Neuroscience Letters 584 (2015) 109–112
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Serotonin transporter polymorphisms predict response inhibition in healthy volunteers N.I. Landrø a,∗ , R. Jonassen a , L. Clark b , K.B. Foss Haug c , M. Aker a , R. Bø a , J.P. Berg c , A. Neumeister d , T.C. Stiles e a
Clinical Neuroscience Research Group, Department of Psychology, Oslo, Norway Department of Psychology, University of Cambridge, Cambridge, UK c Section for Research, Department of Medical Biochemistry, Oslo University Hospital, and Institute of Clinical Medicine, University of Oslo, Oslo, Norway d New York University, Department of Psychiatry, New York, USA e Department of Psychology, The Norwegian University of Science and Technology, Trondheim, Norway b
h i g h l i g h t s • 5-HTTLPR polymorphisms mediate different levels of inhibitory control. • More impulsive behavior in healthy carriers of the low expressive genotype. • These initial observations will allow extensions to patient populations.
a r t i c l e
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Article history: Received 9 July 2014 Received in revised form 30 September 2014 Accepted 5 October 2014 Available online 17 October 2014 Keywords: Serotonin transporter Inhibitory control
a b s t r a c t Serotoninergic transmission is reliably implicated in inhibitory control processes. The aim of this study was to test the hypothesis if serotonin transporter polymorphisms mediate inhibitory control in healthy people. 141 healthy subjects, carefully screened for previous and current psychopathology, were genotyped for the 5-HTTLPR and rs25531 polymorphisms. Inhibitory control was ascertained with the Stop Signal Task (SST) from the Cambridge Neuropsychological Test Automated Battery (CANTAB). The triallelic gene model, reclassified and presented in a biallelic functional model, revealed a dose-dependent gene effect on SST performance with Individuals carrying the low expressive allele had inferior inhibitory control compared to high expressive carriers. This directly implicates serotonin transporter polymorphisms (5-HTTLPR plus rs25531) in response inhibition in healthy subjects. © 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
1. Introduction Impulsivity can be understood as arising from impairment in inhibitory control [1]. Impaired serotonin (5-HT) function [2] has been shown to contribute to the neurobiology of impaired executive control processes [3] and impulsive behaviors [4]. However, the genetic contribution of these behavioral processes is incompletely understood. The 5-HT-transporter-linked polymorphic region (5-HTTLPR) in the promoter region of the human 5-HT transporter (5-HTT) gene (SLC6A4) results in two main alleles or variants [5]; the short (S) and long (L), comprising 14 and 16 copies of a 20–23 nucleotide repeat
∗ Corresponding author at: Department of Psychology, University of Oslo, Norway. Box 1094, Blindern, 0317 Oslo, Norway. Tel.: +47 22845146. E-mail address: [email protected] (N.I. Landrø).
cassettes, respectively. A functional triallelic 5-HTTLPR polymorphism include an additional single nucleotide polymorphism A > G SNP (rs25531) in the first of two 22-bp imperfect repeats that define the 16-repeat L allele. The 5-HTTLPR L allele combined with the major allele A in rs25531 (LA ) is associated with higher expression of the transporter protein compared to the LG allele and the short S allele [6], resulting in altered 5-HT tone and neurotransmission. Few studies have directly studied the potential role of the 5-HTTLPR polymorphisms and inhibitory control in healthy subjects under laboratory conditions and results so far are conflicting. Whereas some studies found no association between 5-HTTLPR variants and measures of inhibitory control and impulsivity [7,8], others reported that the short allele of the 5-HTTLPR may mediate impairments in impulse control [9,10]. Operationalization of inhibitory control varies across studies, with three of the four studies using variants of continuous performance/go-no go tasks, and another study applying the Stop Signal Task [8], which requires
http://dx.doi.org/10.1016/j.neulet.2014.10.006 0304-3940/© 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/3.0/).
