Neurotox Res (2014) 25:161–169 DOI 10.1007/s12640-013-9416-1
SHORT REPORT/RAPID COMMUNICATION
3,4-Methylenedioxymethamphetamine Induces Gene Expression Changes in Rats Related to Serotonergic and Dopaminergic Systems, But Not to Neurotoxicity Elisabet Cuyas • Patricia Robledo • Nieves Pizarro Magı´ Farre´ • Elena Puerta • Norberto Aguirre • Rafael de la Torre
•
Received: 5 June 2013 / Revised: 16 July 2013 / Accepted: 30 July 2013 / Published online: 15 August 2013 Ó Springer Science+Business Media New York 2013
Abstract 3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is an amphetamine derivative widely abused by young adults. Although many studies have reported that relatively high doses of MDMA deplete serotonin (5-HT) content and decrease the availability of serotonin transporters (5-HTT), limited evidence is available as to the adaptive mechanisms taking place in gene expression levels in the brain following a dosing regimen of MDMA comparable to human consumption. In order to further clarify this issue, we used quantitative PCR to study the long-term changes induced by acute administration of MDMA (5 mg/kg 9 3) in the expression of genes related to serotonergic and dopaminergic systems, as well as those related to cellular toxicity in the cortex, hippocampus, striatum, and brain stem of rats. Seven days after MDMA administration, we found a significantly lower expression of the 5-HTT (Slc6a4) and the vesicular Electronic supplementary material The online version of this article (doi:10.1007/s12640-013-9416-1) contains supplementary material, which is available to authorized users. E. Cuyas P. Robledo N. Pizarro M. Farre´ R. de la Torre (&) Human Pharmacology and Clinical Neurosciences Research Group, Neurosciences Research Program, IMIM-Hospital del Mar Medical Research Institute, Parc de Recerca Biome`dica de Barcelona, Doctor Aiguader, 88, 08003 Barcelona, Spain e-mail:
[email protected] P. Robledo R. de la Torre Universitat Pompeu Fabra (CEXS-UPF), Barcelona, Spain E. Puerta N. Aguirre Department of Pharmacology, University of Navarra, Pamplona, Spain R. de la Torre CIBER de Fisiopatologı´a de la Obesidad y Nutricio´n (CB06/03), CIBEROBN, Santiago de Compostela, Spain
monoamine transporter (Slc18a2) genes in the brain stem area. In the hippocampus, monoamine oxidase B (Maob) and tryptophan hydroxylase 2 (Tph2) gene expressions were increased. In the striatum, tyrosine hydroxylase (Th) expression was decreased, and a lower expression of a-synuclein (Snca) was observed in the cortex. In contrast, no significant changes were observed in the genes considered to be biomarkers of toxicity including the glial fibrillary acidic protein (Gfap) and the heat-shock 70 kD protein 1A (Hspa1a) in any of the structures assayed. These results suggest that MDMA promotes adaptive changes in genes related to serotonergic and dopaminergic functionality, but not in genes related to neurotoxicity. Keywords MDMA Ecstasy Serotonin Dopamine Serotonin transporter PCR
Introduction 3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is one of the most popular illegal psychostimulants abused by young adults. There is evidence that MDMA produces long-lasting alterations of serotonergic function in humans (Capela et al. 2009) that can lead to long-term psychological and neurocognitive deficits and increased psychopathology prevalence (Martı´n-Santos et al. 2010; de Sola Llopis et al. 2008; Cuya`s et al. 2011). Administration of MDMA to rats and monkeys results in an acute and rapid release of serotonin (5-HT) (O’Loinsigh et al. 2001; Nichols et al. 1982; Johnson et al. 1986; Fitzgerald and Reid 1990), followed by long-lasting decrements in the concentration of this neurotransmitter and its main metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in the brain (Ricaurte et al. 1988; Mayerhofer et al. 2001; Green
