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Met polymorphism
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Effects of transcranial direct current stimulation (tDCS) on executive functions: Influence of COMT Val/Met polymorphism Christian Plewnia a,*, Bastian Zwissler a, Isabella La¨ngst a, Brigitte Maurer c, Katrin Giel b and Rejko Kru¨ger c a
Department of Psychiatry and Psychotherapy, Neurophysiology & Interventional Neuropsychiatry, University of Tu¨bingen, Germany Department of Psychosomatic Medicine and Psychotherapy, University of Tu¨bingen, Germany c Department for Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, German Center for Neurodegenerative Diseases, University of Tu¨bingen, Germany b
article info
abstract
Article history:
Introduction: Transcranial direct current stimulation (tDCS) is a frequently used technique to
Received 18 July 2012
investigate healthy and impaired neuronal functions. Its modulatory effect on executive
Reviewed 18 August 2012
functions is of particular interest for understanding the mechanisms underlying integration of
Revised 23 September 2012
cognition and behavior. The key role of prefrontal dopamine function for executive functions
Accepted 7 November 2012
suggest that differences of the Val158Met polymorphism of the catechol-O-methyltransferase
Action editor Andreas Meyer-
(COMT) gene would interact with tDCS interventions in this domain. In this study, we hy-
Lindenberg
pothesized that the COMT Met allele homozygosity, associated with higher levels of prefrontal
Published online 15 November 2012
dopamine, would influence the effect of tDCS on higher-level executive functions. Method: Forty-six healthy subjects participated in a double-blind sham-controlled crossover
Keywords:
study and underwent COMT genotyping. Anodal tDCS (20 min, 1 mA) to the left dorsolat-
Dopamine
eral prefrontal cortex (dlPFC) or sham stimulation was applied during the performance of a
Neurophysiology
parametric Go/No-Go (PGNG) test measuring sustained attention, response inhibition and
Brain stimulation
cognitive flexibility as measured by set-shifting.
Executive functions
Results: In COMT Met/Met allele carrier anodal tDCS of the dlPFC was associated with a
Genetics
deterioration of set-shifting ability, which is assessed by the most challenging level of the PGNG. Without regard to the carrier status of the COMT Val158Met polymorphism no effects of anodal tDCS on executive functions could be determined. Conclusions: In line with the model of non-linear effects of L-dopa on cortical plasticity high dopaminergic prefrontal activity mediated by COMT Val158Met polymorphism predicts a detrimental effect of anodal tDCS on cognitive flexibility. Therefore, we suggest that the individual genetic profile may modulate behavioral effect of tDCS. More precise application of brain stimulation techniques according to the individual genetic patterns may support the development of personalized treatment approaches. ª 2012 Elsevier Ltd. All rights reserved.
* Corresponding author. Department of Psychiatry and Psychotherapy, Neurophysiology & Interventional Neuropsychiatry, University of Tu¨bingen, Calwerstrasse 14, D-72076 Tu¨bingen, Germany. E-mail address: [email protected] (C. Plewnia). 0010-9452/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cortex.2012.11.002
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1.
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Introduction
Self-regulation and management of cognitive processes as well as adaptation to environmental changes are fundamental human abilities. Loosely summarized as executive control these functions allow for successful behavior and social interaction. In turn, deficits in executive functions are widely acknowledged as important aspects of major psychiatric illnesses and particularly represent a major cause of functional impairment (Ferreri et al., 2011; Kitchen et al., 2012; Snyder, 2012). The dorsolateral prefrontal cortex (dlPFC) is an essential element in the neural network subserving executive functions like sustained attention (Toichi et al., 2004) inhibition of responses (Fassbender et al., 2004), and cognitive flexibility (Cools et al., 2004). Patients with focal lesions of this area attain lower scores than controls in several executive measures (Barbey et al., 2012). For instance, subjects with schizophrenia show executive dysfunction as well as changes in cognitive control neural circuitry along with deficits in frontal lobe activity (Eisenberg and Berman, 2010; Minzenberg et al., 2009). Major depression is associated with decreased dlPFC activity in response to cognitive tasks (Siegle et al., 2010). Age related cognitive decline has been shown to be associated with reduced neuronal firing in the dlPFC that can be rescued by restoring an optimal neurochemical environment (Wang et al., 2011). Therefore, it is conceivable that the enhancement of activity in the dlPFC may yield an improvement of executive control in healthy subjects and patients with disorders affecting dlPFC function. Transcranial direct current stimulation (tDCS) has been established as a simple, effective and safe method to modulate cortical excitability (Nitsche and Paulus, 2000, 2001) and cognitive functions (Wassermann and Grafman, 2005). Beneficial polarity dependent effects of tDCS to the dlPFC on executive functions have already been demonstrated (Dockery et al., 2009; Fregni et al., 2005; Leite et al., 2011), but the interaction of stimulation polarity, cognitive domain and other intra- and interindividual variables is still largely unexplored (Jacobson et al., 2012). Interestingly, the effects of tDCS require physiological concentrations of dopamine. Just as much as very low and excessive dopamine concentrations compromise cognitive functions (Cools and D’Esposito, 2011; Seamans and Yang, 2004), high doses of L-Dopa turn facilitatory effects of anodal tDCS into inhibition (Kuo et al., 2008). Accordingly, the availability of prefrontal dopamine has a major non-linear influence on the effects of focal and nonfocal brain stimulation techniques (Monte-Silva et al., 2010; Thirugnanasambandam et al., 2011). In this context, the Val-Met polymorphism of the catecholO-methyltransferase (COMT) gene appears to be particularly relevant. The COMT enzyme is responsible for degradation of dopamine and critically involved in the regulation of prefrontal dopamine levels. The activity of the enzyme is tightly regulated by a functional c.1947G>A polymorphism in the COMT gene leading to an amino acid exchange from valine to methionine in position 158 of the peptide sequence. Subjects homozygote for the COMT Met allele (Met/Met) are expected to have a 67e75% decrease in enzymatic activity and thus
reduced dopamine catabolism (Lachman et al., 1996; Lotta et al., 1995). Of note, the role of COMT for dopamine metabolism is pronounced in the prefrontal cortex, where it contributes for more than 60% of the dopamine degradation. This contrasts with other brain regions, i.e., the striatum, where COMT activity accounts for less than 15% of the dopamine metabolism (Karoum et al., 1994). Therefore the Met/Met carrier status is most likely associated with an increase in availability of dopamine in prefrontal cortex of these individuals. Relevant effects of COMT genotype on prefrontal functioning and executive control have been demonstrated in healthy subjects (Wishart et al., 2011) and in schizophrenia spectrum disorders (Ehlis et al., 2007). Compared to Valcarriers, COMT Met-homozygotes show a tendency of better baseline performance in executive functions but less cognitive flexibility when adaptation to changing rules is required (Witte and Floel, 2012). In this study, we tested the hypothesis that prefrontal dopamine as regulated COMT Val/Met polymorphism predicts the effect of anodal tDCS to the left dlPFC on sustained attention, response inhibition and set-shifting ability.
