Results of molecular imaging studies showed quite consistently that SERT binding is lower after use/administration of ecstasy/MDMA, particularly after administration of high dosages, while studies on the 5-HT 2A receptor showed inconsistent results. Results of molecular imaging studies on the DA system are quite consistent in that most molecular imaging studies in humans did not find any significant effect of MDMA on the dopamine system. Here, we will focus primarily on the statistically significant findings reported in Tables 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11.

5-Hydroxytryptamine synthesis

In this review, only one human study on 5-HT synthesis was included (Table 2). The main reason that, until recently, only one study looked into 5-HT synthesis in MDMA users is that the PET radiotracer, alpha-[11C]-methyl-l-tryptophan ([11C]AMT), which is a well-validated radiotracer to measure 5-HT synthesis, is hardly available. In this study, only 17 MDMA users and 18 age-matched controls were included, whereof half of the MDMA users and controls were men. Increases and decreases in [11C]AMT trapping were observed comparing MDMA users with controls; however, decreases were mainly seen in prefrontal–orbital and parietal regions and increases in the brainstem. The differences were more extensive in men than in women. As suggested by the authors, the decreases in the forebrain may reflect 5-HT neurotoxicity and the increases in the brainstem could be explained by an up-regulation of synthesis to compensate the loss of 5-HT neurons. Nevertheless, further research should be performed to draw definitive conclusions whether 5-HT synthesis is altered in MDMA users. Also, it may be relevant to perform studies in small laboratory animals with this radiotracer, to validate whether administration of MDMA is able to induce detectable changes in 5-HT synthesis as assessed by this radiotracer and to study the relationship between 5-HT synthesis and 5-HT neurotoxicity.

Serotonin transporter

Eleven animal studies looked into the effects of MDMA on SERT binding, and all of them showed lower SERT binding, reaching statistical significant effects in ten of these studies (Table 3). The ES were large (ranging from −0.38 to −20.03), which indicates that the effect of MDMA on SERT binding is a robust finding in animals. As compared to human studies, an advantage of animal studies is that the animals were solely treated with MDMA. In humans, however, polydrug use is common, which makes it harder to look at the effects of MDMA per se (Gouzoulis-Mayfrank and Daumann 2006). Consequently, it may be hard to generalize the findings observed in animals to humans. Also, in animal studies, MDMA was administered frequently. Indeed, most of the animal studies administered MDMA twice a day for 4 days in a row, whereas humans typically only use one or two tablets of ecstasy in the weekend. Moreover, relatively high doses within a short interval (e.g. two doses per day for four consecutive days) of MDMA were used in the animal studies (range 20–141 mg/kg), which may explain the large ES, and the drug was administered commonly intraperitoneally. However, some research indicates that due to differences in metabolism, neurotoxic dosages of MDMA are different between small animal species and primates. In rats, only high dosages of at least 20 mg/kg may be neurotoxic (Schmidt 1987). Using differences in clearance and body mass/surface area between monkeys and humans, an estimation of the neurotoxic dosage of MDMA for a human can be made, which was estimated at 1.28 mg/kg by Ricaurte et al. (2000). As mentioned before, humans typically use one or two tablets of ecstasy, each containing approximately 138 mg (reflecting 2–4 mg/kg in a person of 70 kg) (Van Laar et al. 2015). This dosage may be in the neurotoxic range based on the prediction by Ricaurte et al. (2000). In contrast, Baumann et al. (2007) argued that interspecies scaling, which means adjusting doses between species, should not be used, because behavioural, endocrine and neurochemical reactions will occur at corresponding doses, around 1–2 mg/kg. Furthermore, other researchers argued that high doses, i.e. >25 mg/kg, of MDMA produce neurotoxicity to all types of neurons (Jensen et al. 1993). These findings implicate that the doses of MDMA used in most animal research might be too high to compare the results of these studies with human studies.