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the cancelation of a motor response that has already been initiated [11]. The Stop Signal Task offers significant psychometric advantages over conventional Continuous Performance, or Go/No Go Task, since the difficulty of stopping can be adjusted for each individual by manipulating the delay between the Go stimulus and the stop signal. The aim of this study was to test the potential role of the 5HTTLPR polymorphism in mediating inhibitory control in healthy people, specifically if carriers of the low expressive 5-HT transporter variant (5-HTTLPR S and LG ) exhibit less effective inhibitory control relative to carriers of the high expressive variant (LA ). 2. Methods 157 healthy subjects (105 females, 52 males) were recruited from the general public using advertisements in a local newspaper in Oslo. Mean age of the cohort was 36.4 years (SD = 13.1), ranging from 19 to 64 years of age. After giving written informed consent, the participants provided information about their medical status and underwent psychiatric evaluation including the Diagnostic Interview for Genetic Studies [12], the Structural Clinical Interview for DSM-IV, Axis I and II disorders (SCID I and SCID II). Depression and anxiety symptoms were assessed using the Beck Depression Inventory [13] and the Beck Anxiety Inventory [14], respectively. The SCID interviews were administered and recorded by trained clinicians and were subjected to consensus diagnoses. Subjects fulfilling the criteria of any psychiatric diagnosis were excluded, including subjects with current/ongoing drug abuse or dependency. Other exclusion criteria were head trauma during the last year with loss of consciousness greater than 30 min as well as other neurological disorders. Education level was classified by means of the International Standard Classification of Education [15]. General cognitive functioning was estimated from scaled scores of two subtests from the WAIS-III, Picture Completion and Similarities [16]. The subjects were given a $ 50 gift certificate for their participation. The Regional Ethics Committee approved the project. 2.1. Genotyping The biallelic 5-HTTLPR polymorphism, located in the regulatory region of the 5-HT transporter gene (SLC6A4), was genotyped essentially as described in detail elsewhere [17,18]. A real-time fluorescence Light Cycler instrument was used to amplify genomic DNA by polymerase chain reaction (PCR) in a final volume of 20 L using Light Cycler Faststart DNA SYBR Green kit (Roche cat no. 12239264001) with specific primers (0.5 M) [17] generating a long (L) 419 base pair (bp) or a short (S) 375 bp PCR product. Differences in product length depend on the variable number of a 22 bp tandem repeat (VNTR) sequence in the promoter region. Cycle conditions were initiated by 10 min denaturation (95 ◦ C) followed by 45 cycles at 95 ◦ C (10 s), 66 ◦ C (10 s) and 72 ◦ C (10 s). For the detection of the additional A > G SNP (rs25531), the PCR fragments were digested with 1 U MspI restriction enzyme (New England Biolabs, Beverly, Massachusetts) for 2 h at 37 ◦ C. The PCR fragments contain two obligatory MspI sites, whereas the A > G substitution creates an additional MspI site. The PCR reaction followed by restriction digestion and gel electrophoreses provides classification of the S, LA and LG alleles. 2.2. Inhibitory control measure (the Stop Signal Task; SST) The Stop Signal Task was selected from the Cambridge Neuropsychological Test Automated Battery [19]. Trained research assistants administered the SST. This task measures the ability to
inhibit an already-initiated motor response [11]. In a subset of trials (i.e. 25%), an auditory beep occurs (the “stop signal”) to indicate that the response should be withheld on that particular trial. A procedure is applied to track the participants’ performance, by varying the stop signal delay (SSD) parameter after successful and unsuccessful stop attempts. Over time, this tracking procedure stabilizes the probability of successful inhibition around 0.5 for each subject. The Stop signal reaction time (SSRT), calculated by subtracting the SSD50 from the median Go RT, is the main outcome variable. Thus, the SSRT reflects the effectiveness in the ability to inhibit a prepotent response. The total number of Go discrimination errors (i.e. a right button press to a left-facing arrow) was also registered. Sixteen subjects failed to achieve convergence, either through too high (≤60%) or too low (≤40%) levels of successful inhibition. These staircase failures may arise through strategic slowing of the go reaction time, or through inconsistent performance or excessive distraction. They invalidate an assumption of the horse race model that Go-and stop-related processes are independent [11]. Thus, the final group for analysis was a total of 141 participants (94 females, 47 males). 3. Statistical analyses One-way ANOVAs were conducted to explore possible group differences in age, education level (ISCED) and general cognitive functioning (sub-tests Similarities and Picture Completion from WAIS-III), as well as symptoms related to depression (BDI) and anxiety (BAI). A one-way ANOVA was conducted to predict Stop Signal Task performance from 5-HTTLPR genotype combined with the A > G SNP (rs25531). Polynomial contrast was performed to test the dose effects across genotypes. Levene’s test of homogeneity confirmed that the groups were not significantly different in variance, thus validating use of the F test. Finally, a linear regression model was conducted to explore the amount of unique variance explained by genotype after taking variance explained by group differences (from the ANOVAs) into account. 4. Results The triallelic classification was reclassified into a functional model, based on the 5-HTTLPR-directed level of transcriptional activity of the transporter gene as follows: LG /S, LG /LG and S/S genotypes were classified as SS’ (low leveled RNA transcription); LA /S and LA /LG genotypes were classified as LS’ (intermediate leveled); and LA /LA genotype was classified as LL’ (high leveled) [20]. The genotype distribution was LA LA 22.9%, LG LA 10.2%, LA S 39.5%, LG LG 0.6%, SLG 7.6% and SS 19.1%. The genotype distribution was in Hardy-Weinberg equilibrium (x2 = 0, df = 1; p = 0.99). The genotype distribution after exclusion (n = 141) was LA LA 24.1%, LG LA 9.9%, LA S 41.1%, LG LG 0.7%, SLG 7.8% and SS 16.3%. There was a significant difference between the genotype groups in education level (F(2, 138) = 4.168, p = 0.017, 2 = 0.057) and a trending toward significant difference in age (F(2, 138) = 3.006, p = 0.053, 2 = 0.042). There were no statistically significant differences between 5-HTTLPR sub-groups on BDI, BAI, or the two subtests from WAIS. Education level and age was therefore added in the final regression model. There was no statistical significant sex by genotype interactions for any of the SST variables. Because of this, the analyses were collapsed across gender. There was a statistically significant effect of 5-HTTLPR plus A > G SNP on the SSRT variable (F(2, 138) = 3.518, p = 0.032, 2 = 0.049). Polynomial contrast measure revealed a linear effect of the number of low expressive alleles (CE = 21.6, p = 0.009) (Fig. 1). SSRT was predicted by 5-HTTLPR genotype (Beta = 0.195, p = 0.022), but not by age (Beta = 0.157, p = 0.65) or education level (Beta = −0.030,
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Fig. 1. Mean stop signal reaction time in milliseconds. Number of low expressive alleles: LL’ (high leveled RNA transcription), LS’ (intermedium leveled) and SS’ (low leveled). Error bars = 95% CI.
