There has been an explosion in research elucidating the underlying molecular mechanisms of the circadian rhythm and how this influences multi-scale functions in the brain from basic cellular functions to larger scale networks. Recent studies have identified the molecular controls of the circadian clock that is regulated by the CLOCK/Brain and Muscle ARNT-like 1 system. It has been shown that core clock genes, clock circadian regulator, CLOCK and BMAL1 contribute to epileptic excitability.Interestingly, mTOR regulates BMAL1 proteostasis and TSC mouse models show abnormal circadian rhythms that can be corrected following genetic lowering of BMAL1 levels.On the contrary, BMAL1 functions as a translational factor that links circadian timing to the mTOR signaling pathway and BMAL1 knockout mice have lowered seizure thresholds.Decreased CLOCK protein levels were seen in resected brain tissue from patients with intractable epilepsy and that these changes can alter cortical circuits.Tying together BMAL1/CLOCK and mTOR pathways may provide additional insights into epileptogenesis and potential novel drug targets. Sleep is an important process in memory creation through regulation of normal synaptic homeostasis. Animal studies have demonstrated abnormal synaptic potentiation induced by epileptiform activity and seizures. In patients with focal epilepsy, a high density EEG study uncovered a widespread increase in slow wave activity, a marker of increased synaptic strength or excitability,cannabis curing that correlated with the burden of epileptic activity and cognitive impairment.Seizures during sleep can not only impair learning but increase mortality risk in patients with epilepsy. Clinical studies have demonstrated that sudden unexpected death in epilepsy is more common during sleep.
The mechanisms for this increased risk is unclear, but rodent studies suggest that serotonin is important in arousal, in postictal recovery of respiratory function, and as a modulator of seizure severity.Improved understanding of the role of the serotonergic system in sleep, seizure susceptibility, and in the overall circadian regulation will be important in our search for preventative treatments for SUDEP.Serotonin plays a crucial role in emotional processes. A considerable number of imaging studies involving pictures of emotional faces show that changes in serotonergic function are associated with changes in amygdala reactivity when viewing negative facial expressions: Acute tryptophan depletion, which reduces central serotonin synthesis, leads to higher amygdala activity when processing negative face expressions . Several studies have found that acute/sub-acute SSRI intervention leads to a decrease in amygdala activation . Further, when the cerebral serotonin level is pharmacologically enhanced by a three-week intervention with a selective serotonin reuptake inhibitor , the ensuing decreased cerebral [11C]SB207145- PET binding in response to pharmacologically increased brain serotonin levels is associated with lower threat-related amygdala reactivity . Whereas it is relatively clear that induction of acute and sub-acute changes in serotonin neurotransmission leads to changes in emotional processing, less is known about how the neural processing of emotional information is affected by chronic cerebral serotonin depletion. Ecstasy, or 3,4-methylene-dioxymethamphetamine is a widely used recreational drug that has immediate effects interms of improved mood and feelings of empathy . In this paper, we will use the term “ecstasy” when referring to the recreational human, and “MDMA” when referring to experimental human/animal studies. MDMA exerts its primary effects on the serotonin neurotransmitter system, in particular by reversing normal serotonin transporter function and hence releasing serotonin from the storage vesicles into the synaptic cleft .
Animal studies show that repeated exposure to moderate and high doses of MDMA is associated with a reduction in cerebral serotonin levels and a decreased number of SERT binding sites. In humans, prolonged recreational use of ecstasy is also associated with reductions in SERT in both cortical and sub-cortical brain areas . Most , although not all , molecular imaging studies show that the accumulated lifetime intake of ecstasy correlates negatively with SERT binding, supporting a dose-dependent relationship between recreational ecstasy use and reductions in SERT binding. Thus, it also seems plausible that in humans, an ecstasy-associated reduction in SERT is associated with reduced cerebral serotonin levels, and that the SERT changes may even result from chronically reduced serotonin levels. Additional support for serotonin depletion in recreational ecstasy use comes from the finding of increased levels of the post-synaptic serotonin 2A receptor in most , although not all , studies where ecstasy users have had their serotonin 2A receptors measured. Importantly, lowering brain serotonin levels in preclinical models leads to low SERT combined with high serotonin 2A receptor levels, and low SERT has also been found to be associated with high serotonin 2A receptor levels in humans . The reduction in sub-cortical SERT binding in ecstasy users seems to be reversible, since a positive correlation with time of abstinence from ecstasy intake has been observed . Taken together, data from these preclinical and clinical studies indicate that there is a causal relationship between ecstasy intake and effects on the serotonergic system. Reduced SERT and serotonin after MDMA/ecstasy exposure in combination with the observed correlation between serotonin depletion and reduced SERT binding in animal studies makes it plausible that SERT binding can be regarded as a representation of the extent of chronic—but most likely reversible—serotonin depletion in long-term ecstasy users. With MDMA being an interesting and promising candidate as adjunct to psychotherapy for treatment of post-traumatic stress disorder and possibly other conditions as well , it is important to explore the possible long-term impact of this drug on serotonergic neurotransmission, as well as the functional consequences of this. Although the multiple and not always pure MDMA doses used recreationally differ from the only few—and pure—doses employed in therapy, recreational use of MDMA/ ecstasy can serve as a model for the long-term effect of repeated doses. In the present study, we used functional magnetic resonance imaging to investigate the functional effects of long-term recreational ecstasy use,curing cannabis representing a model of long-term serotonin depletion, on the neural basis of emotional responses in the amygdala.