Consistent with findings in animals, 14 out of the 16 SERT studies performed in humans also showed significantly lower SERT binding, particularly in cortical brain areas. However, not all studies corrected for multiple comparisons. Ten studies examined SERT binding in the occipital cortex, and in six of these studies, the decrease of SERT binding was most pronounced in this particular brain area, with ES ranging from −0.21 to −2.17. Several experimental studies have reported that, indeed, high doses of ecstasy affect preferentially 5-HT-ergic projections to the occipital cortex (Oliveri and Calvo 2003). Hadzidimitrou and colleagues (1999) and Molliver et al. (1990) stated that axons with a great length, e.g. axons to the occipital cortex, have a higher sensitivity to neurotoxic substances. Besides the cortical regions, forebrain regions were also examined. Four studies explored hippocampal SERT, and in 3 out of these 4 studies was the SERT binding significantly reduced in MDMA users; however, only 2 were corrected for multiple comparisons. It has been shown that heavy MDMA users have verbal and visuo-spatial memory deficits, and loss of SERT in the hippocampus may contribute to these deficits (Bosch et al. 2013). For cognitive processes like language and memory, the thalamus is also very important (Herrero et al. 2002). Eleven studies found that the SERT binding was lower in this brain area in users with a history of ecstasy use; however, only three studies showed significant effects. It might be that SERT loss in the thalamus plays a key role in verbal memory deficits too.

The study of Urban et al. (2012) showed statistically non-significant decreases of −100 % in SERT binding in the orbitofrontal and parietal cortex. These large percentages can be explained by the fact that the binding of [¹¹C]DASB in these regions is very low, which hampers an accurate quantification of SERT binding.

It should be considered that different radiotracers with different binding characteristics were used in studies on the effects of MDMA use/administration on SERT, which may have influenced outcomes. SPECT studies used the non-selective tracer [123I]β-CIT, while PET studies used selective tracers, e.g. [11C]DASB and [18F]ADAM (Chen et al. 2012; Frankle et al. 2004). Since [123I]β-CIT binds with high affinity to both the DAT and SERT, SERT binding in DAT-rich areas (i.e. striatum) cannot be assessed with this radiotracer. Other methodological issues could have affected the accuracy of the quantitative measurements as well. For example, simple ratio methods were used in the SERT SPECT studies, which are more prone to changes in tracer delivery, whereas modelling time activity curves were used in some PET studies (e.g. the study of Booij et al. (2014)). Finally, the limited spatial resolution of PET scanners, and particularly of clinical SPECT scanners, can lead to an underestimation of the binding potential in small volumes (partial volume effect) (Erlandsson et al. 2012).

In the past 10 years, another technique called pharmacological MRI was evaluated to assess 5-HT dysfunction. This technique measures the hemodynamic response on a pharmaceutical, e.g. a selective serotonin reuptake inhibitor (SSRI). It is a very interesting development; however, more research is necessary to validate this technique (Schouw et al. 2012; Wingen et al. 2008).

Although the results of the included studies may be influenced by differences in tracer and techniques (PET versus SPECT, but also analysis techniques), the findings of imaging studies on SERT were robust. Confirming previous studies, use/administration of MDMA declines SERT binding.