p = 0.724). The overall model explained about seven percent of the total variance (R2 = 0.072). There were no statistically significant differences across the genotype groups on Go Reaction Time (F(2, 138)) = 0.969, p = 0.382) and number of Go Discrimination Errors (F(2, 138) = 0.178), p = 0.837). The Go Reaction Times (means and standard deviations) for the three genotype groups were: LL: 404.0 (93.2), LS: 390.6 (80.2), SS: 396.9 (96.2). Number of Go Discrimination Errors were: LL: 4.5 (5.8), LS: 5.3 (6.3), SS: 6.4 (7.0). There were no significant differences between the genotype groups on Go reaction time or Go Errors, or any SST measure based on the original biallelic gene model (ignoring the A > G rs25531SNP). 5. Discussion The main finding was that 5-HTTLPR polymorphisms predict response inhibition in healthy subjects. Healthy subjects carrying the high expressive allele exhibited significantly faster inhibition of a prepotent response as compared to the low expressive genotype. This suggests more impulsive behavior in carriers of the low expressive genotype. There were no differences between the groups with respect to the basic Go reaction time or number of Go Discrimination Errors, indicating that the genotype effect on response inhibition does not reflect general cognitive slowing or deficit in basic discrimination ability. Furthermore, the association between the 5-HT transporter and the specific ability to inhibit a prepotent response was observed in the context of no differences between the genotype subgroups on indicators of general cognitive abilities or in self-reported depressive mood or anxiety symptoms (Table 1). Both the biallelic- and triallelic 5-HTTLPR model are often dichotomized in dominant gene models, i.e. carriers of the Table 1 Demographic, psychometric and clinical data for genotype sub-groups. Values represent mean and standard deviation. The p-values refer to the results from one-way ANOVAs.
Age Education level (ISCED) Similarities (scaled) Picture completion (scaled) Beck depression inventory Beck anxiety inventory
LL (n = 34)
LS (n = 72)
SS (n = 35)
p Value
32.4 (11.7) 4.7 (0.7) 11.3 (2.6) 13.1 (2.8) 2.6 (2.3) 2.3 (2.3)
34.0 (12.0) 4.6 (0.8) 11.4 (3.4) 13.5 (3.3) 3.2 (5.0) 2.5 (3.4)
39.2 (13.5) 5.1 (0.9) 11.5 (3.0) 12.5 (3.3) 4.1 (6.0) 2.1 (2.2)
0.053 0.017 0.990 0.305 0.418 0.768
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heterozygous LS variant are pooled with the homozygous SS variants However, the rationale for dichotomizing genotypes in a recessive-dominant model based on the triallelic functional gene model, suggested by Hu and collegues [6], is not well founded, as no firm conclusions can be drawn about the functional properties of the triallelic gene model [21–24]. Several studies support a functional dominant – recessive effect for the biallelic gene model but these findings have also been inconclusive [21,25–28]. Therefore, our data add important novel information suggesting a dose-dependent effect for the 5-HTTLPR variants on impulsivity. Our results are in line with work showing that healthy carriers of the long 5-HTTLPR allele exhibit significantly better performance on measures of impulsivity as compared to short allele carriers [9,10]. Short allele carriers also showed impaired post error and post-conflict behavioral adjustment, as compared to long allele carriers, on a modified flanker task [29]. 5-HT influences affective decision making [30] and three studies reported reduced Iowa Gambling Task performance among short allele 5-HTTLPR carriers [31–33]. There are, however, studies including other executive control tasks reporting opposite results. Three studies, none taking the triallelic model into consideration, found that short allele carriers outperformed their long allele counterparts. Strobel and collegues [34] found that SS and LS variants showed higher efficiency of cognitive control processes compared to LL allele carriers. Borg and collegues [35] applied the Wisconsin Card Sorting Test (WCST) and found that short allele carriers had fewer perseverative errors and they needed fewer cards to complete the task. Although typically described as a set shifting task, WCST is complex and the authors state that their study does not resolve which of all component parts of WCST performance the 5-HTTLPR influences. In a recent study on working memory, also in healthy subjects, it was found that short allele carriers performed better than long allele carriers. However, this was a change detection task reflecting primarily storage capacity in working memory and not really cognitive control [36]. Although there are exceptions, most studies reporting less effective cognitive control in short allele as compared to long allele 5-HTTLPR carriers have taken the triallelic model into consideration. Studies reporting no gene effects or the opposite pattern, i.e. better performance on cognitive control tasks in short versus long allele carriers, have in most cases based the analyses on the biallelic model. It seems unlikely that a variation with respect to gene model is the only explanation for discrepant findings when linking 5-HTTLPR and cognitive control. An intriguing idea, and which may be relevant in this context, is that the 5-HTTLPR short allele in general increases sensitivity to the environment, indicating that this gene variant may not be a vulnerability genotype as much as a plasticity genotype [37–39]. Increased neural plasticity or behavioral malleability might implicate that carriers with the short allele polymorphism will benefit more than long allele carriers from tasks with systematic feedback. Thus, this might be a possible explanation for the reported finding of better Wisconsin Card Sorting Test performance in short allele carriers, where the subject receives continuous feedback [35]. In our study, based on the Stop Signal Task, the subjects receive no such feedback. Similarly, in the Iowa Gambling Task there is no contingency with respect to what is the “correct” response. These initial observations will allow extensions to patient populations. Understanding how genetic variation within key transmitter systems contribute to functional component of the phenotypic expression of substance use [40] and mood and anxiety disorders [41] may provide insight into mechanisms that may drive the development, maintenance, and treatment of these disorders. Animal models are also making an increasing contribution to our understanding of response inhibition and impulsivity and should be integrated with human studies in the future. A study that used Stop Signal Task behavioral paradigm in mouse has shown that