2.
Methods
2.1.
Subjects
Forty-six subjects [25 males, 21 females; mean age ¼ 25.87, standard deviation (SD) ¼ 7.29] right-handed healthy volunteers participated in this study and gave written informed consent to the experimental procedure approved by the University of Tuebingen local ethics committee. None of the subjects had a history of physical, mental or neurological illness.
2.2.
Parametric Go/No-Go (PGNG) test
We used the PGNG test (Langenecker et al., 2007) to assess sustained attention, response inhibition and set-shifting ability. In the PGNG, participants are presented with a continuous stream of letters, while each letter is displayed for 500 msec. This stream comprises a small constant set of target letters, while all other letters are distractor stimuli. The PGNG comprises different tasks on three levels of difficulty: on the first level, which primarily addresses sustained attention and establishes reaction tendency to target letters, participants are asked for a “go” response to target letters, that is, to react to them as quickly as possible by pressing a button. On the second and third difficulty level, participants are additionally asked for a “no-go” response, that is, to inhibit depending on a specific context rule a reaction to the target letter. While level 2 requires reaction and inhibition in response to two target letters and primarily addresses response inhibition, the most challenging level 3 requires reaction and inhibition in reaction to three target letters and hence is considered a task primarily assessing set-shifting ability. Each level is presented twice in following order: 1-2-3-1-3-2. Both, speed and accuracy are assessed on each task level. For the PGNG, a satisfactory reliability has been demonstrated, and convergent validity has
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been confirmed using established neuropsychological tests assessing executive functioning (Langenecker et al., 2007). Due to the different levels of difficulty, the PGNG is especially sensitive to detect subtle differences in performance. As in the present study, we investigated executive functioning in healthy individuals, we considered the choice of a sensitive measure especially relevant in order to avoid ceiling effects. In accordance with findings from a validation study (Langenecker et al., 2007), we used the percentage of correct “go” responses (PCTT e percentage correct target trials) on level 1 as a measure of sustained attention, the percentage of correct “no-go” responses (PCIT e percentage correct inhibitory trials) on level 2 as a measure of response inhibition and the percentage of correct “go” responses (PCTT) on level 3 as a measure of set-shifting ability.
2.3.
Experimental procedure Fig. 1 e Experimental design.
The study was designed as a double-blind sham-controlled crossover trial. The participants were sitting comfortably in front of a computer with 60 cm distance to the screen. Twenty minutes of tDCS (1 mA) was applied by a batterydriven stimulator (NeuroConn GmbH, Ilmenau, Germany) via a pair of water-soaked sponge electrodes (35 cm2 surface). To affect the left dlPFC, the anodal electrode was placed on the scalp at F3 according to the international 10e20 system of electrode placement and the reference (cathodal) electrode above the right orbit. At the beginning and the end of stimulation, the current was increased and decrease for 5 sec, respectively. For the sham condition, the electrodes were placed at the same positions but the current was applied only for 40 sec producing the same tingling sensation without sustained effect on cortical activity. Predefined codes assigned to either sham or real stimulation were used to start stimulation allowing for a double-blind study design. After 5 min of stimulation the PGNG was started. Instructions were presented on the screen prior to each level. Performance of the PGNG took approximately 23 min outlasting the stimulation for about 8 min. The order of anodal tDCS and sham stimulation was balanced across the participants and the second session (sham or anodal tDCS) followed 2 days after the first (Fig. 1).
2.4.
Genotyping
Extraction of DNA was performed from ethylenediaminetetraacetic acid (EDTA) blood samples of all probands according standard protocols. Ethical approval was provided by the Ethics Committee of the University of Tu¨bingen. All subjects signed an informed consent. The COMT polymorphism (c.1947G>A) was analyzed as described previously with slight modification (Lachman et al., 1996). Polymerase chain reaction (PCR) was carried out in a thermocycler (MJ Research PTC-220Dyad, Germany) under the following conditions: 50 ng DNA was amplified using forward primer (50 gcc cgc ctg ctg tca cc-30 ) and reverse primer (50 -ctg agg ggc ctg gtg ata gtg-30 ) in a final volume of 15 ml containing GoTaq Reaction Buffer (Promega), 2 mM MgCl2, 500 mM of each dNTP, .5 mM of each PCR primer, 1 M betain and 1.25 Unit GoTaq DNA Polymerase (Promega). The following cycling conditions
were performed with optimized annealing temperatures: 5 min at 95 C; 1 min at 95 C, 45 sec at 59 C, 45 sec at 72 for 39 cycles, followed by a final elongation of 7 min at 72 C. After amplification the product was digested with NlaIII rendering three fragments of 114 bp, 70 and 54 bp in most individuals. The G1947 (valine) allele is characterized by the 114 bp fragment, whereas the A1947 (methionine) allele contains an additional NlaIII restriction site generating fragments of 96 and 18 bp.
2.5.