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et al. 2003; Colado et al. 2004). Furthermore, long-term depletion of tryptophan hydroxylase (TPH) activity, the rate-limiting enzyme in the synthesis of 5-HT, reduction of TPH-immunoreactive fibers, and alterations in TPH mRNA expression have also been observed (Stone et al. 1989; Schmidt and Taylor 1987; Bonkale and Austin 2008; Kovacs et al. 2007). Alterations in the functionality and density of the serotonin transporter (5-HTT) are also associated to MDMA exposure (Bonkale and Austin 2008; Kovacs et al. 2007). Indeed, the 5-HTT is thought to play an important role in long-term MDMA-induced 5-HT depletion. This hypothesis is based on the fact that 5-HTT reuptake inhibitors such as fluoxetine and fluvoxamine prevent 5-HTT loss in rats (Li et al. 2010; Sanchez et al. 2001). In addition, it has been suggested that dopamine (DA) may also contribute to 5-HT terminal damage (Stone et al. 1988; Shankaran et al. 1999). Hence, DA released by MDMA may enter into serotonergic terminals by interacting with 5-HTT, or it may also be formed within those terminals via hydroxylation of tyrosine to L-dihydroxyphenylalanine (LDOPA) and subsequent decarboxylation (Sprague et al. 1998; Breier et al. 2006). Inside the terminal, DA can be deaminated by monoamine oxidase (MAO) resulting in the production of reactive oxygen species, leading to an eventual serotonergic neurotoxicity (Jones et al. 2004; Monks et al. 2004; Hrometz et al. 2004; Alves et al. 2007, 2009). Although it is clear that MDMA induces alterations in serotonergic neurotransmission, there is still controversy as to whether these changes reflect neuronal damage or not. Previous studies postulate that MDMA promotes serotonergic neurodegeneration consisting in anatomical changes of axon terminals (axonopathy) (see Green et al. 2003 for review). However, several investigations in rats using low doses of MDMA, close to those used by humans have not been able to demonstrate such neuroanatomical alterations (Baumann et al. 2007; Wang et al. 2005). Moreover, recent reports suggest that a lower availability of the 5-HTT after MDMA administration may be related to decreased gene expression rather than to 5-HT terminal loss (Biezonski and Meyer 2010, 2011). Therefore, the aim of our study was to investigate the longterm changes induced by acute administration of MDMA in the expression levels of genes related to serotonergic and dopaminergic systems, as well as those related to cellular toxicity in the cortex, hippocampus, striatum, and brain stem of rats.
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cycle (lights on at 7 h). Food and water were available ad libitum. All the procedures applied in the present work were in compliance with the European Community Council Directive (86/609/EEC) and were approved by the Ethical Committee of the Universidad de Navarra. Experimental Procedure Rats received 3 intraperitoneal injections (every 2 h) of saline (n = 6) or MDMA hydrochloride (Lipomed, Arlesheim, Switzerland) at a dose of 5 mg/kg (n = 5). Rectal temperature was measured at ambient temperature (21.5 ± 1 °C) before the first MDMA administration, and thereafter every 60 min up to 8 h using a lubricated digital thermometer probe (Panlab, Barcelona) inserted *3 cm into the rectum. Seven days after treatment, rats were killed by decapitation. Subsequently, brains were rapidly removed and different brain regions, including the hippocampus, the frontal cortex, the striatum, and the brainstem region containing the dorsal raphe nucleus and the substantia nigra (DRN?SN) were dissected, weighed and quickly frozen under dry ice, followed by storage at -80 °C until use. Half of the samples were used for RNA extraction and half for biochemical determinations. Biochemical Measurements Concentrations of 5-HT, 5-HIAA, in striatum, hippocampus, and cortex, were determined by high performance liquid chromatography with electrochemical detection. Concentrations of DA and DOPAC were determined in the striatum only using the same technique. Briefly, tissue samples were homogenized in a solution composed of HClO4 (0.01 M) and sodium metabisulfite (0.2 %) and centrifuged at 20.0009g for 10 min. The pellets were discarded and 20 ll of the supernatants were filtered (0.45 lm pore size) and injected using an automatic sample injector (Waters 717 plus) onto a Spherisorb ODS-2 reverse phase C18 column (5 lm, 150 9 4.6 mm; Teknokroma, Sant Cugat del Valle`s, Spain) connected to a DECADE amperometric detector (Antec Leyden, Zoeterwoude, The Netherlands), with a glassy carbon electrode set at 0.7 V with respect to a Ag/AgCl reference electrode. The mobile phase consisted of citric acid 0.1 M, Na2HPO4 0.1 M, octanesulfonic acid 0.74 mM, EDTA 1 mM, and methanol 16 % (pH 3.4), pumped at a flow rate of 1 ml/min.
