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Keywords:

  • accumbens shell;
  • dopamine;
  • microdialysis;
  • phosphorylated extracellular signal regulated kinase;
  • R;
  • S(±)-methylenedioxymethamphetamine (Ecstasy);
  • stereoselectivity

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

R,S(±)-3,4-methylenedioxymethamphetamine (R,S(±)-MDMA, ‘Ecstasy’) is known to stimulate dopamine (DA) transmission in the nucleus accumbens (NAc). In order to investigate the post-synaptic correlates of pre-synaptic changes in DA transmission and their relationship with MDMA enantiomers, we studied the effects of R,S(±)-MDMA, S(+)-MDMA, and R(−)-MDMA on extracellular DA and phosphorylated extracellular signal regulated kinase (pERK) in the NAc shell and core. Male Sprague–Dawley rats, implanted with a catheter in the femoral vein and vertical concentric dialysis probes in the NAc shell and core, were administered i.v. saline, R,S(±)-MDMA, S(+)-MDMA, or R(−)-MDMA. Extracellular DA was monitored by in vivo microdialysis with HPLC. Intravenous R,S(±)-MDMA (0.64, 1, and 2 mg/kg) increased dialysate DA, preferentially in the shell, in a dose-related manner. S(+)-MDMA exerted similar effects but at lower doses than R,S(±)-MDMA, while R(−)-MDMA (1 and 2 mg/kg) failed to affect dialysate DA. R,S(±)- and S(+)-MDMA but not R(−)-MDMA increased ERK phosphorylation (expressed as density/neuron and number of pERK-positive neurons/area) in both subdivisions of the NAc. The administration of the D1 receptor antagonist, SCH 39166, prevented the increase in pERK elicited by R,S(±)-MDMA and S(+)-MDMA, while the D2/3 receptor antagonist, raclopride, increased pERK in the NAc core per se but failed to affect the R,S(±)-MDMA-elicited stimulation of pERK. The present results provide evidence that the DA stimulant effects of racemic MDMA are accounted for by the S(+)-enantiomer and that pERK may represent a post-synaptic correlate of the stimulant effect of R,S(±)-MDMA on D1-dependent DA transmission.

Abbreviations used
DA

dopamine

ERK

extracellular signal regulated kinase

NAc

nucleus accumbens

PBS

phosphate-buffered saline

pERK

phosphorylated extracellular signal regulated kinase

R,S(±)-MDMA

R,S(±)-3,4-methylenedioxymethamphetamine

R,S(±)-3,4-methylenedioxymethamphetamine [R,S(±)-MDMA, ‘Ecstasy’], one of the most commonly abused amphetamine derivatives, is self-administered by humans as the racemic mixture of S(+)- and R(−)-MDMA. MDMA and its enantiomers are self-administered by rhesus monkeys (Fantegrossi et al. 2002) and by rats (Ratzenboeck et al. 2001; Schenk et al. 2003; Wakonigg et al. 2003), and this property in monkeys involves serotonin 5HT2receptor activation (Fantegrossi et al. 2002). Recent studies have shown that R,S-(±)-MDMA self-administration also depends on a dopaminergic substrate (Daniela et al. 2004). R,S(±)-MDMA releases serotonin in vivo and in vitro from various brain areas (cortex, hippocampus, and striatum) (Gudelsky and Nash 1996; Shankaran and Gudelsky 1998; Mechan et al. 2002). Racemic MDMA also releases dopamine (DA) in vitro (Schmidt 1987; Schmidt et al. 1987) and in vivo (Yamamoto and Spanos 1988; Gudelsky et al. 1994; Sabol and Seiden 1998; Colado et al. 2004), but a limited number of studies have addressed the issue of the stereospecificity of the pre-synaptic actions of R,S(±)-MDMA. Early reports did not observe any difference among the two enantiomers in the ability to release serotonin in vitro (Johnson et al. 1986; Schmidt et al. 1987), whereas the S(+)-MDMA but not the R(−) isomer was reported to release DA in vivo (Johnson et al. 1986; Hiramatsu and Cho 1990). Racemic MDMA depresses the firing activity of dopaminergic neurons in the mesencephalon (Kelland et al. 1989; Matthews et al. 1989) and this effect was accounted for entirely by S(+)-MDMA (Gifford et al. 1996). Racemic MDMA, like most drugs of abuse (Di Chiara 1999), preferentially stimulates DA release in the shell of the nucleus accumbens (NAc) (Cadoni et al. 2005). Therefore, one goal of this study was to characterize the stereospecificity of the pre-synaptic effects of racemic MDMA on DA transmission.