Statistical analysis
All statistical calculations were performed using Statistica 6.1 (StatSoft, Inc. 2003, www.statsoft.com). The behavioral data were analyzed using repeated-measures analyses of variance (ANOVAs) with the within-factor tDCS stimulationsham, VERUM, at first for the overall sample but then introducing the betweenfactor genotypeval, MET/MET. SexMALE/FEMALE was included as covariate because gender ratio differences between the COMT genotype groups approached statistical significance. Post-hoc comparisons were calculated using Fisher’s least significant difference (LSD) test. An alpha level of .05 was used for all calculations. A web-based version of the Genepop software (http://genepop.curtin.edu.au/) was used to test the goodness of fit of the samples to HardyeWeinberg equilibrium by means of the exact test of Guo and Thompson (1992).
3.
Results
3.1.
Genotyping results
Genotyping revealed 36 COMT Val-allele carriers (7 Val/Val, 29 Val/Met) and 10 COMT Met/Met-carriers. Distributions of COMT genotypes were in HardyeWeinberg equilibrium ( p > .05). Both groups did not significantly differ in respect to age (Val: 26.7 8.0, Met/Met: 22.8 2.0; p ¼ .134), gender ratio (Val: 52% female, Met/Met: 20% female; p ¼ .066), and education (100% secondary school education).
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3.2.
tDCS effects
3.2.1.
Sustained attention
On PGNG level 1, no difference between sham and verum stimulation was observed [F(1, 45) ¼ 1.170, p ¼ .285]. Also, introduction of genotype as a between-factor revealed no significant interaction [F(1, 43) ¼ .017, p ¼ .896]. Fig. 2 depicts the stimulationsham, VERUM genotypeval, MET/MET interaction with regard to the PCTT. With regard to reaction time, no significant interaction was found [F(1, 43) ¼ 2.50, p ¼ .121].
3.2.2.
Response inhibition
On PGNG level 2, again no difference between sham and verum stimulation was detected [F(1, 45) ¼ 1.409, p ¼ .242] and also no significant interaction [F(1, 43) ¼ 1.14, p ¼ .24] appeared after genotype had been introduced to the model. Fig. 3 illustrates the stimulationsham, VERUM genotypeval, MET/MET ANOVA with regard to the PCIT. Analysis of reaction times yielded no significant interaction effect [F(1, 43) ¼ 2.06, p ¼ .158].
3.2.3.
Set-shifting ability
Just as on levels 1 and 2, on PGNG level 3, sham and verum stimulation alone did not yield significantly different performances [F(1, 45) ¼ 1.772, p ¼ .190]. However, introduction of genotype as a between-factor revealed a significant interaction [F(1, 43) ¼ 7.16, p ¼ .011]. Post-hoc analysis demonstrated a significantly lower set-shifting ability for Met/Met as compared to Val-carriers under verum stimulation ( p ¼ .03). Also, within the Met/Met group, set-shifting suffered from verum stimulation as compared to sham stimulation ( p ¼ .006). Fig. 4 shows the stimulationsham, VERUM genotypeval, MET/MET interaction with regard to the PCTT. No significant effects on reaction time were observed [F(1, 43) ¼ 2.1, p ¼ .16].
4.
Fig. 3 e Stimulation-by-COMT genotype interaction effect on response inhibition. Difference of correctly inhibited trials before and after anodal tDCS was plotted on the vertical axis for two genotype groups (COMT Methomozygotes and Val-carriers).
Discussion
In the present study, we tested the effects of anodal tDCS to the left dlPFC on different measures of executive function in relation to COMT Met/Met homozygosity that was previously
Fig. 2 e Stimulation-by-COMT genotype interaction effect on sustained attention. Difference of accuracy before and after anodal tDCS was plotted on the vertical axis for two genotype groups (COMT Met-homozygotes and Valcarriers).
suggested to be associated with higher prefrontal dopaminergic activity. The key finding was a deterioration of setshifting ability after anodal tDCS in the COMT Met/Methomozygotes as compared to sham stimulation and Valcarriers. Without including genetic information, no effect of tDCS on executive function was observed. Anodal tDCS has commonly been associated with an enhancement of cortical activity and improvements in cognitive functions (Jacobson et al., 2012; Nitsche and Paulus, 2011). Nevertheless a straightforward linear relationship between the polarity of stimulation (anodal vs cathodal) and the behavioral effect is unlikely. Rather, previous amount of network activity (Fricke et al., 2011), neuropharmacological substances (Nitsche et al., 2009a,b) as well as individual factors like age and gender (Boggio et al., 2010; Chaieb et al., 2008) modulate the effect of brain stimulation interventions. Recently animal (Fritsch et al., 2010) and human studies
Fig. 4 e Stimulation-by-COMT genotype interaction effect on set-shifting ability. Difference of accuracy before and after anodal tDCS was plotted on the vertical axis for two genotype groups (COMT Met-homozygotes and Valcarriers). *p < .05, **p < .01; error bars in the graph represent standard error of mean.