5-HT 2A receptor

Three out of five imaging studies showed an increased 5-HT 2A binding in MDMA users (Table 5). In these three studies, the period of abstinence for ecstasy ranged between 2.0 and 169.8 weeks and ES ranged between 0.14 and 1.84. The other two studies showed a loss of 5-HT 2A receptor binding, and in these studies, the period of abstinence ranged between 1.6 and 36.9 weeks. The [123I]R91150 SPECT study of Reneman et al. (2002c) showed that in recent MDMA users (mean time of abstinence 3.3 weeks), postsynaptic 5-HT 2A receptor binding was significantly lower in all cortical areas studied, while 5-HT 2A receptor densities were significantly higher in the occipital cortex of ex-MDMA users. Moreover, this study showed a significant positive correlation between cortical 5-HT 2A receptor binding and duration of abstinence from MDMA (P < 0.01). Also, the same study showed, using an ex vivo technique in rats and using the same radiotracer, a decrease of binding followed by a time-dependent recovery of cortical 5-HT 2A receptor binding, which was strongly and positively associated with the degree of 5-HT depletion (Reneman et al. 2002c). However, no positive correlation between the 5-HT 2A receptor binding and time of abstinence was found in the other studies (Di Iorio et al. 2012; Erritzoe et al. 2011; Urban et al. 2012). The time of abstinence in the study of Erritzoe et al. (2011) ranged between 1.6 and 36.9 weeks and in the study of Di Iorio et al. (2012) between 34 and 169.8 weeks, so these ranges should be large enough to evaluate a possible correlation between 5-HT 2A receptor binding and time of abstinence. Moreover, the study of Urban et al. (2012) did not show a decrease in receptor binding, although the subjects were also relatively recent MDMA users (mean time of abstinence 5.7 weeks, ranging from 2 to 8 weeks), comparable to the study of Reneman et al. (2002c). So, all in all, findings on 5-HT 2A receptor binding in MDMA users are inconsistent and it is uncertain if there is a relationship between time of abstinence and 5-HT 2A receptor binding.

Dopamine system (dopaminergic vesicular monoamine transporter, D 2/3 receptor and dopamine release, dopamine transporter, decarboxylase activity)

Some experimental studies in animals suggested that administration of MDMA/ecstasy affects not only the 5-HT system, but also the DA system. For example, Commins et al. (1987) showed that when MDMA was given to rats in a high dosage, DA levels were decreased in some brain regions. However, other research showed that treatment with MDMA/ecstasy has limited effect on the dopamine nerve endings in rats (Battaglia et al. 1987; Stone et al. 1986). In mice, MDMA seems to be a selective DA neurotoxin, while in rats a selective 5-HT neurotoxin (Stone et al. 1986). Therefore, Easton and Marsden questioned the ability to translate findings of animal studies on DA neurotoxicity to humans (Easton and Marsden 2006).

In this search, we found one animal study and five human imaging studies that examined the influence of ecstasy on the central DA system and they showed consistently no significant effects of MDMA on the DA system (Tables 8, 9, 10 and 11). One study in monkeys examined the VMAT expression in the basal ganglia but did not find significant differences between the self-administering MDMA group and the polydrug-administering control group. One human study explored the effect of MDMA on baseline DA D 2/3 receptors and DA release and no significant differences were found. Three studies examined striatal DAT binding in MDMA users; however, only one study of Reneman et al. (2002b) showed statistically significant differences. In that particular study, the effects of use of MDMA and amphetamines on striatal DAT binding were assessed. MDMA users were compared to polydrug using controls and the binding ratios in the striatum were significantly increased (striatal binding ratios, increase 13 %, P = 0.045, ES = 2.92). However, comparing MDMA users that used amphetamines less than 3 weeks before the study to MDMA users, it was found that striatal binding ratios were significantly decreased (striatal binding ratios, decrease 20 %, P = 0.007, ES = −4.09). This study concluded that use of amphetamines, and not the use of MDMA, might induce loss of nigrostriatal DA neurons. Because of the polydrug use of many ecstasy users, it is hard to look specifically at the effects of MDMA and they stressed the importance of the inclusion of a proper control group.

Only one study (Tai et al. 2011) looked into decarboxylase activity (using [18F]dopa PET) and found that there was a significant increase in ex-MDMA users compared to drug-naive controls, only in the putamen. However, the ex-MDMA users were polydrug users and when comparing ex-MDMA users to polydrug using controls, there was no significant effect anymore. This study stresses the importance of a well-selected control group as well.

In short, the results on the DA system are quite consistent. Most molecular imaging studies in humans did not find any significant effect of MDMA on the DA system. Further research has to be conducted to draw definite conclusions whether this system is affected in MDMA users.