A second goal of the present study has been that of providing a neurochemical correlate of post-synaptic DA receptor activation. Previously, the expression of the immediate early gene c-fos was thought to provide such an index (Robertson et al. 1992;Morelli et al. 1992); however, c-fos expression is activated both by stimulation and blockade of DA transmission (Dragunow et al. 1990). Another index of in vivo DA transmission might be provided by the phosphorylation of protein substrates related to activation of protein kinases by DA-dependent second messengers. Such substrates are cAMP-response element binding protein (Konradi et al. 1994), DA- and cAMP-regulated neuronal phosphoprotein-32 (Fienberg et al. 1998), and mitogen-activated protein kinases also known as extracellular signal regulated kinases (ERKs) (Berhow et al. 1996; Sweatt 2001). Neuronal ERKs are tyrosine kinases that regulate a number of cellular functions (Sweatt 2004) and have been linked to long-term potentiation (Sweatt 2001; Ying et al. 2002) and associative learning (Atkins et al. 1998; Schafe et al. 2000). The activation of the ERKs cascade has been demonstrated to be involved in the regulation of corticostriatal glutamatergic transmission (Sgambato et al. 1998) and in the mechanism of action of drugs of abuse (Valjent et al. 2000, 2001a).

Given these premises, phosphorylation of ERKs might provide an index of post-synaptic DA transmission. If this prediction is correct, R,S-(±)-, S(+)- ,and R(−)-MDMA administration should elicit parallel changes in extracellular DA and phosphorylated forms of ERK. In order to verify this prediction, we studied in parallel the effects of intravenous R,S-(±)-MDMA and its enantiomers on extracellular DA and on the levels of phosphorylated ERKs (pERK), as estimated by immunohistochemistry in the NAc shell and core.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals

Male Sprague–Dawley rats (275–300 g) (Charles River, Calco, Italy) were housed in groups of four per cage for at least 3 days before use and were maintained on a 12:00/12:00 h light/dark cycle (lights on at 8:00 a.m.) with food and water available ad libitum. After surgery, the rats were individually housed in hemispherical bowls, which also served as the experimental environment. Experiments were carried out between 9:00 a.m. and 5:00 p.m., at least 24 h after the surgical implants. Experimental protocols were approved by the Ethical Committee of the University of Cagliari and performed in strict accordance with the EC regulations for the use of experimental animals (EEC N 86/609) and recommended guidelines approved by the National Institutes of Health (NIH Publications No. 8023, revised 1978).

Surgery

For intravenous administration of drugs, a polyethylene catheter was inserted, under halothane anesthesia (1.5% in therapeutic oxygen), in the left femoral vein, tunneled subcutaneously to exit from the middle scapular region where it was anchored to the skin according to Crane and Porrino (1989). On the same day, rats were anesthetized with ketamine HCl (Ketalar®, Parke Davis, Italy) (100 mg/kg i.p.) and stereotaxically implanted with two concentric microdialysis probes one in the NAc shell (AP = +2.2 mm, DV = −7.8 mm, and ML = −1.2) and the other in the core (AP = +1.6 mm, DV = −7.6 mm, and ML = +1.7) according to Paxinos and Watson (1998).

Microdialysis

Vertical concentric dialysis probes were prepared with AN 69 fibers (Hospal Dasco, Bologna, Italy), according to the method of Di Chiara et al. (1993). The dialyzing membrane was covered with epoxy glue along its whole length except for 1.5 mm corresponding to the area of dialysis for both NAc shell and core. The day of the experiment rats were connected to a perfusion pump (Bee Syringe Pump; BAS, Lafayette, IN, USA) by a polyethylene tubing, connected to a 2.5 mL glass syringe containing normal Ringer (147 mmol/L NaCl, 2.2 mmol/L CaCl2, and 4 mmol/L KCl in double distilled water), with the perfusion flow set at 1.25 µL/min. Samples were collected every 10 min into a 20 µL sample loop and subsequently injected in an HPLC injector valve. DA was separated on a reverse-phase column (Chromolith Performance RP-18e, 100–4.6 mm; Merck, Darmstadt, Germany) and assayed by HPLC coupled with electrochemical detection (ESA Coulochem II, Bedford, MA, USA). The mobile phase was an aqueous sodium phosphate buffer (100 mmol/L NaH2PO4, 0.48 mmol/L sodium octyl sulfate, 0.2 mmol/L Na2-EDTA, and 15% (v/v) methyl-alcohol, pH = 8) delivered at a constant flow of 1 mL/min by an HPLC pump. The detection limit of the assay was about 10 fmol/sample.