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(Cheeran et al., 2008) indicate a relevant influence of genetic factors particularly the BDNF Val/Met polymorphism on the modulatory effect of tDCS with Val/Met carrier showing enhanced plasticity (Antal et al., 2010). Notably, data on the interaction of the functional COMT Val/Met polymorphism and tDCS are not yet available. However, using paired associative stimulation (PAS) a technique to induce motor-cortex plasticity with peripheral somatosensory and transcranial magnetic stimulation, it has been shown that BDNF Val/Met effects are dependent on COMT Val/Met status (Witte et al., 2012). But since the effect of COMT Val/Met alone has not been tested, the role of COMT Val/Met per se remains unknown. Even more importantly, studies using different oral doses of L-dopa suggest that the influence of dopamine varies fundamentally between the different methods of brain stimulation applied (Monte-Silva et al., 2010; Thirugnanasambandam et al., 2011). Particularly, the focal induction of neuroplastic change e.g., by using PAS paradigms is stabilized by L-dopa, whereas it exerts an opposite influence on anodal stimulation by turning excitation into inhibition (Kuo et al., 2008). Moreover, the COMT Val/Met polymorphism is of major interest in respect to the mediation of tDCS effects on cognitive function. First, it was previously shown that the COMT enzyme plays an important role in regulating prefrontal dopamine levels (Karoum et al., 1994) and that the Val/Met polymorphism of the COMT gene predicts the activity of this enzyme (Lachman et al., 1996). Second, the COMT Met carrier status has been linked with specific characteristics in performance of executive functions although these results are not unequivocal (Wishart et al., 2011; Witte and Floel, 2012). Particularly, higher levels of prefrontal dopamine prevalent in Met-carriers seem to be associated with advantages in baseline executive function on the one hand but lower task flexibility on the other hand (Witte and Floel, 2012). Third, oral administration of dopamine (L-dopa) has distinct dosage-dependent effects on plasticity of the human cortex (Monte-Silva et al., 2010; Thirugnanasambandam et al., 2011) and memory formation (Floel et al., 2005). Therefore, although speculative, it is conceivable that the gene regulating prefrontal dopaminergic activity predicts the effect of tDCS on cognitive, particularly executive functions. The presented findings are in line with the concept of an inverted-U-shaped influence of dopamine on learning, memory and their presumed neurophysiological mechanisms of neuroplasticity (MonteSilva et al., 2010). Although Met/Met-homozygotes and Val-carriers display comparable set-shifting ability, adding excitatory brain stimulation appears to shift dopaminergic activity into the downward right slope of the curve below baseline associated with a decrease in cognitive flexibility. In contrast, Val-allele carriers with lower dopamine levels presumably located at the ‘upwards’ side of the inverted-U are not significantly influenced by excitatory tDCS and maintain high cognitive flexibility. Compatible with this model, the difference between the two groups becomes most apparent at the level of highest cognitive load when a shift of dlPFC functioning toward a supraoptimal state at the descending side of the alleged inverted-U-shaped curve is most likely associated with deterioration of performance.
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Of note, our findings using tDCS induced brain activation closely match the interaction of COMT Val/Met genotype and pharmacological COMT inhibition (Farrell et al., 2012). Here, the further increase of dopaminergic activity led to a deterioration of executive function as quantified by an N-back task. Moreover, on the basis of earlier animal studies (Papaleo et al., 2008) a differential responsivity to stress related to a stress-induced increase in prefrontal dopamine levels was observed in an Nback task embedded in a stressful or neutral context, with Methomozygotes showing a deterioration of WM performance in stressful situations (Qin et al., 2012). These data in concert with our findings underline the critical role of dopamine homeostasis in the prefrontal cortex for executive functions particularly and its specific interaction with additional behavioral or brain stimulation. Hence, as predicted earlier (Monte-Silva et al., 2010), magnitude and direction of brain stimulation effects on behavior are dependent on the dopaminergic tone in the human brain as a result of the COMT Val/Met genotype. However, some limitations of this study have to be considered: The reference electrode was located over the contralateral supraorbital area. Relevant effects of this simultaneous cathodal stimulation of the right frontal cortex cannot be excluded (Keeser et al., 2011). We have selected this electrode montage because until now it was used in the majority of studies and larger distances between electrodes are associated with lesser modulatory effects (Moliadze et al., 2010). Nevertheless, further studies should address this question. The contributions of other genetic (Meyer-Lindenberg et al., 2006) or epigenetic (Ursini et al., 2011) sources of COMT variation as well as polymorphisms interacting with dopaminergic genes (Heinzel et al., 2012; Witte et al., 2012) were not considered in this study. However, a rational analysis of these interactions should be based on reliable knowledge about the individual factors as presented here. Since we compared homozygous Met/Met with Val carrier, we were not able to address possible ‘dose effects’. However, earlier studies suggested most prominent differences between Met/Met compared with Val/Val and Val/Met individuals (Wishart et al., 2011). This study was not designed to assess the role of the COMT Val/Met polymorphism for individual differences in executive functions. Nevertheless, the lack of differences between the two genotypes in the sham condition allows for the conclusion that our data provide no clear-cut support for the notion that COMT Met/Met-homozygotes may show a better baseline performance in executive functions. However, this lack of a difference may also be attributed to insufficient sensitivity of the behavioral tests applied (Meyer-Lindenberg et al., 2006). Putting aside the specific impairing effect on set-shifting ability in MeteMet-homozygotes, anodal tDCS did not affect executive functions in a relatively large sample of healthy subjects. This is somewhat surprising since a number of previous studies indicated performance enhancing effects of anodal tDCS on various cognitive functions (Jacobson et al., 2012; Wassermann and Grafman, 2005) and specifically setshifting tasks (Leite et al., 2011). The divergent results may be due to differences in the applied task or the fact that in our study stimulation was applied not prior but during task performance and obtained from a significantly larger sample
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(Leite et al., 2011). However, the interaction of stimulation effect and task proficiency (Dockery et al., 2009) and the evidence for beneficial influence of cathodal stimulation on cognition (Jacobson et al., 2012; Weiss and Lavidor, 2012) suggest a more complex interaction of stimulation polarity and behavioral effects. Therefore, further studies will investigate the effects of cathodal stimulation as well as the stimulation of subjects with specific deficits of prefrontal functions (e.g., patients with depression or schizophrenia). In conclusion, the reported findings (i) support the role of individual genetic determinants for the efficacy of brain stimulation in general and tDCS in particular, (ii) suggest that dopaminergic neurotransmission is critical for the mediation of tDCS effects on cognitive functions, and (iii) may point toward new options for an individualized neurostimulation approach by integrating genetic information in the design of studies and therapeutic interventions.
Disclosures Dr. Plewnia received research grants of the German Research Council (DFG; PL 525/1e1) and the Werner Reichardt Centre for Integrative Neuroscience (CIN, PP2011_11). Dr. Kru¨ger serves as Editor of Parkinsonism and Related Disorders, the European Journal of Clinical Investigation, the Journal of Neural Transmission, and Associate Editor of BMC Neurology; has received research grants of the German Research Council (DFG; KR2119/3-2; KR2119/8-1), the Michael J Fox Foundation, the Fritz Thyssen foundation (10.11.2.153) and the Federal Ministry for Education and Research [BMBF, NGFNplus; 01GS08134], as well as speakers honoraria and/or travel grants from UCB Pharma, Cephalon, Abbott Pharmaceuticals, Takeda Pharmaceuticals and Medtronic.