Materials and Methods
Total RNA Extraction
Animals
Tissue samples were submerged in RNAlaterÒ solution (Ambion) at 4 °C overnight and then stored at -80 °C until tissue homogenization and RNA extraction. Total RNA was extracted from the different homogenized tissues using the RiboPure Kit (Ambion Inc., Austin TX, USA), according to
Male Wistar rats weighting 260–280 g were grouped housed under constant conditions of humidity (55 ± 10 %) and temperature (22 ± 1 °C), with a 12-h/12-h light–dark
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manufacturer’s instructions. RNA samples were stored at -20 °C prior to its use. RNA quality was assessed using the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA), and integrity was evaluated with the RNA integrity number (RIN). RNA concentrations were determined using a ND-1000 spectrophotometer (NanoDropÒ ND-1000, NanoDrop Technologies, USA). RNA samples were stored in aliquots at -20 °C prior to use. The reverse transcription reaction was performed using a High-Capacity cDNA Reverse Transcription Kit with RNase inhibitor (Applied Biosystems) according to manufacturer’s instructions. Gene expression was assessed by quantitative Real-Time PCR (TaqManÒ Low Density Array (TLDA), Applied Biosystems) (see Table S1 for assay ID’s) using the conditions reported in the ABI PRISM 7900HT user’s guide. b-actin and 18s rRNA were used as a reference genes. Data obtained were analyzed by the Relative Quantity Manager 1.2 software (Applied Biosystems). Since the aim of our study was to clarify whether the adaptive changes in gene expression levels induced by MDMA exposure were related or not to neurotoxicity, we specifically focused on examining genes involved in presynaptic dopaminergic and serotonergic function, and those associated to toxic effects. The genes monitored were grouped by functionality: (i) monoamine transporters: Slc6a4 (serotonin transporter), Slc6a3 (dopamine transporter), Slc6a2 (norepinephrine transporter), Slc18a2 (vesicular monoamine transporter-2); (ii) regulation of membrane stability and/or turnover: Snca and Sncg (a and c synuclein); (iii) biosynthesis and biotransformation enzymes: Maoa and Maob (monoamine oxidase A and B), Ddc (aromatic amino acid decarboxylase), Tph1 and Tph2 (tryptophan hydroxylase 1 and 2), Th (tyrosine hydroxylase); and (iv) toxicity biomarkers: Gfap (glial fibrillary acidic protein) and Hspa1a (heat-shock 70 kD protein 1A). Statistical Analysis Shapiro–Wilk test was used to test samples normality. Statistical comparisons between saline and MDMA-treated groups were performed by independent samples Student’s t test. Differences were considered statistical significant at p \ 0.05. Data analyses were performed using the Statistical Program for the Social Sciences (SPSS12 for Windows).