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manipulation of the serotonergic system can have major effects on inhibition [42]. 6. Conclusions Healthy individuals carrying the low expressive 5-HT transporter polymorphisms (5-HTTLPR plus rs25531) exhibit less ability to inhibit a prepotent response as compared to high expressive carriers. This suggests a direct role of the low expressive 5-HT transporter polymorphism in regulating impulsive behavior traits in healthy people. Acknowledgments The present research was supported by a grant from the Research Council of Norway to NIL (project number 175387/V50). The genotyping assistance of Ms Runa M. Grimholt is greatly appreciated. We also thank cand.psychol. Anne Marie Hoel for assistance in data collection. References [1] A.J. Lawrence, J. Luty, N.A. Bogdan, B.J. Sahakian, L. Clark, Impulsivity and response inhibition in alcohol dependence and problem gambling, Psychopharmacology (Berl.) 207 (1) (2009) 163–172. [2] J.W. Dalley, J.P. Roiser, Dopamine, serotonin and impulsivity, Neuroscience 215 (2012) 42–58. [3] T.W. Robbins, Controlling stress: how the brain protects itself from depression, Nat. Neurosci. 8 (3) (2005) 261–262. [4] C.S. Carver, S.L. Johnson, J. Joormann, Two-mode models of self-regulation as a tool for conceptualizing effects of the serotonin system in normal behavior and diverse disorders, Curr. Dir. Psychol. Sci. 18 (4) (2009) 195–199. [5] A. Heils, A. Teufel, S. Petri, G. Stober, P. Riederer, D. Bengel, K.P. Lesch, Allelic variation of human serotonin transporter gene expression, J. Neurochem. 66 (6) (1996) 2621–2624. [6] X.Z. Hu, R.H. Lipsky, G. Zhu, L.A. Akhtar, J. Taubman, B.D. Greenberg, et al., Serotonin transporter promoter gain-of-function genotypes are linked to obsessive-compulsive disorder, Am. J. Hum. Genet. 78 (5) (2006) 815–826. [7] G.M. Lage, L.F. Malloy-Diniz, L.O. Matos, M.A.R. Bastos, S.S.C. Abrantes, H. Correa, Impulsivity and the 5-HTTLPR polymorphism in a non-clinical sample, PLoS ONE 6 (2) (2011), http://dx.doi.org/10.1371/journal.pone.0016927. [8] L. Clark, J.P. Roiser, R. Cools, D.C. Rubinsztein, B.J. Sahakian, T.W. Robbins, Stop signal response inhibition is not modulated by tryptophan depletion or the serotonin transporter polymorphism in healthy volunteers: implications for the 5-HT theory of impulsivity, Psychopharmacology (Berl.) 182 (4) (2005) 570–578. [9] M. Paaver, N. Nordquist, J. Parik, M. Harro, L. Oreland, J. Harro, Platelet MAO activity and the 5-HTT gene promoter polymorphism are associated with impulsivity and cognitive style in visual information processing, Psychopharmacology (Berl.) 194 (4) (2007) 545–554. [10] E. Walderhaug, A.I. Herman, A. Magnusson, M.J. Morgan, N.I. Landro, The short (S) allele of the serotonin transporter polymorphism and acute tryptophan depletion both increase impulsivity in men, Neurosci. Lett. 473 (3) (2010) 208–211. [11] G.D. Logan, W.B. Cowan, K.A. Davis, On the ability to inhibit simple and choice reaction-time responses – a model and a method, J. Exp. Psychol. Hum. Percept. Perform. 10 (2) (1984) 276–291. [12] J.I. Nurnberger Jr., M.C. Blehar, C.A. Kaufmann, C. York-Cooler, S.G. Simpson, J. Harkavy-Friedman, et al., Diagnostic interview for genetic studies Rationale, unique features, and training. NIMH Genetics Initiative, Arch. Gen. Psychiatry 51 (1994) 849–864. [13] A.T. Beck, R.A. Steer, Manual for the Beck Anxiety Inventory, Psychological Corporation, San Antonio, TX, 1993. [14] A.T. Beck, R.A. Steer, G.K. Brown, Manual for the Beck Depression Inventory-II, Psychological Corporation, San Antonio, TX, 1996. [15] UNESCO, I.f.S. International Standard Classification of Education 1997, (ISCED 1997), 1997, Available from: www.unescostat.unesco.org [16] D. Wechsler, Wechsler Adult Intelligence Scale. Manual. Norwegian Version, 3rd ed., The Psychological Corporation, Stockholm, 2003. [17] J. Gelernter, H. Kranzler, J.F. Cubells, Serotonin transporter protein (SLC6A4) allele and haplotype frequencies and linkage disequilibria in African- and European-American and Japanese populations and in alcohol-dependent subjects, Hum. Genet. 101 (1997) 243–246. [18] M.B. Stein, S. Seedat, J. Gelernter, Serotonin transporter gene promoter polymorphism predicts SSRI response in generalized social anxiety disorder, Psychopharmacology (Berl.) 187 (1) (2006) 68–72.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Serotonin transporter polymorphisms predict response inhibition in healthy volunteers N.I. Landrø a,∗ , R. Jonassen a , L. Clark b , K.B. Foss Haug c , M. Aker a , R. Bø a , J.P. Berg c , A. Neumeister d , T.C. Stiles e a
Clinical Neuroscience Research Group, Department of Psychology, Oslo, Norway Department of Psychology, University of Cambridge, Cambridge, UK c Section for Research, Department of Medical Biochemistry, Oslo University Hospital, and Institute of Clinical Medicine, University of Oslo, Oslo, Norway d New York University, Department of Psychiatry, New York, USA e Department of Psychology, The Norwegian University of Science and Technology, Trondheim, Norway b
h i g h l i g h t s • 5-HTTLPR polymorphisms mediate different levels of inhibitory control. • More impulsive behavior in healthy carriers of the low expressive genotype. • These initial observations will allow extensions to patient populations.