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Research report
Effects of transcranial direct current stimulation (tDCS) on executive functions: Influence of COMT Val/Met polymorphism Christian Plewnia a,*, Bastian Zwissler a, Isabella La¨ngst a, Brigitte Maurer c, Katrin Giel b and Rejko Kru¨ger c a
Department of Psychiatry and Psychotherapy, Neurophysiology & Interventional Neuropsychiatry, University of Tu¨bingen, Germany Department of Psychosomatic Medicine and Psychotherapy, University of Tu¨bingen, Germany c Department for Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, German Center for Neurodegenerative Diseases, University of Tu¨bingen, Germany b
article info
abstract
Article history:
Introduction: Transcranial direct current stimulation (tDCS) is a frequently used technique to
Received 18 July 2012
investigate healthy and impaired neuronal functions. Its modulatory effect on executive
Reviewed 18 August 2012
functions is of particular interest for understanding the mechanisms underlying integration of
Revised 23 September 2012
cognition and behavior. The key role of prefrontal dopamine function for executive functions
Accepted 7 November 2012
suggest that differences of the Val158Met polymorphism of the catechol-O-methyltransferase
Action editor Andreas Meyer-
(COMT) gene would interact with tDCS interventions in this domain. In this study, we hy-
Lindenberg
pothesized that the COMT Met allele homozygosity, associated with higher levels of prefrontal
Published online 15 November 2012
dopamine, would influence the effect of tDCS on higher-level executive functions. Method: Forty-six healthy subjects participated in a double-blind sham-controlled crossover
Keywords:
study and underwent COMT genotyping. Anodal tDCS (20 min, 1 mA) to the left dorsolat-
Dopamine
eral prefrontal cortex (dlPFC) or sham stimulation was applied during the performance of a
Neurophysiology
parametric Go/No-Go (PGNG) test measuring sustained attention, response inhibition and
Brain stimulation
cognitive flexibility as measured by set-shifting.
Executive functions
Results: In COMT Met/Met allele carrier anodal tDCS of the dlPFC was associated with a
Genetics
deterioration of set-shifting ability, which is assessed by the most challenging level of the PGNG. Without regard to the carrier status of the COMT Val158Met polymorphism no effects of anodal tDCS on executive functions could be determined. Conclusions: In line with the model of non-linear effects of L-dopa on cortical plasticity high dopaminergic prefrontal activity mediated by COMT Val158Met polymorphism predicts a detrimental effect of anodal tDCS on cognitive flexibility. Therefore, we suggest that the individual genetic profile may modulate behavioral effect of tDCS. More precise application of brain stimulation techniques according to the individual genetic patterns may support the development of personalized treatment approaches. ª 2012 Elsevier Ltd. All rights reserved.
* Corresponding author. Department of Psychiatry and Psychotherapy, Neurophysiology & Interventional Neuropsychiatry, University of Tu¨bingen, Calwerstrasse 14, D-72076 Tu¨bingen, Germany. E-mail address: [email protected] (C. Plewnia). 0010-9452/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cortex.2012.11.002
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Introduction
Self-regulation and management of cognitive processes as well as adaptation to environmental changes are fundamental human abilities. Loosely summarized as executive control these functions allow for successful behavior and social interaction. In turn, deficits in executive functions are widely acknowledged as important aspects of major psychiatric illnesses and particularly represent a major cause of functional impairment (Ferreri et al., 2011; Kitchen et al., 2012; Snyder, 2012). The dorsolateral prefrontal cortex (dlPFC) is an essential element in the neural network subserving executive functions like sustained attention (Toichi et al., 2004) inhibition of responses (Fassbender et al., 2004), and cognitive flexibility (Cools et al., 2004). Patients with focal lesions of this area attain lower scores than controls in several executive measures (Barbey et al., 2012). For instance, subjects with schizophrenia show executive dysfunction as well as changes in cognitive control neural circuitry along with deficits in frontal lobe activity (Eisenberg and Berman, 2010; Minzenberg et al., 2009). Major depression is associated with decreased dlPFC activity in response to cognitive tasks (Siegle et al., 2010). Age related cognitive decline has been shown to be associated with reduced neuronal firing in the dlPFC that can be rescued by restoring an optimal neurochemical environment (Wang et al., 2011). Therefore, it is conceivable that the enhancement of activity in the dlPFC may yield an improvement of executive control in healthy subjects and patients with disorders affecting dlPFC function. Transcranial direct current stimulation (tDCS) has been established as a simple, effective and safe method to modulate cortical excitability (Nitsche and Paulus, 2000, 2001) and cognitive functions (Wassermann and Grafman, 2005). Beneficial polarity dependent effects of tDCS to the dlPFC on executive functions have already been demonstrated (Dockery et al., 2009; Fregni et al., 2005; Leite et al., 2011), but the interaction of stimulation polarity, cognitive domain and other intra- and interindividual variables is still largely unexplored (Jacobson et al., 2012). Interestingly, the effects of tDCS require physiological concentrations of dopamine. Just as much as very low and excessive dopamine concentrations compromise cognitive functions (Cools and D’Esposito, 2011; Seamans and Yang, 2004), high doses of L-Dopa turn facilitatory effects of anodal tDCS into inhibition (Kuo et al., 2008). Accordingly, the availability of prefrontal dopamine has a major non-linear influence on the effects of focal and nonfocal brain stimulation techniques (Monte-Silva et al., 2010; Thirugnanasambandam et al., 2011). In this context, the Val-Met polymorphism of the catecholO-methyltransferase (COMT) gene appears to be particularly relevant. The COMT enzyme is responsible for degradation of dopamine and critically involved in the regulation of prefrontal dopamine levels. The activity of the enzyme is tightly regulated by a functional c.1947G>A polymorphism in the COMT gene leading to an amino acid exchange from valine to methionine in position 158 of the peptide sequence. Subjects homozygote for the COMT Met allele (Met/Met) are expected to have a 67e75% decrease in enzymatic activity and thus
reduced dopamine catabolism (Lachman et al., 1996; Lotta et al., 1995). Of note, the role of COMT for dopamine metabolism is pronounced in the prefrontal cortex, where it contributes for more than 60% of the dopamine degradation. This contrasts with other brain regions, i.e., the striatum, where COMT activity accounts for less than 15% of the dopamine metabolism (Karoum et al., 1994). Therefore the Met/Met carrier status is most likely associated with an increase in availability of dopamine in prefrontal cortex of these individuals. Relevant effects of COMT genotype on prefrontal functioning and executive control have been demonstrated in healthy subjects (Wishart et al., 2011) and in schizophrenia spectrum disorders (Ehlis et al., 2007). Compared to Valcarriers, COMT Met-homozygotes show a tendency of better baseline performance in executive functions but less cognitive flexibility when adaptation to changing rules is required (Witte and Floel, 2012). In this study, we tested the hypothesis that prefrontal dopamine as regulated COMT Val/Met polymorphism predicts the effect of anodal tDCS to the left dlPFC on sustained attention, response inhibition and set-shifting ability.