Results Temperature and Biochemical Measurements MDMA treatment produced a significant raise in core temperature with respect to saline injections (mean core temperature for controls: 37.9 ± 0.2, and treated animals:
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40.8 ± 0.9, p \ 0.001). Determinations of amine concentrations in the different brain regions studied showed statistically significant reductions in 5-HT and 5-HIAA content in cortex, striatum, and hippocampus of treated animals compared to controls. In the striatum, only DOPAC content was significantly reduced with respect to controls (see Table 1 for details). Quality Parameters for RNA Samples RIN values for all the samples were within the acceptable values for most applications (between 1.6 and 2.0). Since RNA samples were frozen at -20 °C prior to their use for reverse transcription, six random samples were selected and thawed in order to check their integrity before further use. All samples selected had similar values of integrity to those obtained before freezing, suggesting that the freeze– thaw cycle did not affect the integrity of RNA (data not shown). Therefore, we considered that all the samples were suitable for their inclusion in the gene expression assay. Gene Expression Assays in Rat Brain Four different brain regions (hippocampus, striatum, cortex, and the DRN?SN) were selected to carry out gene expression studies. These brain areas were chosen since our aim was to examine MDMA-induced changes in the expression levels of genes related to serotonergic and dopaminergic systems. Thus, the brainstem region containing the DRN?SN was selected since the DRN is the largest brain region containing serotonergic cell bodies (Bonvento et al. 1991), and the SN is a region rich in dopaminergic neurons (Purves 2004). The hippocampus was chosen based on its extensive serotonergic innervations with sparse dopaminergic afferents, and its vulnerability to MDMA-induced neurotoxicity (Green et al. 2003), and the frontal cortex was chosen because it receives extensive projections from both the DRN (Purves 2004) and mesencephalic dopamine neurons (Lindvall et al. 1974). Initially, two reference genes were selected for data normalization: 18s rRNA, and b-actin. However, amplification of 18s rRNA started approximately at cycle 5, whereas most of the genes of interest (and also b-actin) started between cycles 20 and 30. Accordingly, b-actin was selected as the most suitable reference gene for all the analyses. The relative quantification (RQ) values for all genes studied in the different structures of rats treated with MDMA and saline are shown in Table 2. Significant changes were observed in gene expression levels for MDMA-treated rats in all the brain areas sampled (Fig. 1). In the cortex, a lower expression level (20 % less) was observed for the Snca gene (p \ 0.01) in MDMAtreated animals as compared to controls (Fig. 1a). In this
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Table 1 Amine concentrations (pg/mg tissue) determined in different brain regions 7 days after treatment with MDMA (5 mg/kg 9 3) or saline (control) Striatum Control 5-HT 5-HIAA
413 ± 39 452 ± 26
Hippocampus MDMA 237 ± 45 250 ± 51
DA
93,928 ± 1,049
93,364 ± 442
DOPAC
12,627 ± 427
10,395 ± 543
HVA
793 ± 60
747 ± 44
Cortex
p value
Control
MDMA
p value
Control
MDMA
p value
0.05
352 ± 14
115 ± 26
0.001
304 ± 23
135 ± 24
0.01
0.01
311 ± 22
106 ± 29
0.001
281 ± 29
147 ± 19
0.01
0.657 0.01 0.561
Bold values indicate statistically significant differences (p \ 0.05 vs. control) Results are presented as mean ± SEM
structure, no amplification was obtained for the Slc6a2, Slc6a3, and Tph1 genes. In the hippocampus, an increase in the expression levels of the Maob (p \ 0.01), and the Tph2 (p \ 0.01) genes was observed in MDMA-treated animals with respect to controls (9 % and 42 % more, respectively) (Fig. 1b). No amplification was observed for the Slc6a2 gene, and for the Slc6a3 and Tph1 genes, amplification started later than cycle 35, therefore the differences observed for these genes were not considered. In the striatum, the level of expression for the Th gene was decreased (20 % less) in MDMA-treated rats versus control animals (p \ 0.05) (Fig. 1c), and no amplification was obtained for the Tph1 and Slc6a2 genes. In DRN?SN, all selected genes except SLC6A3 were amplified. A decrease in the expression levels of the Slc6a4 (p \ 0.05) and the Slc18a2 (p \ 0.01) genes was observed (Fig. 1d). In addition, nonsignificant changes in the expression levels of the Tph2 and Sncg genes were also observed. In clear contrast to these results, no significant changes in the expression levels of genes considered biomarkers of neurotoxicity (Gfap or Hspa1a) were observed between MDMA- and saline-treated rats in any of the structures assayed (Table 2).