a r t i c l e
i n f o
Article history: Received 9 July 2014 Received in revised form 30 September 2014 Accepted 5 October 2014 Available online 17 October 2014 Keywords: Serotonin transporter Inhibitory control
a b s t r a c t Serotoninergic transmission is reliably implicated in inhibitory control processes. The aim of this study was to test the hypothesis if serotonin transporter polymorphisms mediate inhibitory control in healthy people. 141 healthy subjects, carefully screened for previous and current psychopathology, were genotyped for the 5-HTTLPR and rs25531 polymorphisms. Inhibitory control was ascertained with the Stop Signal Task (SST) from the Cambridge Neuropsychological Test Automated Battery (CANTAB). The triallelic gene model, reclassified and presented in a biallelic functional model, revealed a dose-dependent gene effect on SST performance with Individuals carrying the low expressive allele had inferior inhibitory control compared to high expressive carriers. This directly implicates serotonin transporter polymorphisms (5-HTTLPR plus rs25531) in response inhibition in healthy subjects. © 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
1. Introduction Impulsivity can be understood as arising from impairment in inhibitory control [1]. Impaired serotonin (5-HT) function [2] has been shown to contribute to the neurobiology of impaired executive control processes [3] and impulsive behaviors [4]. However, the genetic contribution of these behavioral processes is incompletely understood. The 5-HT-transporter-linked polymorphic region (5-HTTLPR) in the promoter region of the human 5-HT transporter (5-HTT) gene (SLC6A4) results in two main alleles or variants [5]; the short (S) and long (L), comprising 14 and 16 copies of a 20–23 nucleotide repeat
∗ Corresponding author at: Department of Psychology, University of Oslo, Norway. Box 1094, Blindern, 0317 Oslo, Norway. Tel.: +47 22845146. E-mail address: [email protected] (N.I. Landrø).
cassettes, respectively. A functional triallelic 5-HTTLPR polymorphism include an additional single nucleotide polymorphism A > G SNP (rs25531) in the first of two 22-bp imperfect repeats that define the 16-repeat L allele. The 5-HTTLPR L allele combined with the major allele A in rs25531 (LA ) is associated with higher expression of the transporter protein compared to the LG allele and the short S allele [6], resulting in altered 5-HT tone and neurotransmission. Few studies have directly studied the potential role of the 5-HTTLPR polymorphisms and inhibitory control in healthy subjects under laboratory conditions and results so far are conflicting. Whereas some studies found no association between 5-HTTLPR variants and measures of inhibitory control and impulsivity [7,8], others reported that the short allele of the 5-HTTLPR may mediate impairments in impulse control [9,10]. Operationalization of inhibitory control varies across studies, with three of the four studies using variants of continuous performance/go-no go tasks, and another study applying the Stop Signal Task [8], which requires
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the cancelation of a motor response that has already been initiated [11]. The Stop Signal Task offers significant psychometric advantages over conventional Continuous Performance, or Go/No Go Task, since the difficulty of stopping can be adjusted for each individual by manipulating the delay between the Go stimulus and the stop signal. The aim of this study was to test the potential role of the 5HTTLPR polymorphism in mediating inhibitory control in healthy people, specifically if carriers of the low expressive 5-HT transporter variant (5-HTTLPR S and LG ) exhibit less effective inhibitory control relative to carriers of the high expressive variant (LA ). 2. Methods 157 healthy subjects (105 females, 52 males) were recruited from the general public using advertisements in a local newspaper in Oslo. Mean age of the cohort was 36.4 years (SD = 13.1), ranging from 19 to 64 years of age. After giving written informed consent, the participants provided information about their medical status and underwent psychiatric evaluation including the Diagnostic Interview for Genetic Studies [12], the Structural Clinical Interview for DSM-IV, Axis I and II disorders (SCID I and SCID II). Depression and anxiety symptoms were assessed using the Beck Depression Inventory [13] and the Beck Anxiety Inventory [14], respectively. The SCID interviews were administered and recorded by trained clinicians and were subjected to consensus diagnoses. Subjects fulfilling the criteria of any psychiatric diagnosis were excluded, including subjects with current/ongoing drug abuse or dependency. Other exclusion criteria were head trauma during the last year with loss of consciousness greater than 30 min as well as other neurological disorders. Education level was classified by means of the International Standard Classification of Education [15]. General cognitive functioning was estimated from scaled scores of two subtests from the WAIS-III, Picture Completion and Similarities [16]. The subjects were given a $ 50 gift certificate for their participation. The Regional Ethics Committee approved the project. 