We consider this less likely because, as discussed already, interventional animal studies have shown that administration of MDMA lowers cerebral SERT levels, and data from our group and others support the presence of an ecstasy dose–response relationship and recovery of SERT binding with abstinence from ecstasy . As for all investigations of the long-term consequences of illicit drug use, especially the use of ecstasy, there will be uncertainties about the precision of the users’ reporting of drug use and actual content of substance taken. As explained in more details elsewhere , hair analysis for MDMA, use of systematic semi-structured questionnaires, and access to systematically acquired data on the content of Danish ecstasy pills in the period of data collection was employed to minimize these factors. MDMA has several effects on the serotonergic system, such as inhibiting tryptophan hydroxylase, the rate-limiting enzyme for serotonin synthesis, and serotonin degradation by monoamine oxidase B . It is possible that it is not the specific effect of MDMA on SERT, but other effects of prolonged MDMA use on the serotonergic neurotransmitter system that mediate the effect of MDMA use on brain responses to emotional faces. MDMA also has noradrenergic and dopaminergic effects that could affect amygdala activation. An additional limitation of our study is that we did not record hormonal contraception or menstrual-cycle phase for the two females in each group. These factors have been shown to affect face processing . However, we do not have any reason to suspect differences in contraceptive use or cycle phase between groups, why the lack of this information is considered as added noise, potentially reducing the power of the study. In conclusion,ebb and flow flood table these results emphasize the important role of serotonergic neurotransmission in the amygdala for processing angry face expressions.

We show that long-term ecstasy use has a dose-dependent effect on the amygdala response to angry faces. Importantly, on the basis of earlier work on amygdala responses to emotional face stimuli after manipulation of serotonin levels, this finding is in support of the hypothesis that recreational use of ecstasy can cause serotonin depletion. The decreased SERT binding among ecstasy users in the current as well as in several previous samples further supports this notion. The fact that changes observed in the current study showed signs, although at a trend level, of reversibility with sustained abstinence is also in agreement with previous PET/SPECT imaging studies. With the recent focus on MDMA as a potential therapeutic tool in psychiatry , it is important to emphasize that heavy use of the same substance in a recreational setting is associated with functional and molecular—possibly reversible—changes related to serotonergic neurotransmission.Oleoylethanolamide is a fatty acid ethanolamide and a natural analog of the endogenous cannabinoid anandamide. There is no detailed research on the role of endocannabinoids in sleep in humans. Anandamide is known to engross slow-wave sleep by increasing adenosin levels in the forebrain of rodents . This is blocked by the cannabinoid CB1-receptor antagonist rimonabant. Oleamide is an endogenous sleepinducing lipid with putative cannabinomimetic properties . Murillo-Rodriguez et al. supposed oleoylethanolamide, which—unlike oleamide— activates the nuclear peroxisome proliferator-activated receptor-a to increase alertness and to participate in the regulation of waking. Up to now, elevated levels of oleoylethanolamide and anandamide were found in human microdialysates within the first day of ischemia as well as following neural injuryor other stressors associated with necrosis. Furthermore, massive increases in FAEs and their precursor phospholipids have been found during the acute phase of stroke in the adult rat brain .

Thereby it was suggested that increases of brain FAE levels serve a neuroprotective function mediated by CB1-receptors. Oleoylethanolamide does not bind to cannabinoid CBreceptors but to PPAR-a, thereby activating a different neuroprotective mechanism. Sleep deprivation has been hypothesized to represent an oxidative challenge for the brain and that sleep may have a protective role against oxidative damage. While Gopalakrishnan et al. found no change in antioxidant enzymatic activities or increased oxidant production in the brain or in peripheral tissues after prolonged sleep deprivation, other studies suppose that sleep deprivation may result in a condition of oxidative stress including a reduction in glutathione levels in whole brains of rats . Ramanathan et al. showed that prolonged sleep deprivation results in significant decreases in the activity of superperoxide-dismutase in rat hippocampus and brainstem. This effect may be due to the degradation of antioxidative enzymes after prolonged activation during wakefulness, which suggests an alteration in the metabolism resulting in oxidative stress. Even if sleep deprivation might not show extensive effects comparable to cerebral injury or tissue necrosis, FAEs have been shown previously to be elevated during situations of cellular stress and oxidative stress factors are elevated following sleep deprivation . Therefore, in this study, we investigated whether the levels of the endogenous PPAR-a agonist oleoylethanolamide and the endocannabinoid anandamide were elevated in CSF and serum of healthy individuals after acute sleep deprivation.The Ethics Committee of the Medical Faculty of the University of Cologne reviewed and approved the protocol of this study and the procedures for sample collection and analysis. All volunteers gave written informed consent twice at least 1 day prior to each lumbar puncture after extensive introduction into the procedures and goal of this study. The healthy subjects received an allowance for the participation in this trial.