Discussion The main finding of this study was that a dosing regimen of MDMA comparable to the human recreational dose induced significant long-lasting changes in gene expression levels in several brain structures related to dopaminergic and serotonergic neurotransmission, but no significant alterations in genes associated with neuronal damage. Exposure to MDMA (5 mg/kg 9 3) induced hyperthermia and significant decreases in 5-HT and 5-HIAA tissue content in the striatum, hippocampus, and cortex, as has been previously described for single or repeated administration of MDMA in rats (see Puerta and Aguirre 2011 for review). Studies evaluating the neurotoxic effects
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of MDMA have often associated the relatively long-lasting depletions observed in brain TPH, 5-HT, 5-HIAA, and 5-HTT with neuronal damage (Green et al. 2003; Xie et al. 2006; Goni-Allo et al. 2008; Puerta and Aguirre 2011). However, increasing evidence has been put forward suggesting that these alterations might not be absolute indicators of toxicity (Kovacs et al. 2007; Biezonski and Meyer 2010; Wang et al. 2004, 2005). In fact, inconsistent data indicative of neuronal damage have been reported following MDMA exposure in rats. Thus, no astrogliosis has been observed as measured by changes in Gfap protein following MDMA administration of intermediate and high doses (O’Callaghan and Miller 1993; Pubill et al. 2003; Wang et al. 2004, 2005). In addition, other indications of MDMA-induced activation of astroglia or microglia have been difficult to establish, including changes in the expression of heat-shock protein 27, or OX-6 (Pubill et al. 2003; Wang et al. 2004, 2005). In contrast, other investigators describe increases in brain Gfap after single low (15 mg/kg) (Adori et al. 2006), intermediate (20 mg/kg) (Aguirre et al. 1999), or a high dose of MDMA (40 mg/kg) (Sharma and Ali 2008). In this study, we found that MDMA induced decrements of about 20 % in the expression levels of the Slc6a4 gene selectively in the brainstem region. In agreement, one study using in situ hybridization in rats exposed to a single dose (15 mg/kg) of MDMA found decreases in Slc6a4 gene expression in the dorsal/median raphe nuclei 3 weeks following treatment (Kirilly et al. 2008). In our study, the decrease in the expression of the Slc6a4 gene was not associated to significant changes in the expression of genes related to neuronal toxicity (Gfap or Hspa1a) in any of the brain structures assayed. These findings are in accordance with previous studies showing that in Dark Agouti rats, a single dose of MDMA (15 mg/kg) induces serotonergic depletion and changes in 5-HTT mRNA expression in the raphe nucleus 1 week after treatment with no indication of axotomy (Kovacs et al. 2007). Similarly, another study using qRT-PCR has shown larger reductions (50 %) in
1.152 ± 0.205
1.115 ± 0.125
1.364 ± 0.339 1.276 ± 0.182
Gfap Hspa1a
1.417 ± 0.379 1.320 ± 0.136
0.745 ± 0.109
1.328 ± 0.366
–
1.450 ± 0.290
* p \ 0.05; ** p \ 0.01 versus control
Results are presented as mean ± SEM
Bold values indicate statistically significant differences
1.130 ± 0.149
0.738 ± 0.214
Tph2
Th
Tph1
–
1.044 ± 0.123
Maob
Ddc
1.104 ± 0.169
1.409 ± 0.226
Maoa
0.965 ± 0.036
1.107 ± 0.192 1.195 ± 0.165
1.201 ± 0.121
0.999 ± 0.207
–
1.224 ± 0.155
1.074 ± 0.060
1.105 ± 0.095
1.004 ± 0.206
0.834 – 0.105** 0.825 ± 0.080
1.023 ± 0.081
0.852 ± 0.133
Snca
1.218 ± 0.140
–
–
1.028 ± 0.145
0.978 ± 0.344
–
–
1.538 ± 0.305
Sncg
1.036 ± 0.273
–
Slc6a2
Slc6a18
–
1.452 ± 0.439
Slc6a3
Slc6a4
Control
Control
MDMA
Hippocampus
Cortex
1.105 ± 0.089 1.331 ± 0.170
1.383 ± 0.229
1.455 – 0.249**
–
1.363 ± 0.113
1.039 ± 0.204 1.314 ± 0.352
0.936 ± 0.075
1.1226 ± 0.190
–
1.147 ± 0.210
1.108 ± 0.063 1.040 ± 0.061
1.122 ± 0.087
1.021 ± 0.091
1.053 ± 0.063
1.375 ± 0.372
–
1.288 ± 0.493
0.825 ± 0.259
Control
1.165 – 0.030*
1.227 ± 0.174
1.028 ± 0.072
1.106 ± 0.165
–
–