2.1. Genotyping The biallelic 5-HTTLPR polymorphism, located in the regulatory region of the 5-HT transporter gene (SLC6A4), was genotyped essentially as described in detail elsewhere [17,18]. A real-time fluorescence Light Cycler instrument was used to amplify genomic DNA by polymerase chain reaction (PCR) in a final volume of 20 L using Light Cycler Faststart DNA SYBR Green kit (Roche cat no. 12239264001) with specific primers (0.5 M) [17] generating a long (L) 419 base pair (bp) or a short (S) 375 bp PCR product. Differences in product length depend on the variable number of a 22 bp tandem repeat (VNTR) sequence in the promoter region. Cycle conditions were initiated by 10 min denaturation (95 ◦ C) followed by 45 cycles at 95 ◦ C (10 s), 66 ◦ C (10 s) and 72 ◦ C (10 s). For the detection of the additional A > G SNP (rs25531), the PCR fragments were digested with 1 U MspI restriction enzyme (New England Biolabs, Beverly, Massachusetts) for 2 h at 37 ◦ C. The PCR fragments contain two obligatory MspI sites, whereas the A > G substitution creates an additional MspI site. The PCR reaction followed by restriction digestion and gel electrophoreses provides classification of the S, LA and LG alleles. 2.2. Inhibitory control measure (the Stop Signal Task; SST) The Stop Signal Task was selected from the Cambridge Neuropsychological Test Automated Battery [19]. Trained research assistants administered the SST. This task measures the ability to
inhibit an already-initiated motor response [11]. In a subset of trials (i.e. 25%), an auditory beep occurs (the “stop signal”) to indicate that the response should be withheld on that particular trial. A procedure is applied to track the participants’ performance, by varying the stop signal delay (SSD) parameter after successful and unsuccessful stop attempts. Over time, this tracking procedure stabilizes the probability of successful inhibition around 0.5 for each subject. The Stop signal reaction time (SSRT), calculated by subtracting the SSD50 from the median Go RT, is the main outcome variable. Thus, the SSRT reflects the effectiveness in the ability to inhibit a prepotent response. The total number of Go discrimination errors (i.e. a right button press to a left-facing arrow) was also registered. Sixteen subjects failed to achieve convergence, either through too high (≤60%) or too low (≤40%) levels of successful inhibition. These staircase failures may arise through strategic slowing of the go reaction time, or through inconsistent performance or excessive distraction. They invalidate an assumption of the horse race model that Go-and stop-related processes are independent [11]. Thus, the final group for analysis was a total of 141 participants (94 females, 47 males). 3. Statistical analyses One-way ANOVAs were conducted to explore possible group differences in age, education level (ISCED) and general cognitive functioning (sub-tests Similarities and Picture Completion from WAIS-III), as well as symptoms related to depression (BDI) and anxiety (BAI). A one-way ANOVA was conducted to predict Stop Signal Task performance from 5-HTTLPR genotype combined with the A > G SNP (rs25531). Polynomial contrast was performed to test the dose effects across genotypes. Levene’s test of homogeneity confirmed that the groups were not significantly different in variance, thus validating use of the F test. Finally, a linear regression model was conducted to explore the amount of unique variance explained by genotype after taking variance explained by group differences (from the ANOVAs) into account. 4. Results The triallelic classification was reclassified into a functional model, based on the 5-HTTLPR-directed level of transcriptional activity of the transporter gene as follows: LG /S, LG /LG and S/S genotypes were classified as SS’ (low leveled RNA transcription); LA /S and LA /LG genotypes were classified as LS’ (intermediate leveled); and LA /LA genotype was classified as LL’ (high leveled) [20]. The genotype distribution was LA LA 22.9%, LG LA 10.2%, LA S 39.5%, LG LG 0.6%, SLG 7.6% and SS 19.1%. The genotype distribution was in Hardy-Weinberg equilibrium (x2 = 0, df = 1; p = 0.99). The genotype distribution after exclusion (n = 141) was LA LA 24.1%, LG LA 9.9%, LA S 41.1%, LG LG 0.7%, SLG 7.8% and SS 16.3%. There was a significant difference between the genotype groups in education level (F(2, 138) = 4.168, p = 0.017, 2 = 0.057) and a trending toward significant difference in age (F(2, 138) = 3.006, p = 0.053, 2 = 0.042). There were no statistically significant differences between 5-HTTLPR sub-groups on BDI, BAI, or the two subtests from WAIS. Education level and age was therefore added in the final regression model. There was no statistical significant sex by genotype interactions for any of the SST variables. Because of this, the analyses were collapsed across gender. There was a statistically significant effect of 5-HTTLPR plus A > G SNP on the SSRT variable (F(2, 138) = 3.518, p = 0.032, 2 = 0.049). Polynomial contrast measure revealed a linear effect of the number of low expressive alleles (CE = 21.6, p = 0.009) (Fig. 1). SSRT was predicted by 5-HTTLPR genotype (Beta = 0.195, p = 0.022), but not by age (Beta = 0.157, p = 0.65) or education level (Beta = −0.030,
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Fig. 1. Mean stop signal reaction time in milliseconds. Number of low expressive alleles: LL’ (high leveled RNA transcription), LS’ (intermedium leveled) and SS’ (low leveled). Error bars = 95% CI.
