In this study we evaluated the transcriptional consequences three weeks after a single neurotoxic dose of MDMA in DA rats with gene expression arrays. MDMA’s effects on the transcriptional level suggest alterations in cognition and memory related processes with the possible involvement of the CB1 and ephrin receptors in the HC. On the other hand, FC region exhibits more wide-scale changes in basic catabolic processes within FC cells and the upregulation of the 'dendrite development’ , 'regulation of synaptic plasticity’ and 'positive regulation of synapse assembly’ gene sets suggest a partial new synapse formation/synaptic reorganization in this region. These differences between the HC and FC indicate markedly different transcriptional responses of these two brain regions three weeks after a single dose MDMA administration.

Hippocampus

In the HC we observed an upregulation of CB1 receptor mRNA. Nawata et al. also investigated CB1 receptor mRNA levels in the HC regions of mice up to 7 days following the cessation from repeated MDMA administration and they reported an increase 7 days, but not 1 day after the last treatment [43]. Our study shows that an increase of CB1 receptor levels can be caused even by a single-dose of MDMA and can be detected three weeks after the drug administration in rats. The presence of elevated CB1 receptor mRNA levels in both rats and mice, which have markedly different reactions to MDMA on the long-run [44], raises the possibility of such effects in human ecstasy users alike.

Selective serotonin reuptake inhibitor (SSRI) treatment is known to cause alterations in CB1 receptor levels in the HC [45, 46] and these alterations might be the consequences of the altered serotonergic tone. Activation of serotonin 2C (5-HT 2C) receptors increases endocannabinoid production in the postsynaptic HC and amygdala neurons via the downstream activation of diacylglycerol (DAG) lipase [45]. The released 2-arachidonoil glycerol (2-AG) acts on the presynaptic neurons, and inhibits serotonin (5-HT) release through CB1 receptor activation thus forming a negative feedback loop [45]. MDMA treatment leads to a long-term serotonergic deficiency and to the damage of serotonergic axon terminals [2, 5–7]. Hence, the result of the decreased endocannabinoid release from postsynaptic neurons might result in the observed upregulation of the CB1 receptor.

Cannabinoid agonists impair working memory and short term memory [36, 39–41] and it has been also reported that MDMA can cause impairments in cognitive functions in humans, rats and mice [2, 6, 8–10, 43]. Nawata et al. also showed that CB1 receptor antagonist attenuated the MDMA-induced cognitive deficit in mice [43]. Accordingly, we observed in the present study that genes involved in the regulation of memory and cognitive processes were downregulated after MDMA treatment in DA rats. The latter findings and the fact that both CB1 receptor elevations and cognitive deficits are present in multiple species suggest a central role of CB1 receptor and thus cannabinoid signaling in the reduced cognitive and memory functions following MDMA administration.

CB1 receptors exert their effects in the cells via calcium-signaling, thus upregulation of some of the calcium/calmodulin-dependent kinases (CAMK), calcium transporters, an inositol transporter and the phosphorylation gene set may be in association with CB1 receptor signaling. In addition, enhancement in cannabinoid signaling usually indirectly reduces glutamate release by acting on GABA-ergic interneurons in the HC (for a review see [47]). Accordingly, genes belonging to the regulation of the glutamatergic synaptic transmission were downregulated in the present study. Finally, elevated CB1 receptor signaling has been shown to impair neurite growth and arborization in developing rodent brain and cannabinoid agents in adult mice were able to modulate synaptic plasticity [48–50]. In accordance, we also observed downregulations in 'neuron projection development’ and synaptic plasticity related gene sets.

Here we also show upregulations of the mRNA levels of Epha4, Epha5 and Epha6 receptors in the HC after MDMA administration. These receptors have been reported to modulate synapse formation and glutamatergic long-term potentiation (LTP) (for a review see [51]). Since ephrin receptors are bidirectional receptors and regulate both presynaptic and postsynaptic neurons, the up- or downregulation of these receptors does not result in consequent elevation or suppression of synapse formation. Instead, a proper level of these receptors is required for accurate neuronal projection termination [52]. While Epha4 and Epha6 are widely expressed in the HC, Epha5 is weakly labeled in this region under physiological conditions [53–55]. Epha4 was found to suppress synapse development in the HC [56], while Epha5 knock-out (KO) mice showed decreased spine density in other brain regions [42] and may be necessary for proper hippocampal projections [57, 58]. In another study, Epha6 KO mice showed impaired cognitive functions [59]. Thus, it is clear that ephrin receptors modulate synapse formation in the HC and related cognitive functions.

Frontal cortex

While alterations in the expression of 5-HT markers are well-defined, studies examining other effects of MDMA on gene expression are scarce. Thiriet et al. examined 1176, toxicology-related genes in adult Sprague–Dawley rats and followed expression patterns up to 7 days after a 20 mg/kg single-dose MDMA administration in the FC [60]. They found nerve growth factor alterations and suggested cytoskeletal reorganization while in another study by Fernandez-Castillo et al. emphasized neuroinflammatory responses in MDMA-effects 8 hours after repeated-administration in adult mice [61]. Martinez-Turillas et al. investigated brain-derived neurotrophic factor augmentations in the FC region of Wistar rats up to 7 days after drug administration [34]. In our present study we examined the gene expression patterns longer time (3 weeks) after a single neurotoxic dose of MDMA in the vulnerable DA rat strain. We report wide-scale downregulation of genes involved in chromatin organization, nucleocytoplasmic transport, ribosome-related functions, protein synthesis/folding and transmembrane transport processes in the FC region (Figure 5). It seems reasonable that the observed changes are the long-term consequence of the acute general neurotoxic processes, like toxic metabolite formation, hyperthermic effect or free radical production or the impairment in the autoregulation of cerebral blood flow [5, 11–19]. The latter is even further supported by the upregulation of the response to hyperoxia gene set in the present study. We could not confirm neuroinflammatory changes [61] or BDNF dysregulation [34] observed by other authors in our experimental setup. In comparison with the previous study of Thiriet et al. [60], the growth factor activity gene set was upregulated, while some gene sets, related to neuronal cytoskeletal transport were downregulated in line with the previous study. However, this similarity existed only on the level of the mentioned biological processes, and was a result of the dysregulation of other genes, a possible result of the differences in the strain, time-scale and dosage regimen between the two studies.