We hypothesized that amygdala engagement to aversive stimuli would show a positive correlation with accumulated lifetime ecstasy use; a negative correlation with SERT binding, as assessed with positron emission tomography imaging, in the amygdala; and a negative correlation with time since last use of ecstasy, suggesting recovery of amygdala response.Fourteen users of ecstasy and 12 non-using control subjects were recruited by flyers, advertisements posted on relevant websites, and word of mouth. Potential candidates were invited to a face to-face screening that involved assessment of history of alcohol, tobacco, and illicit drug use and Lifetime Drinking History, as well as screening of current and previous psychiatric symptoms using the Schedules for Clinical Assessment in Neuropsychiatry interview . All participants included in the present study, which focuses on fMRI investigations not presented previously, constitute of individuals who took part in a simultaneously conducted study with PET measurements of serotonergic markers—the PET results obtained in the larger sample have been published previously . To avoid acute drug effects in the ecstasy users, use of drugs was not allowed seven days before the PET and MRI scans, which were conducted on separate days at the Neurobiology Research Unit at Rigshospitalet and at the Danish Research Centre for Magnetic Resonance at Hvidovre Hospital, respectively. Abstinence was confirmed by urine screen on the PET and MRI days. Control individuals were excluded if they reported more than 15 lifetime exposures to cannabis or had any history of use of other illegal drugs, and urine screen on the day of the scan was carried out. Demographic and drug data are presented in Table 1. No present or prior neurological or psychiatric disorders were allowed for any of the subjects, and all subjects had a normal neurological examination and were lifetime naïve to antidepressants and antipsychotics. The study was approved by the local Ethics Committee, Copenhagen and Frederiksberg, Denmark 01-124/04 with amendment 11-283038, and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants.Participants were presented with pictures of faces from the Pictures of Facial Affect Series . The following types of affect were presented: neutral , anger , disgust , fear and sadness . Using a blocked design, eight pictures of each emotion were presented for three seconds, with an interval of 0.75 seconds between stimuli presentations and emotion blocks. Sixteen blocks were presented in an ABACADAE design, each letter designating a block. Each block included eight trials and lasted 30 seconds.The responses were registered during an interval starting 50 ms and ending 3750 ms from each stimuli presentation.
Regression analysis was used to investigate possible correlations between reaction times and log2 to the lifetime intake of ecstasy tablets or log2 to the number of days since the last use of ecstasy. Due to our previous findings that the effects of cumulative ecstasy lifetime intake and time since last use of ecstasy on SERT binding follow a log2distribution, we expected a log2-linear relationship between RT and lifetime intake and time since last use . Analysis and results that include lifetime intake and/or number of days since last use of ecstasy will in this manuscript refer to “log2 to” the number of accumulated lifetime doses/time since last use, but in order to improve readability, in the Results and Discussion sections, we have left out “log2 to” from the text. Differences in RT were assessed using a repeated measures analysis of variance model, with group as between-subject factor and emotion as within-subject factor . The significance level was set to α=0.05. Post hoc t-tests were carried out in order to test paired comparisons between facial expressions. The Greenhouse–Geisser method was used to correct for non-sphericity.The degree of serotonin depletion as reflected by cerebral SERT binding was assessed with PET and the selective SERT radioligand, 11C-DASB, as described in more detail by Erritzoe et al. . In short, PET data were acquired on a GE-Advance scanner as a dynamic 90-minute emission recording after intravenous injection of the radiotracer, 11C-DASB. There was maximum of 1.6 months between the 11C-DASB PET and the fMRI experiment ; approximately half of the group had PET before MRI, and vice versa. The vast majority was scanned within one to two weeks; only two were scanned with an interval of more than a month. For each participant, a mean SERT binding value from the amygdala was calculated. To confirm previous results obtained in the overlapping study sample , we used linear regression analysis to test whether the log2 to the accumulated lifetime intake of ecstasy tablets correlated with SERT binding from the amygdala. Since effects of lifetime ecstasy intake on SERT become smaller with increasing abstinence from ecstasy , we controlled for this by also including the log2 to the number of days since the last use of ecstasy in the analysis. Also using a linear regression analysis, we tested whether the log2 to the number of days since the last use of ecstasy correlated with SERT binding from the amygdala . The possible difference between ecstasy users and controls in amygdala SERT binding was tested with an independent samples t-test.Functional data were preprocessed and analyzed using the statistical parametric mapping software package . Preprocessing included spatial realignment, co-registration to the anatomical image, segmentation, and normalization to the standard Montreal Neurological Institute template, and smoothing using a symmetric 6-mm Gaussian kernel. Subject-level models were constructed using five emotional face regressors , together with regressors modulating the events by their RT values. In addition, the subject-level models included a regressor for incorrect sex discrimination answers, and 24 nuisance regressors to correct for movement artifacts, including first- and second order movement parameters and spin history effects . T-contrasts comparing BOLD responses to emotional and neutral images were created for each participant. These maps were used in group-level models assessing: the main effects of emotional face processing across all participants, differences in BOLD responses between ecstasy users and control subjects, correlations between BOLD responses and the log2 to the accumulated lifetime ecstasy intake in ecstasy users, correlations between brain activity and regional SERT binding in the amygdala in ecstasy users and control subjects, and correlations between BOLD responses and the log2 to the number of days since the last use of ecstasy in ecstasy users.