Our findings demonstrated significantly reduced reciprocal effective connectivity in the facial emotion-processing circuit in the carriers of low activity 5-HTTLPR
S' and COMT
met alleles, relative to those with high activity homozygotes 5-HTTLPR
L'L' and COMT
val/val. Furthermore, there was a significant interaction between 5-HTTLPR and COMT polymorphisms upon effective connectivity in the face emotion-processing circuit. Effective connectivity was lower in val/met or met/met carriers who at the same time were either L'/S' or S'/S' carriers, compared with individuals who were homozygous for either val or L' alleles, or had both val and L' homozygotes. The above effects were observed in a fear condition, in the circuit comprising bilateral fusiform/occipital regions, bilateral inferior prefrontal cortex, right superior temporal gyrus/superior temporal sulcus and the right amygdala.
The combined effect of 5-HTTLPR
polymorphisms has been studied previously, albeit only with regard to the BOLD response to emotional pictures.12
Our results concur, with the proposal of the above study,12
that the joint effect of low activity 5-HTTLPR
polymorphisms confers inefficient emotion processing. The novelty of our findings is that the interactive effect of 5-HTTLPR
polymorphisms upon effective connectivity provides an insight into the genetically determined differences in emotion regulation, rather than emotion processing per se
. We emphasize that above polymorphisms modulated connectivity in a distributed neural circuit, rather than connectivity between a-priori
selected prefrontal regions and amygdala. Importantly, although this circuit has been determined empirically, it is fully consistent with existing neural models for face processing.44, 45
Our results are in line with the findings of decreased connectivity (uncoupling) between anterior prefrontal cortical regions and amygdala in healthy carriers of low activity alleles of 5-HTTLPR
in response to emotionally aversive stimuli.14
Some previous studies have reported increased cortico–limbic connectivity in 5-HTTLPR
allele carriers21, 46
We believe that our results do not contradict earlier results, given that it has previously been shown that connectivity between the brain regions depends on the precise prefrontal cortical regions examined. For example, one study14
demonstrated both increased connectivity between ventro-medial prefrontal cortex and amygdala and decreased connectivity between subgenual ACC and amygdala in the S
allele carriers vs L
allele homozygotes of 5-HTTLPR
. We note here that the prefrontal cortical regions of the emotional circuit identified in our study were located dorsally and laterally to the ventro-medial regions of prefrontal cortex of other studies.5, 21, 22, 46
Our finding that low activity alleles of both 5-HTTLPR
genes (that is, alleles that are thought to confer risk for emotional disorders) are associated with reduced connectivity within a distributed face emotion-processing network are consistent with findings of reduced connectivity between prefrontal and ventral limbic regions in individuals with MDD,16, 17, 18, 19, 29
or pathological anxiety,47
and social anxiety disorder.49
What are the implications of the reduced connectivity within the emotion-processing circuit? The converging evidence from animal research,50, 51
neuroimaging data on healthy individuals14, 15, 52
and the above studies in patients with depression and anxiety disorders indicate that reduced prefrontal-limbic connectivity may underlie inefficient emotion regulation during the processing of negative stimuli. We suggest that our findings of reduced connectivity within the emotion-processing neural circuit may be a necessary, although not sufficient component pathophysiological process in MDD. Indeed, the MDD is known to be associated with cellular and structural abnormalities in prefrontal53
cortices and/or abnormal reductions in prefrontal cortical activity.55
We briefly consider here the issue of directionality as examined with the Granger connectivity analysis. Neuroimaging genetic studies that have explored functional connectivity, found genetic modulation of connectivity between medial prefrontal and limbic regions.14, 21
This was suggested to indicate a modulation of top-down effect on emotion regulation. However, the assumption has not been formally tested, as functional connectivity is of correlational rather than a causal character. Using Granger effective connectivity, we specifically tested the potentially top-down relationships within the emotion-processing circuit.
Our data demonstrated that the frontal regions did not simply impact on limbic areas in a top-down manner, but there was a rather more complex functional organization that involved reciprocal feed-forward and feedback relationships.
This included reduced bi-directional connectivity between the prefrontal cortex and amygdala in individuals with low activity COMT and 5HTTLPR alleles. We suggest that the finding of reduced bi-directionality does not contradict the notion of reduced top-down emotion regulation in low activity carriers as it includes both top-down and bottom-up mechanisms. Thus, our data add a new aspect (that is, feedback mechanisms) to the frontal-limbic relationships.
Therefore, our results are in an agreement with the above studies that were based on correlational methods.
In contrast to the above studies that were testing a coupling between the two a-priori defined regions, we were able to study effective connectivity within the facial emotion-processing circuit that was established empirically. Our findings add to the existing knowledge by showing that the observed reduced connectivity is not just a correlational in nature but involves both top-down and feedback projections.