All investigations were conducted in accordance with the Declaration of Helsinki. This study was integral to a larger project on endocannabinoid levels in CSF and serum of healthy volunteers and patients suffering psychiatric disorders. As part of that, volunteers were investigated to establish baseline levels of endocannabinoids in a healthy control population . Twenty healthy volunteers with no family history and no clinical indication for any relevant medical, psychiatric or neurological disturbances were included in our study to investigate effects of sleep withdrawal on endocannabinoid levels. Necessary criteria for inclusion were absence of cannabis use within the last year and lifetime cannabis use not exceeding five times. No positive urine drug screening for illicit drugs was accepted at time of the lumbar puncture . Living circumstances for those volunteers selected did not change substantially during this period. Subjects were lumbar-punctured twice during the course of our study using a nontraumatic LP procedure. The first LP was done under regular sleep condition. The second LP took place after 24 h of sleep deprivation about 1 year later to avoid seasonal influences and artificial alterations in cerebrospinal fluid due to a previous LP in the near past. All subjects spent the nights before both LPs at home; sleep quality before the first lumbar puncture was assessed by the self-rating questionnaire for fatigue and for sleep and awakening quality . The night of sleep deprivation was spent in the habitual environment of the volunteers. Alertness was monitored by wrist actigraphy,hydroponic drain table using the ‘‘Actiwatch2000,’’ a piezoelectric transductor recording a maximum of 240 wrist motions per minute. Actiwatch was given to the volunteers 24 h before the second lumbar puncture. Volunteers showing a mosaic of inactivation at the actiwatch-scan of more than 5 min during the 24-h period were excluded from the experiment. This approach allowed us to continuously monitor the subjects during the night of sleep withdrawal . Sleep habits in volunteers were assessed for 7 days before sleep deprivation using a sleep protocol to exclude interferences of the sleep rhythm. For sleep deprivation, volunteers were requested to stay awake for one night without consuming any coffee or alcohol during that night; intake of food was allowed. All LPs were done between 10:00 and 12:00 AM, and each time peripheral blood was collected. All samples revealed no pathognomonic cell counts, CSF/serum albumin ratios or oligoclonal bands. Virological and microbiological testing of the CSF was negative in all cases. In all subjects, we measured CSF and serum levels of oleoylethanolamide along with anandamide. To quantify FAEs, CSF aliquots and serum were spiked with 25 pmol of [2 H4]-anandamide and [2 H4]-oleoylethanolamide and prepared for further analysis as described previously . Fatty acid ethanolamides were purified and quantified by isotope dilution high performance liquid chromatography/massspectrometry using a HP 1100 Series HPLC/ MS system equipped with a octadecylsilica Hypersil column . MS analyses were performed with an electro-spray ion source as previously described . Statistical analyses were performed using SPSS and R software . Because of apparent non-normality of empirical data distributions, location differences in measurements were assessed by Wilcoxon signed rank or rank sum test .Our results show a significant increase of oleoylethanolamide in human CSF after 24 h of sleep deprivation, whereas the levels of the endocannabinoid anandamide remain unaffected. Interestingly, there is no indication for a functional role of anandamide in sleep induction in humans as hypothesized previously . In rats, Murillo-Rodriguez et al. showed that anandamide increases slow wave sleep, which can be blocked by administration of the cannabinoid CB1-receptor antagonist rimonabant, indicating that the endocannabinoid system is involved in sleep regulation . Further, recent data in rats indicate diurnal variations of anandamide and oleoylethanolamide in CSF with maximum values for oleoylethanolamide during the late light phase, decreasing in the dark phase .

This research was done in rats, which are awake in the lights-off period. Translating these results to the human sleep/wake cycle it may be hypothesized that the concentration of oleoylethanolamide in CSF may decrease during daytime and increase during sleep. Given these assumptions, the increase of oleoylethanolamide is not very likely the result of an accumulation, particularly with regard to the very limited extracellular life span of endocannabinoids . As in our study the second lumbar puncture was done at the same time of the day as the first one; circadian rhythms should not confound our results. Oleoylethanolamide is renowned for modulation of feeding, body weight and lipid metabolism, as its levels decrease during food deprivation and increase upon feeding . This should not be a confounder as well, as oleoylethanolamide plays its role as a local satiety hormone in the intestine , and the feeding conditions were not changed before and after sleep-withdrawal in this study. Like anandamide, oleoylethanolamide is synthesized by neurons and other cells in a stimulus-dependent manner and is rapidly eliminated by enzymatic hydrolysis. Oleoylethanolamide binds with high affinity to the PPAR-a, a nuclear receptor that is known to regulate several aspects of lipid metabolism and induces satiety by activating PPAR-a . PPAR-a is also involved in neuroprotection through activation of antioxidant and anti-inflammatory mechanisms. It has been shown that pretreatment with the synthetic PPAR-a agonist fenofibrate is neuroprotective against cerebral injury . Furthermore, treatment with PPAR-a agonists as well as monounsaturated and polyunsaturated fatty acids induces an increase in activity of major antioxidant enzymes in the brain .Moreno et al. have provided an overview on the distribution of PPARs in the adult rat central nervous system . They reported the highest densities of PPAR-a in the hippocampal dentate gyrus and the granular cell layer of the cerebellar cortex. Furthermore, they observed PPAR expression in previously unreported locations, such as the basal ganglia, the reticular formation, some thalamic, mesencephalic and cranial motor nuclei and the large motor neurons of the spinal cord . This widespread distribution in the CNS also includes brain areas involved in sleep regulation such as the thalamus and the reticular formation. It has been hypothesized that sleep may serve an antioxidant function by removing free radicals or oxygen reactive species produced during waking time and that restoration of antioxidant balance is a property of recovery sleep . However, these protective mechanisms, especially in the brain, are still poorly understood. Interestingly, several studies have found that sleep deprivation is a stressful condition, which is associated with the disruption of various physiological processes . Reduced glutathione levels have been found in different brain regions, such as thalamus and hypothalamus, as well as in different organs of sleep deprived animals . Additionally, markers of generalized cell injury accompanied these decreases and decreased superoxide-dismutase activity has been shown in hippocampus and brainstem after prolonged sleep deprivation . The activation of these components of the antioxidant defense system suggests that sleep deprivation is a stressful condition for the entire body and for the brain in particular. Several studies have shown that PPAR-a activation enhances antioxidative enzyme activities .