0.606 ± 0.362
MDMA
Striatum
1.169 ± 0.330 1.459 ± 0.367
1.571 ± 0.471 1.054 ± 0.143
0.981 ± 0.190 1.159 ± 0.336
1.438 ± 0.232
0.983 ± 0.194
0.989 ± 0.113
1.002 ± 0.086
0.992 ± 0.055
0.793 ± 0.119
0.998 ± 0.102
1.093 ± 0.130
0.939 ± 0.513
–
1.141 ± 0.176
Control
DRN?SN
0.812 – 0.103*
–
1.002 ± 0.144
0.991 ± 0.047
1.142 ± 0.054
0.955 ± 0.069
1.045 ± 0.075
1.175 ± 0.262
–
1.235 ± 0.456
0.719 ± 0.261
MDMA
Table 2 Gene expression levels (RQ) in different brain regions 7 days after treatment with MDMA (5 mg/kg 9 3) or saline (control)
1.537 ± 0.576 1.251 ± 0.302
0.903 ± 0.388
0.806 ± 0.132
1.221 ± 0.527
0.844 ± 0.124
1.019 ± 0.092
0.955 ± 0.094
0.684 ± 0.050
0.906 ± 0.138
0.827 – 0.150*
0.469 ± 0.137
–
0.880 – 0.089**
MDMA
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166
Neurotox Res (2014) 25:161–169 Cortex
% of Control
160
C 160
% of Control
120
**
*
80 40
40 0
**
160
control MDMA
120 80
Hippocampus
B
A
Snca Striatum
120
*
0
D 160
Tph2
DRN+SN
120
80
80
40
40
0
Maob
**
*
0
Th
Slc18a2
Slc6a4
Fig. 1 Significant changes in gene expression levels in different brain areas including a-synuclein (Snca) in the cortex (a), monoamine oxidase B (Maob) and tryptophan hydroxylase 2 (Tph2) in the hippocampus (b), tyrosine hydroxylase (Th) in the striatum (c), and vesicular monoamine transporter-2 (Vmat2) and serotonin transporter (Slc6a4) in the brain stem containing the dorsal raphe nucleus and the substantia nigra (DRN?SN) (d) following MDMA (5 mg/kg 9 3) as compared to saline administration. The data represent changes in RQ values as % of controls ? SEM. * p \ 0.05; ** p \ 0.01 versus control
Slc6a4 gene expression in dorsal/median raphe nucleus 2 weeks after a higher dose (10 mg/kg 9 4) of MDMA (Biezonski and Meyer 2010). Together, these findings suggest that the MDMA-induced depletion of serotonergic markers occurs with neuroadaptations in gene expression that do not necessarily corroborate neurotoxicity. In parallel, we also found a significant decrease in Slc18a2 gene expression in the brainstem. This result is in line with the study by Biezonski and Meyer (2010) showing reduced Slc18a2 gene expression in the dorsal/ median raphe 2 weeks after a high MDMA binge regimen. These changes were observed in the absence of a significant decrease in VMAT-2 protein expression in the hippocampus, suggesting that even at this high dose, MDMA does not produce a massive loss of serotonergic terminals (Biezonski and Meyer 2010). We found no alterations in Slc18a2 gene expression in serotonergic nerve terminal regions such as the hippocampus and cortex. Therefore, it is possible that the decrease in Slc18a2 gene expression observed in the brainstem region, which in our conditions contained both serotonergic and dopaminergic cell bodies, was mostly due to MDMA-induced neuroadaptive changes at the level of dopaminergic neurotransmission. Interestingly, the expression levels of other genes related to serotonergic function in the brainstem, such as Tph2 were decreased marginally. Marked increases in Tph2 mRNA expression have been observed in the DRN
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2 weeks after repeated treatment with high doses (20 mg/ kg twice a day for 4 days) of MDMA (Bonkale and Austin 2008). On the other hand, we observed a large increase in the expression level of the TPH2 mRNA in the hippocampus of MDMA-treated rats. TPH, the rate-limiting enzyme in the synthesis of 5-HT, is prominently inhibited by MDMA (Stone et al. 1988). Therefore, increased Tph2 gene expression may be considered a compensatory mechanism in order to overcome the decrease in 5-HT induced by MDMA treatment. Likewise, the increase in Maob gene expression observed in the hippocampus could be thought of as an adaptive change related to the increase in dopamine release produced by MDMA. Our results showed specific changes in the expression of dopaminergic markers in the striatum, where rats treated with MDMA