Behavioral measures

Behavioral scoring was performed during the course of the dialysis experiments. Behavioral analysis started 10 min before vehicle or drug administration and lasted for 60 min. Behavioral items were scored, by an experimenter unaware of the treatment, as the percentage of time spent by each rat, performing one of the following behaviors. The following behavioral items were recorded: Active, upward sniffing and rearing, locomotor activity accompanied by sniffing (exploratory behavior), wet-dog shakes, digging under the bedding, grooming, and, at the highest intensity, circling; Still-aroused, eyes wide open, movements of the head and of the whiskers, chewing, and teeth chattering; Still-resting, resting, and lying down with eyes closed or half open.

Histology

At the end of each experiment, rats were anesthetized with chloral hydrate (450 mg/kg i.p.) and subjected to a transcardiac perfusion with 50 mL of saline (NaCl 0.9%) and 100 mL of a 10% formaldehyde solution. The probes were removed and brains were cut on a vibratome (Vibratome 1000 Plus; The Vibratome Company, St Louis, MO, USA) in serial coronal slices of 150 μm oriented according to the atlas of Paxinos and Watson (1998). The location of the probes was reconstructed and referred to the atlas of Paxinos and Watson (1998). Data from animals with incorrect probe placement were excluded.

Immunohistochemistry

All rats were administered saline, SCH 39166 (50 µg/kg s.c.) or raclopride (300 µg/kg s.c.) 10 or 30 min before R,S(±)-MDMA, respectively, saline or SCH 39166 (50 µg/kg s.c.) 10 min before S(+)-MDMA, and saline 10 min before R(−)-MDMA. Five minutes (but in some experiments also 2.5 or 10 min) after i.v. administration, rats were deeply anesthetized with chloral hydrate (450 mg/kg i.p.) before transcardiac perfusion with ice-cold phosphate-buffered saline (PBS: 137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Na2HPO4, and 2 mmol/L KH2PO4, pH 7.4) and 4%p-formaldehyde. After perfusion, the brains were removed and post-fixed overnight in 4%p-formaldehyde. Brain sections (40 µm) were cut on ice-cold PBS with a vibratome (Vibratome 1000 Plus), kept in ice-cold PBS, and immediately processed for immunohistochemistry according to a protocol for free-floating sections. After an incubation of 30 min in 1% H2O2 and after three rinses, sections were incubated for 1 h with 3% BSA. The incubation with the primary antibody [anti pERK cat. no 9101 (1 : 300) and anti-total ERK cat. no 9102 (1 : 200); Cell Signaling Technology, Beverly, MA, USA)] was conducted overnight. On the following day, after rinsing, sections were incubated for 1 h with the biotinylated secondary antibody (1 : 800). After three rinses, the sections were incubated in avidin–biotin peroxidase complex prepared according to the manufacturer’s suggestions (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) and a 3,3′-diaminobenzidine solution (10 mg/mL) was added until development of brown staining. Sections were rinsed and mounted onto gelatine-coated slides and processed through alcohol–xylene for light microscopy examination. pERK-positive neurons were identified in the regions of interest at the lowest magnification (10×) and quantitative analysis was performed using a Leica TCS 4D light microscope equipped with PL Floutar 10× (Leica, Heidelberg, Germany) (numerical aperture = 0.3), 40× oil (numerical aperture = 1.00–0.5), and 100× oil (numerical aperture = 1.3) coupled with a digital camera (Canon Power Shot G2 with 4.0 MPixels; Canon Inc., Tokyo, Japan). The images generated with a PL Floutar 100× were used for densitometric analysis (Fig. 1, panels c–f). To this end, the software Bioscan Optimas, version 6.5.1, was used (Media Cybernetics Inc., Silver Spring, MD, USA). pERK-positive neurons were obtained by marking cells’ profile and excluding all dendritic trunks. The optical density of neurons in each microscopic field was corrected for background interference by dividing it for optical density of the background. Images of the NAc, obtained at the lowest magnification (10×) (Fig. 1, panels a–b), were used to count the number of pERK-positive neurons in its shell and core subdivisions by application of the software Bioscan Optimas.

image

Figure 1.  Representative images of phosphorylated extracellular signal regulated kinase-positive neurons from the nucleus accumbens (NAc) shell and core at low magnification (10×) (panels a and b) after intravenous administration of saline + saline (panel a) and saline + R,S(±)-methylenedioxymethamphetamine (2 mg/kg) (panel b) and at the highest magnification (100×) after intravenous administration of saline + saline and saline + R,S(±)-methylenedioxymethamphetamine (2 mg/kg) from NAc core (panels c and d) and from the NAc shell (panels e and f). AC: anterior commissure.

