clinical characteristics were explorative and some would not survive alpha correction for multiple comparisons. In sum, preliminary findings in clinically depressed patients are presented, providing evidence that a genetic susceptibility for major depression might be transported via dysfunctional neural activity during emotion processing. The present findings hopefully stimulate further investigations of depressed patients using the endophenotype approach. Future studies should address the predictive effect of increased amygdala excitability on the course of depression by investigating unmedicated patients in longitudinal designs.
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Acknowledgments We acknowledge the skillful technical support of Ms Su¨nke Mortensen and Ms Kathrin Schwarte. This study was supported by an IMF grant (AR 510403) of the Medical Faculty of the University of Mu¨nster. All the authors declare that they have no competing financial interests.
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