p = 0.724). The overall model explained about seven percent of the total variance (R2 = 0.072). There were no statistically significant differences across the genotype groups on Go Reaction Time (F(2, 138)) = 0.969, p = 0.382) and number of Go Discrimination Errors (F(2, 138) = 0.178), p = 0.837). The Go Reaction Times (means and standard deviations) for the three genotype groups were: LL: 404.0 (93.2), LS: 390.6 (80.2), SS: 396.9 (96.2). Number of Go Discrimination Errors were: LL: 4.5 (5.8), LS: 5.3 (6.3), SS: 6.4 (7.0). There were no significant differences between the genotype groups on Go reaction time or Go Errors, or any SST measure based on the original biallelic gene model (ignoring the A > G rs25531SNP). 5. Discussion The main finding was that 5-HTTLPR polymorphisms predict response inhibition in healthy subjects. Healthy subjects carrying the high expressive allele exhibited significantly faster inhibition of a prepotent response as compared to the low expressive genotype. This suggests more impulsive behavior in carriers of the low expressive genotype. There were no differences between the groups with respect to the basic Go reaction time or number of Go Discrimination Errors, indicating that the genotype effect on response inhibition does not reflect general cognitive slowing or deficit in basic discrimination ability. Furthermore, the association between the 5-HT transporter and the specific ability to inhibit a prepotent response was observed in the context of no differences between the genotype subgroups on indicators of general cognitive abilities or in self-reported depressive mood or anxiety symptoms (Table 1). Both the biallelic- and triallelic 5-HTTLPR model are often dichotomized in dominant gene models, i.e. carriers of the Table 1 Demographic, psychometric and clinical data for genotype sub-groups. Values represent mean and standard deviation. The p-values refer to the results from one-way ANOVAs.
Age Education level (ISCED) Similarities (scaled) Picture completion (scaled) Beck depression inventory Beck anxiety inventory
LL (n = 34)
LS (n = 72)
SS (n = 35)
p Value
32.4 (11.7) 4.7 (0.7) 11.3 (2.6) 13.1 (2.8) 2.6 (2.3) 2.3 (2.3)
34.0 (12.0) 4.6 (0.8) 11.4 (3.4) 13.5 (3.3) 3.2 (5.0) 2.5 (3.4)
39.2 (13.5) 5.1 (0.9) 11.5 (3.0) 12.5 (3.3) 4.1 (6.0) 2.1 (2.2)
0.053 0.017 0.990 0.305 0.418 0.768
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heterozygous LS variant are pooled with the homozygous SS variants However, the rationale for dichotomizing genotypes in a recessive-dominant model based on the triallelic functional gene model, suggested by Hu and collegues [6], is not well founded, as no firm conclusions can be drawn about the functional properties of the triallelic gene model [21–24]. Several studies support a functional dominant – recessive effect for the biallelic gene model but these findings have also been inconclusive [21,25–28]. Therefore, our data add important novel information suggesting a dose-dependent effect for the 5-HTTLPR variants on impulsivity. Our results are in line with work showing that healthy carriers of the long 5-HTTLPR allele exhibit significantly better performance on measures of impulsivity as compared to short allele carriers [9,10]. Short allele carriers also showed impaired post error and post-conflict behavioral adjustment, as compared to long allele carriers, on a modified flanker task [29]. 5-HT influences affective decision making [30] and three studies reported reduced Iowa Gambling Task performance among short allele 5-HTTLPR carriers [31–33]. There are, however, studies including other executive control tasks reporting opposite results. Three studies, none taking the triallelic model into consideration, found that short allele carriers outperformed their long allele counterparts. Strobel and collegues [34] found that SS and LS variants showed higher efficiency of cognitive control processes compared to LL allele carriers. Borg and collegues [35] applied the Wisconsin Card Sorting Test (WCST) and found that short allele carriers had fewer perseverative errors and they needed fewer cards to complete the task. Although typically described as a set shifting task, WCST is complex and the authors state that their study does not resolve which of all component parts of WCST performance the 5-HTTLPR influences. In a recent study on working memory, also in healthy subjects, it was found that short allele carriers performed better than long allele carriers. However, this was a change detection task reflecting primarily storage capacity in working memory and not really cognitive control [36]. Although there are exceptions, most studies reporting less effective cognitive control in short allele as compared to long allele 5-HTTLPR carriers have taken the triallelic model into consideration. Studies reporting no gene effects or the opposite pattern, i.e. better performance on cognitive control tasks in short versus long allele carriers, have in most cases based the analyses on the biallelic model. It seems unlikely that a variation with respect to gene model is the only explanation for discrepant findings when linking 5-HTTLPR and cognitive control. An intriguing idea, and which may be relevant in this context, is that the 5-HTTLPR short allele in general increases sensitivity to the environment, indicating that this gene variant may not be a vulnerability genotype as much as a plasticity genotype [37–39]. Increased neural plasticity or behavioral malleability might implicate that carriers with