2.
Methods
2.1.
Subjects
Forty-six subjects [25 males, 21 females; mean age ¼ 25.87, standard deviation (SD) ¼ 7.29] right-handed healthy volunteers participated in this study and gave written informed consent to the experimental procedure approved by the University of Tuebingen local ethics committee. None of the subjects had a history of physical, mental or neurological illness.
2.2.
Parametric Go/No-Go (PGNG) test
We used the PGNG test (Langenecker et al., 2007) to assess sustained attention, response inhibition and set-shifting ability. In the PGNG, participants are presented with a continuous stream of letters, while each letter is displayed for 500 msec. This stream comprises a small constant set of target letters, while all other letters are distractor stimuli. The PGNG comprises different tasks on three levels of difficulty: on the first level, which primarily addresses sustained attention and establishes reaction tendency to target letters, participants are asked for a “go” response to target letters, that is, to react to them as quickly as possible by pressing a button. On the second and third difficulty level, participants are additionally asked for a “no-go” response, that is, to inhibit depending on a specific context rule a reaction to the target letter. While level 2 requires reaction and inhibition in response to two target letters and primarily addresses response inhibition, the most challenging level 3 requires reaction and inhibition in reaction to three target letters and hence is considered a task primarily assessing set-shifting ability. Each level is presented twice in following order: 1-2-3-1-3-2. Both, speed and accuracy are assessed on each task level. For the PGNG, a satisfactory reliability has been demonstrated, and convergent validity has
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been confirmed using established neuropsychological tests assessing executive functioning (Langenecker et al., 2007). Due to the different levels of difficulty, the PGNG is especially sensitive to detect subtle differences in performance. As in the present study, we investigated executive functioning in healthy individuals, we considered the choice of a sensitive measure especially relevant in order to avoid ceiling effects. In accordance with findings from a validation study (Langenecker et al., 2007), we used the percentage of correct “go” responses (PCTT e percentage correct target trials) on level 1 as a measure of sustained attention, the percentage of correct “no-go” responses (PCIT e percentage correct inhibitory trials) on level 2 as a measure of response inhibition and the percentage of correct “go” responses (PCTT) on level 3 as a measure of set-shifting ability.
2.3.
Experimental procedure Fig. 1 e Experimental design.
The study was designed as a double-blind sham-controlled crossover trial. The participants were sitting comfortably in front of a computer with 60 cm distance to the screen. Twenty minutes of tDCS (1 mA) was applied by a batterydriven stimulator (NeuroConn GmbH, Ilmenau, Germany) via a pair of water-soaked sponge electrodes (35 cm2 surface). To affect the left dlPFC, the anodal electrode was placed on the scalp at F3 according to the international 10e20 system of electrode placement and the reference (cathodal) electrode above the right orbit. At the beginning and the end of stimulation, the current was increased and decrease for 5 sec, respectively. For the sham condition, the electrodes were placed at the same positions but the current was applied only for 40 sec producing the same tingling sensation without sustained effect on cortical activity. Predefined codes assigned to either sham or real stimulation were used to start stimulation allowing for a double-blind study design. After 5 min of stimulation the PGNG was started. Instructions were presented on the screen prior to each level. Performance of the PGNG took approximately 23 min outlasting the stimulation for about 8 min. The order of anodal tDCS and sham stimulation was balanced across the participants and the second session (sham or anodal tDCS) followed 2 days after the first (Fig. 1).
2.4.
Genotyping
Extraction of DNA was performed from ethylenediaminetetraacetic acid (EDTA) blood samples of all probands according standard protocols. Ethical approval was provided by the Ethics Committee of the University of Tu¨bingen. All subjects signed an informed consent. The COMT polymorphism (c.1947G>A) was analyzed as described previously with slight modification (Lachman et al., 1996). Polymerase chain reaction (PCR) was carried out in a thermocycler (MJ Research PTC-220Dyad, Germany) under the following conditions: 50 ng DNA was amplified using forward primer (50 gcc cgc ctg ctg tca cc-30 ) and reverse primer (50 -ctg agg ggc ctg gtg ata gtg-30 ) in a final volume of 15 ml containing GoTaq Reaction Buffer (Promega), 2 mM MgCl2, 500 mM of each dNTP, .5 mM of each PCR primer, 1 M betain and 1.25 Unit GoTaq DNA Polymerase (Promega). The following cycling conditions
were performed with optimized annealing temperatures: 5 min at 95 C; 1 min at 95 C, 45 sec at 59 C, 45 sec at 72 for 39 cycles, followed by a final elongation of 7 min at 72 C. After amplification the product was digested with NlaIII rendering three fragments of 114 bp, 70 and 54 bp in most individuals. The G1947 (valine) allele is characterized by the 114 bp fragment, whereas the A1947 (methionine) allele contains an additional NlaIII restriction site generating fragments of 96 and 18 bp.
2.5.