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Drugs

R,S(±)-3,4-MDMA HCl, R(−)-3,4-MDMA HCl, and S(+)-3,4-MDMA HCl (National Institute of Drug Abuse, NIDA, Baltimore, MD, USA) were dissolved in saline (0.64, 1, and 2 mg/mL) and administered intravenously in a volume of 1 mL/kg of body weight with all doses expressed as free base. SCH 39166 (Schering-Plough, Milan, Italy) and raclopride tartrate (Astra, Sodertalje, Sweden) were dissolved in saline and injected subcutaneously.

Statistics

Microdialysis values are expressed as percentage changes with respect to baseline (100%). Baseline was set as the average of the last six pre-treatment samples, not differing by more than 15%. Behaviors, scored every 10 min, starting at the first pre-treatment sample are expressed as the average of the percent of the time spent by rats, performing behaviors of each of the three categories. One-, two- and three-way anova, with time as the repeated measure, was used to analyze the statistical significance of treatment effects.

Normalized optical density measures (>40 cells/group) were used for statistical comparison by two- and three-way anova. Average optical densities/neuron of each treatment was calculated as change percentage with respect to the average optical density/neuron of the saline + saline groups, set as 100%. Similarly, average numbers of pERK-positive neurons/area of each treatment were calculated as percentage change with respect to the average number of pERK-positive neurons/area of the saline + saline groups. Tukey’s and Newman–Keuls post hoc analyses were applied for multiple comparisons, with the statistical significance set at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Basal DA release and effect of R,S(±)-MDMA on NAc shell and core DA and behavior

Basal DA concentrations, expressed as fmol/sample (mean ± SEM), were 41 ± 3 and 54 ± 3 in NAc shell (n = 60) and core (n = 67) dialysates, respectively. Figure 2 shows the effect of the intravenous administration of saline (1 mL/kg) and R,S(±)-MDMA (0.64, 1, and 2 mg/kg) on dialysate DA in the NAc shell and core. Three-way anova revealed a main effect of area [F(1,36) = 4.92, p < 0.03], dose [F(1,36) = 9.81, p < 0.00007], and time [F(6,216) = 29.02, p < 0.00001] and a significant area ×dose × time interaction [F(18,216) = 2.72, p < 0.0003]. Tukey’s post hoc test showed that R,S(±)-MDMA dose-dependently increased DA release in NAc shell (2 mg/kg > 1 mg/kg > 0.64 mg/kg) and NAc core (2 mg/kg > 1 mg/kg = 0.64 mg/kg) and this effect was greater in the shell than in the core at doses of 1 and 2 mg/kg but not at the dose of 0.64 mg/kg.

image

Figure 2.  Effect of intravenous administration of saline on dopamine (DA) release from nucleus accumbens shell (n = 5) and core (n = 5) and effect of intravenous administration of R,S(±)-methylenedioxymethamphetamine (MDMA) on DA release from nucleus accumbens shell and core (top right). Top right panel shows the effect of R,S(±)-MDMA at the dose of 0.64 mg/kg on shell (n = 6) and core (n = 4) DA release; bottom left panel shows the effect of R,S(±)-MDMA at the dose of 1 mg/kg on shell (n = 6) and core (n = 6) DA release. Bottom right panel shows the effect R,S (±)-MDMA at the dose of 2 mg/kg on shell (n = 7) and core (n = 5) DA release. Arrows indicate the last pre-treatment sample. Values are expressed as percentage with respect to basal values. Vertical bars represent SEM. Asterisks indicate time points that differ significantly from the corresponding one of the other curve. Bottom section of each panel indicates the behavioral effects expressed as percentage of time spent at each time point in one of the following: active, still-resting, and still-aroused. *indicates p < 0.05 (Tukey’s post hoc test) versus the corresponding time point after the dose of 2 mg/kg; **indicates p < 0.05 (Tukey’s post hoc test) versus the corresponding time point after the dose of 0.64 mg/kg (active) and versus the corresponding time point after the dose of 2 mg/kg (still-resting); #indicates p < 0.05 (Tukey’s post hoc test) versus the corresponding time point after the dose of 1 mg/kg.