the short allele polymorphism will benefit more than long allele carriers from tasks with systematic feedback. Thus, this might be a possible explanation for the reported finding of better Wisconsin Card Sorting Test performance in short allele carriers, where the subject receives continuous feedback [35]. In our study, based on the Stop Signal Task, the subjects receive no such feedback. Similarly, in the Iowa Gambling Task there is no contingency with respect to what is the “correct” response. These initial observations will allow extensions to patient populations. Understanding how genetic variation within key transmitter systems contribute to functional component of the phenotypic expression of substance use [40] and mood and anxiety disorders [41] may provide insight into mechanisms that may drive the development, maintenance, and treatment of these disorders. Animal models are also making an increasing contribution to our understanding of response inhibition and impulsivity and should be integrated with human studies in the future. A study that used Stop Signal Task behavioral paradigm in mouse has shown that
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manipulation of the serotonergic system can have major effects on inhibition [42]. 6. Conclusions Healthy individuals carrying the low expressive 5-HT transporter polymorphisms (5-HTTLPR plus rs25531) exhibit less ability to inhibit a prepotent response as compared to high expressive carriers. This suggests a direct role of the low expressive 5-HT transporter polymorphism in regulating impulsive behavior traits in healthy people. Acknowledgments The present research was supported by a grant from the Research Council of Norway to NIL (project number 175387/V50). The genotyping assistance of Ms Runa M. Grimholt is greatly appreciated. We also thank cand.psychol. Anne Marie Hoel for assistance in data collection. References [1] A.J. Lawrence, J. Luty, N.A. Bogdan, B.J. Sahakian, L. Clark, Impulsivity and response inhibition in alcohol dependence and problem gambling, Psychopharmacology (Berl.) 207 (1) (2009) 163–172. [2] J.W. Dalley, J.P. Roiser, Dopamine, serotonin and impulsivity, Neuroscience 215 (2012) 42–58. [3] T.W. Robbins, Controlling stress: how the brain protects itself from depression, Nat. Neurosci. 8 (3) (2005) 261–262. [4] C.S. Carver, S.L. Johnson, J. Joormann, Two-mode models of self-regulation as a tool for conceptualizing effects of the serotonin system in normal behavior and diverse disorders, Curr. Dir. Psychol. Sci. 18 (4) (2009) 195–199. [5] A. Heils, A. Teufel, S. Petri, G. Stober, P. Riederer, D. Bengel, K.P. Lesch, Allelic variation of human serotonin transporter gene expression, J. Neurochem. 66 (6) (1996) 2621–2624. [6] X.Z. Hu, R.H. Lipsky, G. Zhu, L.A. Akhtar, J. Taubman, B.D. Greenberg, et al., Serotonin transporter promoter gain-of-function genotypes are linked to obsessive-compulsive disorder, Am. J. Hum. Genet. 78 (5) (2006) 815–826. [7] G.M. Lage, L.F. Malloy-Diniz, L.O. Matos, M.A.R. Bastos, S.S.C. Abrantes, H. Correa, Impulsivity and the 5-HTTLPR polymorphism in a non-clinical sample, PLoS ONE 6 (2) (2011), http://dx.doi.org/10.1371/journal.pone.0016927. [8] L. Clark, J.P. Roiser, R. Cools, D.C. Rubinsztein, B.J. Sahakian, T.W. Robbins, Stop signal response inhibition is not modulated by tryptophan depletion or the serotonin transporter polymorphism in healthy volunteers: implications for the 5-HT theory of impulsivity, Psychopharmacology (Berl.) 182 (4) (2005) 570–578. [9] M. Paaver, N. Nordquist, J. Parik, M. Harro, L. Oreland, J. Harro, Platelet MAO activity and the 5-HTT gene promoter polymorphism are associated with impulsivity and cognitive style in visual information processing, Psychopharmacology (Berl.) 194 (4) (2007) 545–554. [10] E. Walderhaug, A.I. Herman, A. Magnusson, M.J. Morgan, N.I. Landro, The short (S) allele of the serotonin transporter polymorphism and acute tryptophan depletion both increase impulsivity in men, Neurosci. Lett. 473 (3) (2010) 208–211. [11] G.D. Logan, W.B. Cowan, K.A. Davis, On the ability to inhibit simple and choice reaction-time responses – a model and a method, J. Exp. Psychol. Hum. Percept. Perform. 10 (2) (1984) 276–291. [12] J.I. Nurnberger Jr., M.C. Blehar, C.A. Kaufmann, C. York-Cooler, S.G. Simpson, J. Harkavy-Friedman, et al., Diagnostic interview for genetic studies Rationale, unique features, and training. NIMH Genetics Initiative, Arch. Gen. Psychiatry 51 (1994) 849–864. [13] A.T. Beck, R.A. Steer, Manual for the Beck Anxiety Inventory, Psychological Corporation, San Antonio, TX, 1993. [14] A.T. Beck, R.A. Steer, G.K. Brown, Manual for the Beck Depression Inventory-II, Psychological Corporation, San Antonio, TX, 1996. [15] UNESCO, I.f.S. International Standard Classification of Education 1997, (ISCED 1997), 1997, Available from: www.unescostat.unesco.org [16] D. Wechsler, Wechsler Adult Intelligence Scale. Manual. Norwegian Version, 3rd ed., The Psychological Corporation, Stockholm, 2003. [17] J. Gelernter, H. Kranzler, J.F. Cubells, Serotonin transporter protein (SLC6A4) allele and haplotype frequencies and linkage disequilibria in African- and European-American and Japanese populations and in alcohol-dependent subjects, Hum. Genet. 101 (1997) 243–246. [18] M.B. Stein, S. Seedat, J. Gelernter, Serotonin transporter gene promoter polymorphism predicts SSRI response in generalized social anxiety disorder, Psychopharmacology (Berl.) 187 (1) (2006) 68–72.
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