We emphasize that finding of bi-directional relationship within cortico–limbic network is in agreement with existing experimental literature,56, 57, 58
supporting ‘longitudinal' rather than a rigidly hierarchical network models. It has been shown59
that cortico–striato–pallido–thalamo–cortical circuitry was arranged as a series of circuits (closed loops).
The recent studies based on Granger connectivity have also demonstrated resting state60, 61
uni- and bidirectional effective connectivity.
Another important issue that deserves consideration is the emotion-specificity of the genetic effect.
In our study, all emotional expressions, that is, angry, sad, fearful and happy faces activated emotion-processing circuit; however, the genetic effect on connectivity within this circuit was observed in the fearful condition only. As both angry and fearful facial expressions represent threat-related cues, it is important to consider the possible reasons for lack of the genetic effect in angry faces condition.
Although angry faces clearly provide information about the presence of threat, it has been found that the fearful facial expressions (as signaling more ambiguous threat) have been consistently associated with the strongest amygdala activation. This is in accord with the conceptualization of amygdala as a component of vigilance system responding to ambiguous situations of biological relevance.63
This was evident in a study combining fMRI with skin conductance recording,64
where the fearful (but not angry) faces elicited activation in amygdala-dependent arousal system for fight/flight, which is recognizable fear network. Given a crucial role of serotonin in modulation of the brain processes underlying responses to potential environmental threats, it is conceivable that the processing of fearful faces would be modulated by the serotonin neurotransmission. Indeed, the processing of fearful but not angry faces has been consistently associated with the serotonin metabolism (see review).65
In support of our results, the recognition of fearful but not angry faces was modulated by the 5-HTTLPR
S vs L
There is little evidence of a differential effect of dopaminergic transmission on angry vs fearful faces processing. The investigators report the COMT
effect on fear processing in general, for example, the COMT
homozygosity was associated with the lack of ability to extinguish conditioned fear, whereas 5-HTTLPR
homozygosity underlied a potentiation of startle reactions.67
The authors concluded that that the combination of a 5-HTTLPR
allele and COMT
-homozygosity conferred an enhanced risk for acquiring fear that resisted extinction.
These data support our findings of a gene–gene effect of COMT and 5-HTTLPR genotypes on fear processing.
The absence of an effect of low activity 5-HTTLPR
polymorphisms on BOLD signal in limbic regions needs consideration. The effect of 5-HTTLPR
on BOLD signal in the amygdala has been replicated in most of the studies,2
although not all of them had sufficient power—as indicated in a meta-analysis.68
The effect of COMT
polymorphisms on amygdala activation has not been consistently replicated, for example, it was reported by some4
but not other investigators.5
The authors highlighted an importance of baseline stimuli that was not controlled for in some previous studies. Thus, an exaggerated BOLD signal in amygdala to emotional vs neutral stimuli could have been accounted for by significantly greater amygdala response to the baseline, that is, fixation cross, perceived as an ambiguous signal by S
-allele carriers.69, 70
However, a recent study that directly tested the effect of a fixation cross on BOLD response71
did not replicate the findings of greater activation to fixation cross relative to the neutral faces.
Dynamic facial stimuli used in our study represent relatively new type of experimental stimuli. Although they provide for closest possible analogy to the socially occurring events, developing an adequate baseline condition proved to be a challenging task. There is little knowledge regarding the perceptual effect of the baseline condition—moving ovals—which may come across as emotionally ambiguous signals. This may have resulted in a greater amygdala activity in S- and met-allele carriers such that the net BOLD signal changes to emotional faces vs baseline condition was not significantly exaggerated in these individuals. Thus, this should be tested in further research. Alternatively, the absence of a genetic effect on BOLD response in amygdala should not be regarded as a false negative, but rather genuine, statistically plausible negative result. Indeed, a proportion of studies with negative results is expected even if there is a true relationship between COMT (or 5-HTTLPR) and the BOLD response in amygdala to aversive signals. For instance if the statistical power of neuroimaging genetic studies was as high as 90%, still, 1 out of 10 studies should not detect the effect. Therefore, we suggest that the negative results have to be reported in order to avoid the publication bias, which distorts the real state of neuroimaging research.
We emphasize that the main focus of this study was on effective connectivity within the emotion-processing circuit, the measure of which (Granger causality) is based on temporal precedence and thus is not directly related to magnitude of the BOLD effect.
As mentioned above, due to some missing data, we have used the imputation method. To exclude any false positive results due to the imputation, we re-analyzed the data using the data only pertaining to the 84 individuals with full genotyping information. The interaction remained significant: F=6.4, df=3.79; P=0.0006, adjusted R2=16%.