Limitations

Several limitations of this review should be recognized. In this review, we did not find imaging studies that assessed other neurotransmitter systems than the 5-HT or DA system that might be affected by MDMA. There is little imaging research available on other receptors/transporters that may be influenced by MDMA due to a lack of well-validated radiotracers for every transporter/receptor of interest. Moreover, most of the included studies used a very small number of subjects; the number of subjects in animal studies was ranging from 1 to 26 animals and in human studies from 14 to 116 subjects. Another limitation is the washout period used. A reasonable period of abstinence of MDMA/ecstasy is necessary to exclude direct pharmacological effects of MDMA on the neurotransmitter systems; this is of particular importance in studies on the 5-HT and DA system. However, some studies in this review used a minimal period of abstinence for ecstasy of only 1 week. Furthermore, the purity of ecstasy tablets varies and the amount of MDMA in a tablet changed over the years; consequently, there are limitations in comparing the results of the human studies over time (Sherlock et al. 1999). Also, not all studies were corrected for multiple comparisons, and therefore, some significant findings could be explained by chance.

3,4-Methylenedioxymethamphetamine and additional drug use

MDMA users are likely to be polydrug users. Several studies attempted to look specifically at the effects of MDMA by including polydrug using control groups. The study of Tai et al. (2011) (Table 10) showed the importance of a polydrug using control group, because there was no significant difference left in decarboxylase activity when the data of the MDMA group were compared to the data obtained in the polydrug control group. Different subgroups of polydrug users were also analysed by two studies to investigate the effects of some commonly used drugs in combination with MDMA, e.g. cannabis, cocaine and hallucinogens, on the binding of several transporters/receptors. This approach can be useful, because it may assess the influence of those drugs on the outcome of studies that included drug-naive controls instead of polydrug controls.

First, the study of de Win et al. (2008b) assessed the specific/independent neurotoxic effects of heavy ecstasy use and contributions of amphetamine, cocaine and cannabis. They concluded that use of cannabis and cocaine did not have any significant effect on the effects of MDMA on SERT binding as measured with [123I]β-CIT SPECT, comparing MDMA users with polydrug using controls. In the second study of Erritzoe et al. (2011), reductions were seen in the cerebral SERT binding in MDMA-preferring users, but not in hallucinogen-preferring users, and they concluded that not hallucinogens, but MDMA alters the presynaptic 5-HT-ergic transmitter system. Taken these studies into account, use of cannabis, cocaine and hallucinogens may not influence the effects of MDMA on the SERT significantly.

Age-of-first exposure

One study (Klomp et al. 2012) looked into the effects of age-of-first exposure on SERT binding in humans and rats. In the early-exposed group, they found a significant inverse relationship between age-at-first ecstasy use and [123 I]β-CIT binding ratios in the SERT-rich midbrain; however, in the late-exposed group, no significant relationship was seen. They stated that, particularly, the developing brain might be sensitive to the potential neurotoxic effects of MDMA use. In early-exposed rats and humans however, they did not find lower SERT binding ratios in the midbrain. A likely explanation may be that the midbrain of rats is already matured very early in the maturation process; consequently, the effects of MDMA are less pronounced. These results suggest that in future studies, age-of-first exposure should be taken into account. Animal studies already concluded that the maturing brain is affected differently by the administration of MDMA/ecstasy (Broening et al. 1994; Meyer and Ali 2002); however, no animal studies on this topic were included. Only one human in vivo imaging study passed our inclusion criteria; therefore, more research has to be done to draw valid conclusions about what the role of age-of-first exposure is on changes to neurotransmitter systems in humans.

Gender differences

Reneman et al. (2001a) reported about gender differences in susceptibility to possible neurotoxic effects of MDMA use. Several studies looked into this topic and came to different conclusions. Buchert et al. (2004) confirmed the association between sex and reduction of SERT availability. However, de Win et al. (2008b) did not find a gender effect on SERT availability. The only study that looked into 5-HT synthesis reported on a decreased [11C]AMT trapping in frontal regions in males, but not in women (Booij et al. 2014). In this study, men seemed to be more susceptible to the effects of polydrug use. In conclusion, whether gender plays an important role in susceptibility to the effects of MDMA use is not completely clear and further research on this topic should be undertaken.