Motor regions in the FC are targets of thalamical inputs and contribute to motor system functions [62]. Studies in DA rats with the same MDMA administration protocol like in the recent experiment indicated chronic changes in motor activity [63–65]. Additionally, Karageorgiou et al. reported alterations in right supplementary motor area activation in human MDMA users in an fMRI study [66]. These results might reflect subsequent impairments in motor functions on the long-run and are in accordance with the observed wide-scale changes in the recent experiment.

As from another functional perspective FC and prefrontal cortical regions (PFC) are not only responsible for motor functions, but are also closely related to different cognitive tasks, e.g. working memory, goal-directed behavior, and executive functioning in rats [26, 67–69]. In our experiment FC samples contained regions from primary and secondary (supportive) motor cortices principally and likely some parts of the PFC [69]. Thus the inhibition of certain biosynthetic processes found in the present study may even participate in the cognitive decline of heavy MDMA users.

At the same time, however, upregulation of neurite formation related gene sets and thus a partial reinstatement of FC networks is also suggested by our present data. Additionally, the upregulation of HSP-related genes in the present study also suggests different extent in recovery processes. The latter results and the lack of similar processes in the HC might point out to different severity of damage of different memory types. Indeed, the only study investigating such differences following binge administration of MDMA, reported rats learning working-memory related tasks (mainly FC mediated) faster on the long-run compared to spatial reference memory (mainly HC mediated) in an 8-arm radial maze challenge [70].

Taken together, the downregulation of almost 50 gene sets related to biosynthetic processes in the FC may reflect transcriptional adaptations to well-known general neurotoxic effects not related to specific pathways 3 weeks after the drug administration. At the same time, the upregulation of the gene sets responsible for synapse/dendrite formation in this brain region may point to a starting new synapse formation/synaptic reorganization and might be a sign of a compensatory mechanism ameliorating MDMA’s acute effects 3 weeks after the administration.

Dorsal raphe

The changes in the DR region were mild in line with our previous results suggesting that MDMA-caused damage to these neurons are restricted to serotonergic axon terminals instead of neuronal cell bodies directly [71]. The caspase activation gene set significantly changed in the present study was not supported by individual genes, or other gene sets related to apoptotical processes. The downregulation in 11beta-hydroxysteroid dehydrogenase type 1 mRNA levels might suggest a possible role of the hypothalamic-pituitary-adrenal axis in MDMA’s long-term consequences, an effect also proposed by others [72, 73]. However, the DR region seems to be mostly unaffected 3 weeks after a single-dose of MDMA administration in DA rats.

Limitations

In the present study we have not elucidated the temporal patterns of the mRNAs. Further studies are needed addressing the time course of the described alterations to elicit the causative relations of these transcriptional processes in details.

We could not confirm the decreased expression of serotonergic markers in the present study. Both 5-HTT and TPH mRNA levels were unaltered in the treatment group, which is in conflict with previous results: well established prolonged serotonergic depletion and decreased expression of serotonergic markers in both protein and mRNA levels after MDMA-treatment were demonstrated by our group earlier [5, 6]. Here we can assume that the collection of DR samples was not precise enough and as we did not apply laser capture microdissection in this case, significant amount of surrounding tissue was perhaps cut out together with the DR and it may result a bias in the measurement of serotonergic markers. Notably, the decrease of 5-HTT expression, measured by quantitative in situ hybridization, was approximately -20% in the same animal model 3 weeks after the MDMA treatment, compared to the control level, and this moderate alteration was significant only in case of the fine measurement of grain densities of individual cells but not with the measuring of the autoradiography signal on film [6].

On the other hand, microarray method has well-known drawbacks, when compared to polymerase chain reaction methods or immunohistochemistry used in our earlier papers. Namely, the limited amount of probes on the microarray may result in smaller fold change values, when compared to polymerase chain reaction methods (PCR), which can also be noticed on Figure 7. On the other hand, the shorter oligomers used can result in more mismatch hybridization, which can overcome smaller changes in gene expressions, like that in the case of 5-HTT. However, for this very reason we assume that the results presented here, with our strict significance criterion, are robust enough to overcome this bias.

Figure 7 Correlation between the microarray and the Fluidigm GEx PCR array data. The figure summarizes the correlation between the logarithmic microarray data and the validation results for both the 200 ng and 500 ng samples. For validation purposes we used the polymerase chain reaction-based (PCR-based) Fluidigm GEx array with 19 genes and upon receiving the results, the numerical analysis was performed with the inbuilt corr.test function of the R statistical computing program. The results are in agreement with the commonly observed decreasing accuracy of the fold change values of the microarray method at the higher and lower ends. The correlation coefficients were 0.619 and 0.610 for the 200 ng and 500 ng samples, respectively. Red line represents the regression line. Full size image

We did not find alterations in the BDNF gene expression, which is in agreement with our previous study where we demonstrated that (after a slight transient acute decrease) BDNF protein level was increased only 8 weeks after the same MDMA dosage regimen in the same rat strain [35].

We must also note the major limitation of transcriptomic studies, namely, mRNA levels do not necessarily reflect for the appropriate protein levels.