Statistical analysis
All statistical calculations were performed using Statistica 6.1 (StatSoft, Inc. 2003, www.statsoft.com). The behavioral data were analyzed using repeated-measures analyses of variance (ANOVAs) with the within-factor tDCS stimulationsham, VERUM, at first for the overall sample but then introducing the betweenfactor genotypeval, MET/MET. SexMALE/FEMALE was included as covariate because gender ratio differences between the COMT genotype groups approached statistical significance. Post-hoc comparisons were calculated using Fisher’s least significant difference (LSD) test. An alpha level of .05 was used for all calculations. A web-based version of the Genepop software (http://genepop.curtin.edu.au/) was used to test the goodness of fit of the samples to HardyeWeinberg equilibrium by means of the exact test of Guo and Thompson (1992).
3.
Results
3.1.
Genotyping results
Genotyping revealed 36 COMT Val-allele carriers (7 Val/Val, 29 Val/Met) and 10 COMT Met/Met-carriers. Distributions of COMT genotypes were in HardyeWeinberg equilibrium ( p > .05). Both groups did not significantly differ in respect to age (Val: 26.7 8.0, Met/Met: 22.8 2.0; p ¼ .134), gender ratio (Val: 52% female, Met/Met: 20% female; p ¼ .066), and education (100% secondary school education).
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3.2.
tDCS effects
3.2.1.
Sustained attention
On PGNG level 1, no difference between sham and verum stimulation was observed [F(1, 45) ¼ 1.170, p ¼ .285]. Also, introduction of genotype as a between-factor revealed no significant interaction [F(1, 43) ¼ .017, p ¼ .896]. Fig. 2 depicts the stimulationsham, VERUM genotypeval, MET/MET interaction with regard to the PCTT. With regard to reaction time, no significant interaction was found [F(1, 43) ¼ 2.50, p ¼ .121].
3.2.2.
Response inhibition
On PGNG level 2, again no difference between sham and verum stimulation was detected [F(1, 45) ¼ 1.409, p ¼ .242] and also no significant interaction [F(1, 43) ¼ 1.14, p ¼ .24] appeared after genotype had been introduced to the model. Fig. 3 illustrates the stimulationsham, VERUM genotypeval, MET/MET ANOVA with regard to the PCIT. Analysis of reaction times yielded no significant interaction effect [F(1, 43) ¼ 2.06, p ¼ .158].
3.2.3.
Set-shifting ability
Just as on levels 1 and 2, on PGNG level 3, sham and verum stimulation alone did not yield significantly different performances [F(1, 45) ¼ 1.772, p ¼ .190]. However, introduction of genotype as a between-factor revealed a significant interaction [F(1, 43) ¼ 7.16, p ¼ .011]. Post-hoc analysis demonstrated a significantly lower set-shifting ability for Met/Met as compared to Val-carriers under verum stimulation ( p ¼ .03). Also, within the Met/Met group, set-shifting suffered from verum stimulation as compared to sham stimulation ( p ¼ .006). Fig. 4 shows the stimulationsham, VERUM genotypeval, MET/MET interaction with regard to the PCTT. No significant effects on reaction time were observed [F(1, 43) ¼ 2.1, p ¼ .16].
4.
Fig. 3 e Stimulation-by-COMT genotype interaction effect on response inhibition. Difference of correctly inhibited trials before and after anodal tDCS was plotted on the vertical axis for two genotype groups (COMT Methomozygotes and Val-carriers).
Discussion
In the present study, we tested the effects of anodal tDCS to the left dlPFC on different measures of executive function in relation to COMT Met/Met homozygosity that was previously
Fig. 2 e Stimulation-by-COMT genotype interaction effect on sustained attention. Difference of accuracy before and after anodal tDCS was plotted on the vertical axis for two genotype groups (COMT Met-homozygotes and Valcarriers).
suggested to be associated with higher prefrontal dopaminergic activity. The key finding was a deterioration of setshifting ability after anodal tDCS in the COMT Met/Methomozygotes as compared to sham stimulation and Valcarriers. Without including genetic information, no effect of tDCS on executive function was observed. Anodal tDCS has commonly been associated with an enhancement of cortical activity and improvements in cognitive functions (Jacobson et al., 2012; Nitsche and Paulus, 2011). Nevertheless a straightforward linear relationship between the polarity of stimulation (anodal vs cathodal) and the behavioral effect is unlikely. Rather, previous amount of network activity (Fricke et al., 2011), neuropharmacological substances (Nitsche et al., 2009a,b) as well as individual factors like age and gender (Boggio et al., 2010; Chaieb et al., 2008) modulate the effect of brain stimulation interventions. Recently animal (Fritsch et al., 2010) and human studies
Fig. 4 e Stimulation-by-COMT genotype interaction effect on set-shifting ability. Difference of accuracy before and after anodal tDCS was plotted on the vertical axis for two genotype groups (COMT Met-homozygotes and Valcarriers). *p < .05, **p < .01; error bars in the graph represent standard error of mean.