Alteration in receptor binding and neurotoxicity

The main outcome of imaging research is commonly expressed in terms of increased or decreased receptor/transporter binding; however, the cause of the alteration remains unclear in these studies. There are at least four explanations for the observed decrease in receptor/transporter binding: down-regulation and/or endocytosis of the receptor/transporter, neuronal damage resulting in loss of receptors/transporters which are expressed on this particular neuron, decreased expression of protein levels of the receptor and endogenous neurotransmitter release induced by the drug which could reduce the binding of the radiotracer (e.g. administration of MDMA/ecstasy can induce 5-HT release, which can lead to lower 5-HT 2A receptor availability). In this regard, it is of interest that a study by Quelch et al. (2012) showed a significant reduction in the ability of the radioligand [3H]DASB to bind to the SERTs that are located intracellularly (as compared to binding on the SERT expressed on the cell membrane) and they speculate that down-regulation could (partly) explain the reductions in SERT binding in MDMA studies with the radioligand [11C]DASB, since MDMA has been shown to redistribute SERT into intracellular compartments (Kivell et al. 2010). To distinguish between causes of lower receptor/transporter binding, further research in animal brains, e.g. using electron microscopy (to assess internalization of receptor binding) or high-performance liquid chromatography to assess neurotransmitter concentrations and determination of B max (number of binding sites) and K d (affinity for the receptor), would be helpful.

Also, more translational research is necessary to examine in which conditions lower SERT binding may reflect neurotoxicity. This is relevant since it is still debated whether ecstasy use/administration is indeed neurotoxic. There are several techniques developed that claim to measure neurotoxicity, e.g. immunocytochemistry, immunohistochemistry, reactive gliosis and silver staining. However, these techniques differ in sensitivity and specificity and it can be questioned whether they all can demonstrate 5-HT neurotoxicity. Immunocytochemistry can be used to look at the structural and functional integrity of the assessed neurotransmitter system. Immunohistochemistry can be used to assess 5-HT axon degeneration; with this technique concentrations of 5-hydroxyindoleacetic acid (5-HIAA), 5-HT and the SERT can be measured (Battaglia et al. 1987; Commins et al. 1987). Glial activation (reactive gliosis) is a response to all nervous system injury, and silver staining is a direct way to stain degenerating neurons (O’Callaghan and Sriram 2005). However, there are several limitations of these techniques. It is argued that immunohistochemistry should be validated by other means, because the neurotransmitter levels could be unmeasurable due to pharmacological depletions, while the neuron itself can be intact (Baumann et al. 2007; Chang and Slikker Jr 1995). Silver staining is not selective for damage to serotonergic axons but also measures loss of other types of neurons. However, this technique is very useful for measuring neuronal loss (Jensen et al. 1993; O’Callaghan and Miller 1993). In fact, no SPECT or PET tracer is available that can directly assess serotonergic/dopaminergic toxicity or degeneration per se. So, until yet, whether or not a lowered receptor/transporter binding as assessed by PET/SPECT studies in humans represents neurotoxicity is still a matter of interpretation. Importantly, MDMA is not only used recreationally. Some researchers proposed to treat patients with MDMA, e.g. as a catalyst in psychotherapy for PTSD patients (Amoroso 2015; Doblin et al. 2014; White 2014), which further highlights the need to be able to assess whether or not the use of ecstasy is neurotoxic to humans.