exhibited both a decrease in DA turnover and lower Th gene expression with respect to controls. These results contrast with previous in situ hybridization studies showing no changes in Th mRNA expression in mid-brain dopaminergic nuclei following repeated high doses of MDMA in rats (Wotherspoon et al. 1994). However, while it is well accepted that MDMA exposure in rats predominantly depletes serotonergic markers in the brain, longterm changes in dopaminergic function have also been reported following MDMA administration in rats (Commins et al. 1987; McGregor et al. 2003; Able et al. 2006; Kindlundh-Hogberg et al. 2006), monkeys (Ricaurte et al. 2000), and humans (Kish et al. 2000). In line with these data, our results indicate that MDMA induces adaptive changes in dopaminergic markers in the striatum of rats. MDMA-treated rats showed a significant decrease in expression levels of the a-synuclein gene (Snca) only in the cortex, while no changes were observed in c-synuclein in any of the structures assayed. Synucleins (a-synuclein, and c-synuclein) are proteins primarily localized in the presynaptic compartment of neurons, where they regulate monoamine transporter trafficking to-or-away from the cell (Wersinger et al. 2006). Specifically, a-synuclein is highly expressed in brain areas such as the neocortex, hippocampus, and amygdala, where it may mediate synaptic plasticity. Alternatively, a role for a-synuclein in neurodegeneration has also been put forward (Mladenovic et al. 2007), and amphetamine derivatives have been shown to increase a-synuclein protein levels in the substantia nigra of mice at doses that reduce DA content in the striatum (Fornai et al. 2005). On the other hand, c-synuclein expression occurs mainly in serotonergic neurons of the raphe nuclei, where it plays an important role in the regulation of the 5-HTT (Wersinger and Sidhu 2009). Our results showing a specific reduction of a-synuclein in the cortex suggest that MDMA induces adaptive changes that may be important for the maintenance of functional synapses in this brain structure.
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Our data add to the growing literature implicating modulatory mechanisms underlying MDMA-induced longterm serotonergic deficits. Nevertheless, there are several issues that still need to be addressed. Thus, a time course analysis aimed at establishing the correlation between neurochemical data (5-HT and 5-HTT levels) and gene expression changes after MDMA is warranted. Moreover, different drugs or non-pharmacological manipulations known to decrease or to exacerbate MDMA-induced 5-HT deficits (Puerta and Aguirre 2011), should provoke gene expression changes in a similar fashion. Finally, adaptations in 5-HT receptor gene expression should also be investigated, especially for 5-HT2A since these receptors show adaptive changes after MDMA administration in animals and humans (Reneman et al. 2002; Di Iorio et al. 2012), and have been involved in the cognitive and emotional alterations induced by MDMA. Such issues are the focus of on-going studies in our laboratory. In conclusion, this study confirms previous data showing that MDMA treatment produces changes in the expression level of genes related to serotonergic function. Moreover, it extends these findings by demonstrating that a dosing regimen of MDMA comparable to the human recreational dose induces alterations in the expression level of genes related to dopaminergic neurotransmission in specific brain regions. Finally, our data indicate that these adaptive changes in gene expression are not consistent with damage to serotonergic terminals since no significant changes were observed in genes related to neurotoxicity. Acknowledgments This work was supported by grants from DIUE de la Generalitat de Catalunya (2009 SGR 718) and ISCIII-Red de Trastornos Adictivos (RTA: RD12/0028/0009).
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