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(Cheeran et al., 2008) indicate a relevant influence of genetic factors particularly the BDNF Val/Met polymorphism on the modulatory effect of tDCS with Val/Met carrier showing enhanced plasticity (Antal et al., 2010). Notably, data on the interaction of the functional COMT Val/Met polymorphism and tDCS are not yet available. However, using paired associative stimulation (PAS) a technique to induce motor-cortex plasticity with peripheral somatosensory and transcranial magnetic stimulation, it has been shown that BDNF Val/Met effects are dependent on COMT Val/Met status (Witte et al., 2012). But since the effect of COMT Val/Met alone has not been tested, the role of COMT Val/Met per se remains unknown. Even more importantly, studies using different oral doses of L-dopa suggest that the influence of dopamine varies fundamentally between the different methods of brain stimulation applied (Monte-Silva et al., 2010; Thirugnanasambandam et al., 2011). Particularly, the focal induction of neuroplastic change e.g., by using PAS paradigms is stabilized by L-dopa, whereas it exerts an opposite influence on anodal stimulation by turning excitation into inhibition (Kuo et al., 2008). Moreover, the COMT Val/Met polymorphism is of major interest in respect to the mediation of tDCS effects on cognitive function. First, it was previously shown that the COMT enzyme plays an important role in regulating prefrontal dopamine levels (Karoum et al., 1994) and that the Val/Met polymorphism of the COMT gene predicts the activity of this enzyme (Lachman et al., 1996). Second, the COMT Met carrier status has been linked with specific characteristics in performance of executive functions although these results are not unequivocal (Wishart et al., 2011; Witte and Floel, 2012). Particularly, higher levels of prefrontal dopamine prevalent in Met-carriers seem to be associated with advantages in baseline executive function on the one hand but lower task flexibility on the other hand (Witte and Floel, 2012). Third, oral administration of dopamine (L-dopa) has distinct dosage-dependent effects on plasticity of the human cortex (Monte-Silva et al., 2010; Thirugnanasambandam et al., 2011) and memory formation (Floel et al., 2005). Therefore, although speculative, it is conceivable that the gene regulating prefrontal dopaminergic activity predicts the effect of tDCS on cognitive, particularly executive functions. The presented findings are in line with the concept of an inverted-U-shaped influence of dopamine on learning, memory and their presumed neurophysiological mechanisms of neuroplasticity (MonteSilva et al., 2010). Although Met/Met-homozygotes and Val-carriers display comparable set-shifting ability, adding excitatory brain stimulation appears to shift dopaminergic activity into the downward right slope of the curve below baseline associated with a decrease in cognitive flexibility. In contrast, Val-allele carriers with lower dopamine levels presumably located at the ‘upwards’ side of the inverted-U are not significantly influenced by excitatory tDCS and maintain high cognitive flexibility. Compatible with this model, the difference between the two groups becomes most apparent at the level of highest cognitive load when a shift of dlPFC functioning toward a supraoptimal state at the descending side of the alleged inverted-U-shaped curve is most likely associated with deterioration of performance.
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Of note, our findings using tDCS induced brain activation closely match the interaction of COMT Val/Met genotype and pharmacological COMT inhibition (Farrell et al., 2012). Here, the further increase of dopaminergic activity led to a deterioration of executive function as quantified by an N-back task. Moreover, on the basis of earlier animal studies (Papaleo et al., 2008) a differential responsivity to stress related to a stress-induced increase in prefrontal dopamine levels was observed in an Nback task embedded in a stressful or neutral context, with Methomozygotes showing a deterioration of WM performance in stressful situations (Qin et al., 2012). These data in concert with our findings underline the critical role of dopamine homeostasis in the prefrontal cortex for executive functions particularly and its specific interaction with additional behavioral or brain stimulation. Hence, as predicted earlier (Monte-Silva et al., 2010), magnitude and direction of brain stimulation effects on behavior are dependent on the dopaminergic tone in the human brain as a result of the COMT Val/Met genotype. However, some limitations of this study have to be considered: The reference electrode was located over the contralateral supraorbital area. Relevant effects of this simultaneous cathodal stimulation of the right frontal cortex cannot be excluded (Keeser et al., 2011). We have selected this electrode montage because until now it was used in the majority of studies and larger distances between electrodes are associated with lesser modulatory effects (Moliadze et al., 2010). Nevertheless, further studies should address this question. The contributions of other genetic (Meyer-Lindenberg et al., 2006) or epigenetic (Ursini et al., 2011) sources of COMT variation as well as polymorphisms interacting with dopaminergic genes (Heinzel et al., 2012; Witte et al., 2012) were not considered in this study. However, a rational analysis of these interactions should be based on reliable knowledge about the individual factors as presented here. Since we compared homozygous Met/Met with Val carrier, we were not able to address possible ‘dose effects’. However, earlier studies suggested most prominent differences between Met/Met compared with Val/Val and Val/Met individuals (Wishart et al., 2011). This study was not designed to assess the role of the COMT Val/Met polymorphism for individual differences in executive functions. Nevertheless, the lack of differences between the two genotypes in the sham condition allows for the conclusion that our data provide no clear-cut support for the notion that COMT Met/Met-homozygotes may show a better baseline performance in executive functions. However, this lack of a difference may also be attributed to insufficient sensitivity of the behavioral tests applied (Meyer-Lindenberg et al., 2006). Putting aside the specific impairing effect on set-shifting ability in MeteMet-homozygotes, anodal tDCS did not affect executive functions in a relatively large sample of healthy subjects. This is somewhat surprising since a number of previous studies indicated performance enhancing effects of anodal tDCS on various cognitive functions (Jacobson et al., 2012; Wassermann and Grafman, 2005) and specifically setshifting tasks (Leite et al., 2011). The divergent results may be due to differences in the applied task or the fact that in our study stimulation was applied not prior but during task performance and obtained from a significantly larger sample
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(Leite et al., 2011). However, the interaction of stimulation effect and task proficiency (Dockery et al., 2009) and the evidence for beneficial influence of cathodal stimulation on cognition (Jacobson et al., 2012; Weiss and Lavidor, 2012) suggest a more complex interaction of stimulation polarity and behavioral effects. Therefore, further studies will investigate the effects of cathodal stimulation as well as the stimulation of subjects with specific deficits of prefrontal functions (e.g., patients with depression or schizophrenia). In conclusion, the reported findings (i) support the role of individual genetic determinants for the efficacy of brain stimulation in general and tDCS in particular, (ii) suggest that dopaminergic neurotransmission is critical for the mediation of tDCS effects on cognitive functions, and (iii) may point toward new options for an individualized neurostimulation approach by integrating genetic information in the design of studies and therapeutic interventions.
Disclosures Dr. Plewnia received research grants of the German Research Council (DFG; PL 525/1e1) and the Werner Reichardt Centre for Integrative Neuroscience (CIN, PP2011_11). Dr. Kru¨ger serves as Editor of Parkinsonism and Related Disorders, the European Journal of Clinical Investigation, the Journal of Neural Transmission, and Associate Editor of BMC Neurology; has received research grants of the German Research Council (DFG; KR2119/3-2; KR2119/8-1), the Michael J Fox Foundation, the Fritz Thyssen foundation (10.11.2.153) and the Federal Ministry for Education and Research [BMBF, NGFNplus; 01GS08134], as well as speakers honoraria and/or travel grants from UCB Pharma, Cephalon, Abbott Pharmaceuticals, Takeda Pharmaceuticals and Medtronic.
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