Recovery

Not only the causes, but also the duration of the effects of MDMA/ecstasy on receptor/transporter binding is important to be further explored. As mentioned earlier, a number of studies have investigated the effect of the duration of ecstasy abstinence on the SERT binding by examining the reversibility of the SERT binding in relation to period of abstinence from MDMA use/administration. Scheffel et al. (1998) already showed in a baboon study that SERT binding was increased from 40 days to 9 months after MDMA administration in the pons, midbrain and hypothalamus, whereas it remained decreased in cortical regions (pons: increase 35.7 %, midbrain: increase 95 %, hypothalamus: increase 168.5 %). In human studies, similar results were found. Reneman et al. (2001b) concluded that SERT binding of ex-MDMA users that stopped using MDMA for more than a year was similar to that binding of MDMA-naive controls. Moreover, Buchert et al. (2004) found a significant positive correlation between SERT binding and period of abstinence. Two years later, the same research group found a significant increase over the course of time of SERT binding of MDMA users and of SERT binding in the thalamus of ex-MDMA users, respectively (MDMA: P < 0.01; ex-MDMA: thalamus P = 0.006) (Buchert et al. 2006). Selvaraj et al. (2009) further supported the idea of SERT recovery; there was no difference in SERT binding between former ecstasy users and drug-naive controls after 1 year of abstinence. Moreover, Erritzoe et al. (2011) concluded that the duration of abstinence was positively related to SERT binding in pallidostriatum, amygdala and thalamus, but not in the neocortex. According to their data, recovery of the pallidostriatal SERT binding takes 200 days. In conclusion, there seems to be some evidence that there is a recovery of SERT binding. If there is indeed recovery of SERT binding over time, the relevant question is then whether this recovery represents functionally intact 5-HT neurons. The study of Reneman et al. (2001b) suggests that this may not be the case, because SERT binding in ex-MDMA users, who had stopped using MDMA for more than 1 year, was similar to control levels but demonstrated similar deficits on the RAVLT memory test as current MDMA users. Indeed, a couple of studies showed that even small doses of MDMA could lead to cognitive impairments, e.g. in verbal memory, and these impairments persist over time (Quednow et al. 2006; Schilt et al. 2007). A review of Parrott (2013) confirms that different cognitive functions can be affected by ecstasy use. There are deficits found not only in retrospective and prospective memory but also in higher cognition, complex visual processing, sleep architecture, sleep apnoea, pain, neurohormonal activity and psychiatric status. Therefore, recovery of SERT binding may reflect sprouting of 5-HT neurons or reduced endogenous neurotransmitter release after 5-HT toxicity has occurred, instead of recovery of the functional integrity of the 5-HT neurons.

However, another study of Halpern et al. (2011) found little evidence in ecstasy users for cognitive impairments. This study was designed to minimize methodological limitations and concluded that studies on cognitive function should be interpreted with caution. If it is true that use of ecstasy does not lead to persistent cognitive impairments, the recovery of SERT binding may simply reflect normalization of the adaptation (e.g. down-regulation), which may occur initially after MDMA use. In sum, several studies have shown that there is a recovery in SERT binding after MDMA use/administration, but it is not clear whether this is the result of recovery of the 5-HT neurons or other causes. Future fundamental studies on this topic are therefore recommended.

Implications for practice

Selection criteria for the inclusion of subjects are very important for the quality of a given study. Research has shown that regular ecstasy users are polydrug users, so controls have to be matched on polydrug intake to rule out the effects of other drugs. As mentioned earlier, some studies showed that results were not significant anymore when polydrug-using controls were used instead of controls without a history of other drugs. The studies in this review used a great diversity of criteria to select subjects.

To generalize findings from animal studies to the human context, animal studies have to mimic the human context as accurately as possible. In many animal studies on the effects of MDMA, MDMA was administered passively. However, animal studies in which MDMA was self-administered may best reflect the human situation. The effects found on SERT binding were less pronounced in studies which used MDMA self-administration compared to studies which treated the animals passively with MDMA (ES ranged from −0.72 to 5.82 in SERT studies with self-administration and 0.69 to 20.03 in other studies), although the accumulated lifetime intake was higher in the studies with self-administering animals (97–141 mg/kg lifetime intake compared to 40–80 mg/kg in other studies). However, the number of animals used in these studies was relatively small; four MDMA self-administering monkeys and four controls were used in both studies.