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Figure 2 (histograms) also shows the effect of the administration of R,S(±)-MDMA on rat’s spontaneous behavior. Two-way anova revealed significant effects of dose and time × dose interactions for active [F(3,28) = 19.64, p < 0.0001 and F(15,40) = 2.12, p < 0.01], respectively, still-aroused [F(3,28) = 3.52, p < 0.03 and F(15,40) = 3.12, p < 0.002], respectively, and still-resting [F(3,28) = 21.72, p < 0.0001 and F(15,40) = 2.98, p < 0.0004], respectively.

Effect of S(+)-MDMA on NAc shell and core DA and behavior

Figure 3 shows the effects of the intravenous administration of S(+)-MDMA on DA release in the NAc shell and core. Three-way anova revealed a main effect of area [F(1,53) = 22.58, p < 0.000001], dose [F(3,53) = 18.13, p < 0.00001], and time [F(6,318) = 41.97, p < 0.00001] and a significant area × dose × time interaction [F(18,318) = 1.68, p < 0.04]. Tukey’s post hoc test (p < 0.05) showed that S(+)-MDMA dose-dependently increased DA release in the NAc shell (2 mg/kg > 1 mg/kg > 0.64 mg/kg) and core (2 mg/kg >1 mg/kg > 0.64 mg/kg) and that this effect was significantly greater in the NAc shell than in the core at 0.64, 1, and 2 mg/kg.

image

Figure 3.  Effect of intravenous administration of saline and S(+)-methylenedioxymethamphetamine (MDMA) on dopamine (DA) release from nucleus accumbens shell and core. Top right panel shows the effect of S(+)-MDMA at the dose of 0.64 mg/kg on shell (n = 7) and core (n = 12) DA release; bottom left panel shows the effect of S(+)-MDMA at the dose of 1 mg/kg on shell (n = 11) and core (n = 12) DA release and bottom right panel shows the effect S(+)-MDMA at the dose of 2 mg/kg on shell (n = 6) and core (n = 6) DA release. Values are expressed as percentage with respect to basal values. Arrows indicate the last pre-treatment sample. Vertical bars represent SEM. Asterisks indicate time points that differ significantly from the corresponding one of the other curve. Bottom section of each panel indicates the behavioral effects expressed as percentage of time spent at each time point in one of the following: active, still-resting, still-aroused. **indicates p < 0.05 (Tukey’s post hoc test) versus the corresponding time point after the dose of 0.64 mg/kg (active) and versus the corresponding time point after the dose of 2 mg/kg (still-aroused and still-resting); #indicates p < 0.05 (Tukey’s post hoc test) versus the corresponding time point after the dose of 1 mg/kg (still-resting) and versus the corresponding time point after the dose of 0.64 mg/kg (still-aroused).

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Figure 3 (histograms) also shows the effects of S(+)-MDMA on rats’ spontaneous behavior. Two-way anova revealed significant effects of dose and time × dose interactions for active [F(3,60) = 22.71, p < 0.0001 and F(15,235) = 2.57, p < 0.014], respectively, still-aroused [F(3,36) = 20.73, p < 0.0001 and F(15,235) = 5.38, p < 0.0001], respectively, and still-resting [F(3,36) = 53.17, p < 0.0001 and F(15,235) = 3.81, p < 0.0001], respectively.

Effect of R(−)-MDMA on NAc shell and core DA and behavior

Figure 4 shows the effect of the intravenous administration of R(−)-MDMA on DA release in the NAc shell and core. Three-way anova showed a main effect of time [F(6,198) = 4.28, p < 0.0004] but did not reveal any effect of area [F(1,33) = 0.11, NS] and dose [F(2,33) = 1.0, NS], nor any significant area × dose × time interaction [F(12,198) = 1.03, NS].

image

Figure 4.  Effect of intravenous administration of saline (left panel) and R(−)-methylenedioxymethamphetamine (MDMA) on dopamine (DA) release from nucleus accumbens shell and core. Middle panel shows the effect of R(−)-MDMA at the dose of 1 mg/kg on shell (n = 7) and core (n = 6) and right panel shows the effect of R(−)-MDMA at the dose of 2 mg/kg on shell (n = 5) and core (n = 6) DA release. Values are expressed as percentage with respect to basal values. Arrows indicate the last pre-treatment sample. Vertical bars represent SEM. Asterisks indicate time points that differ significantly from the corresponding one of the other curve. Bottom section of each panel indicates the behavioral effects expressed as percentage of time spent at each time point in one of the following: active, still-resting, and still-aroused.

