In this pilot study, using citalopram phMRI we demonstrated a significant difference in temporal activation pattern of the ACC between healthy control participants and migraine without aura patients during the acutely increased extracellular 5-HT level in the brain. The intravenous citalopram elicited increased activation over time in two peaks within the pregenual part of the right ACC in migraine patients when compared to healthy controls. This increased activation in migraine subjects was more pronounced in the first 10 min after the start of the citalopram. In addition, increased activation was found in one cluster in the left rostral ACC in migraine patients. Though this latter activation did not survive the correction for multiple comparisons, our results suggests a bilateral effect of increased brain 5-HT level on the ACC in the migraine patients.
The altered functions of the pgACC in migraine have been reported in previous fMRI studies. Increased activation was found in migraine patients to trigemino-nociceptive stimulation compared to controls also in the rostral part of the ACC . Emphasizing the importance of our results, previous fMRI studies using trigeminal heat stimulation to induce acute pain in migraine patients, found increased brain activation in the pgACC with almost identical MNI coordinates as reported here [8, 31]. One of these studies showed that this enhanced activation to noxious heat reduced after a 60 day successful treatment with external trigeminal neurostimulation . The authors suggested that the pgACC could be involved in the antinociceptive effect of the anti-migraine treatment . Our results are in line with these results as we found that this region is more active in migraine patients after a small increase of 5-HT levels compared to pain-free healthy controls. These observations together suggest that increased sensitivity of the ACC in migraine patients might be related to altered serotonergic control of the incoming excitatory signals.
According to literature the most consistently reported area of cingulate cortex to painful stimuli is the dorsal ACC or anterior middle cingulate cortex (aMCC). The activation of this area is one of the earliest responses to pain as this region has an impact in cortical nociception . A recent meta-analysis showed a significant likelihood of activation to painful stimuli in this brain area in healthy subjects . In case of our study we did not find any activation of this area, however, we have not used any painful stimuli during the scanning sessions and we investigated the difference between migraine patients and healthy controls.
On the basis of the functional imaging studies of the last 20 years, the role of pgACC in many aspects of emotional processing seems to be established. In a PET study the pgACC was active during the assessment of internal emotional state induced by emotional pictures with different valences . Another study with fMRI showed that the subjective ratings of pleasantness or aversiveness of sensory stimuli correlated with the activation of this area during a decision making task . In addition, the pregenual part of ACC is a central node of the default mode network, a task-negative network that is consistently active during mind-wandering and it is implicated in the affective network . The pgACC is the only area in the cingulate gyrus that has connection with all other regions of the cingulate cortex therefore this ACC subregion could be considered as the anterior cingulate association area , which is involved – along with other brain areas – in integrating information across the brain . These observations point to the essential role of the pgACC in emotional processing, specifically in emotional awareness, e.g. the fundamental role of this area for individuals to assess their own emotional experience . Thus, our results may suggest that migraine patients have exaggerated responses to interoceptive, e.g. emotional or visceroceptive stimuli that might be related to altered serotonergic neurotransmission in migraine. However, taking into account the rich brain network of the pgACC further studies are needed to determine whether the increased 5-HT level in the brain directly sensitizes the pgACC in migraineurs or the increased activation of the ACC is secondary to a complex interplay between the increased 5-HT level and those subcortical and other cortical areas that are important in processing different sensory and interoceptive signals.
Nevertheless, the pgACC is the ACC subregion with the highest opioid receptor density  and it also plays an important role in opioid analgesia and opioid placebo effect . In addition, the increased pgACC signal during noxious stimuli may reflect the attention to unpleasantness of pain [41, 42]. The recurring headache attacks and the increased attention to unpleasantness of pain in migraine patients may cause altered pgACC functions. Our results extend this observation and suggest that this phenomenon might be related to altered serotonergic neurotransmission in migraine.
A recent systematic review of electrophysiological and neuroimaging studies of serotonergic system in migraine confirmed the altered 5-HT neurotransmission which has been a main area of interest in migraine research for decades . The authors supported the notion of suddenly increasing 5-HT levels during migraine headache . Based on our results we speculate that during migraine attack the pgACC reacts to the spontaneously elevated 5-HT levels the same way as in our study by citalopram challenge i.e. with increased activation. If so, it may contribute to the development of migraine attack by facilitating the pain transmission at the trigeminal level  similarly to that observed at the spinal cord .
In line with previous studies, we can conclude that migraine patients are more sensitive to acute increase in 5-HT levels and that this phenomenon can be observed in a subregion of the ACC which is involved in emotional aspects and suffering elements of pain and may be involved in the modulation and/or chronification of migraine. However, the direction of the connection between migraine attacks and the steep increase of 5-HT levels during headache remains unclear.
Interestingly, a previous study of peripherial neurochemical changes in migraine showed that slight release of platelet 5-HT after a nitroglycerin test could be protective against migraine development . The authors reported that no migraine attack developed in migraine patients who responded with increased peripherial 5-HT level to nitroglycerin . However, this study investigated only the peripherial changes of 5-HT level. In addition, nitroglycerine induced migraine may develop through other mechanisms than migraine attacks caused by 5-HT releasing agents. As 5-HT releasing agents e.g. reserpine can provoke migraine , it is possible that migraine attacks caused by extensive increase in brain 5-HT levels in sensitive patients may be related to the increased activation and altered pain-modulation of the pgACC.
Nevertheless, it has to be mentioned that none of our 6 migraine patients developed headache during or immediately after the citalopram administration. As we used a relatively low dose of citalopram the increase in 5-HT levels probably did not reach the level that occurs during migraine attack.
Our major limitation in this study is the relatively low number of participants in the patient group. Despite the low sample size, we found significant activation difference between the two groups. It would be fruitful however to investigate the acute effect of 5-HT level changes on the ACC activation in more migraine patients in the future to replicate and confirm our results. It would be also important to investigate the activation differences between migraine patients and controls in other important pain processing areas or even in the whole brain with higher sample size.
Furthermore, it has to be mentioned that in contrast to PET phMRI is not suitable to detect specific receptor activation changes, therefore we were not able to determine which serotonergic receptor or receptors are responsible for the observed activation changes. However, our aim was not to detect changes in receptor activation, rather to investigate the general sensitivity to increased 5-HT levels in migraine.
In addition, we have not corrected our analysis for potential individual differences of the grey matter density in the ACC that might influence the BOLD signal changes.
Finally, in spite of the fact that the pgACC is highly involved in emotional processing, we did not find any difference between citalopram and placebo sessions and between migraine and control groups in the subjective states. Thus, the difference between the two groups to citalopram could not be detected at subjective behavioral level, only at neural level. However, the lack of changes at behavioral level could be related to the dichotomous characteristic of the answers or to the relatively low dosage of citalopram.