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Figure 4 (histograms) also shows the effect of R(−)-MDMA on rats’ spontaneous behavior. Two-way anova revealed significant effects of dose for active [F(2,24) = 4.51, p < 0.02], still-aroused [F(2,24) = 4.06, p < 0.004], and still-resting [F(2,24) = 7.04, p < 0.0001] and a significant time × dose interaction for still-resting [F(10,120) = 2.05, p < 0.04] but not for active [F(10,120) = 1.24, NS] nor still-aroused [F(10,120) = 1.08, NS].

Table 1 provides a summary of the peak effects of racemic MDMA and its different enantiomers on dialysate DA from the NAc shell and core.

Table 1.   Percent maximal increases above baseline of DA transmission in the NAc shell and core after Saline, R,S(±)-MDMA, S(+)-MDMA and R(−)-MDMA. Data, (average ± SEM) shown here for comparison, are from Figs 1, 2, and 3
 Dose (mg/kg i.v.)NAc shell: maximal increase % above baseline (mean ± SEM)NAc core: maximal increase % above baseline (mean ± SEM)
  1. MDMA, 3,4-methylenedioxymethamphetamine; NAc, nucleus accumbens; DA, dopamine.

Saline+13 ± 5+17 ± 6
R,S(±)-MDMA0.64+25 ± 3+29 ± 6
1.0+45 ± 4+15 ± 4
2.0+74 ± 7+44 ± 3
(+)-MDMA0.64+71 ± 8+33 ± 7
1.0+99 ± 9+46 ± 7
2.0+114 ± 7+55 ± 10
R(−)-MDMA1.0+30 ± 11+20 ± 7
2.0+34 ± 9+19 ± 4

Effect of R,S(±)-MDMA on pERK immunoreactivity

Figure 5 shows the effect of R,S(±)-MDMA (1 and 2 mg/kg) on pERK density/neuron (top panel), on pERK-positive neurons/area (bottom panel) in the NAc shell and core.

image

Figure 5.  Effect of intravenous administration of saline, R,S (±)-methylenedioxymethamphetamine (MDMA) (1 and 2 mg/kg), S (+)-MDMA (0.64 and 1 mg/kg), R (−)-MDMA (1 and 2 mg/kg), and of pre-treatment with SCH 39166 (50 µg/kg s.c.) before saline or R,S-(±)-MDMA (2 mg/kg i.v.) or S(+)-MDMA (1 mg/kg i.v.) and of raclopride (300 µg/kg s.c.) before saline or R,S-(±)-MDMA (2 mg/kg i.v.) on percentage phosphorylated extracellular signal regulated kinase (pERK) density/neuron (top panel) and on percentage number of pERK-positive neurons/area (bottom panel) in the nucleus accumbens (NAc) shell and core. Histograms represent the average ± SEM of at least 40 neurons for each treatment (top panel), the average ± SEM of percentage numbers of pERK-positive neurons/area (bottom panel). **indicates p < 0.05 between shell and core (same treatment); ##indicates p < 0.05 between sal/R,S-(±)-MDMA (1 versus 2 mg/kg) (same area); *indicates p < 0.05 with respect to sal/sal (same area); #indicates p < 0.05 with respect to the saline + R,S-(±)-MDMA (2 mg/kg) or saline + S(+)-MDMA (1 mg/kg) (same area) groups.

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Figure 5 also shows the effects of pre-treatment with the DA D1 receptor antagonist, SCH 39166 (50 µg/kg s.c.) on R,S(±)-MDMA (2 mg/kg) and S(+)-MDMA (1 mg/kg), and the DA D2/3 receptor antagonist, raclopride (300 µg/kg s.c.), on R,S(±)-MDMA (2 mg/kg) induced increases of pERK density/neuron (top panel), of pERK-positive neurons/area (bottom panel) in the NAc shell and core.

pERK density/neuron

Two-way anova revealed a main effect of area [F(1,313) = 32.90, p < 0.00001], dose [F(2,313) = 71.17, p < 0.0001], and a significant area × dose interaction [F(2,313) = 12.73, p < 0.00001]. Tukey’s post hoc test (p < 0.05) showed that R,S(±)-MDMA increased pERK density/neuron with respect to saline in the NAc shell and core, at 1 and 2 mg/kg, and that after R,S(±)-MDMA (1 and 2 mg/kg) the increase was greater in the shell than in the core. Three-way anova, of the results of SCH 39166 and R,S(±)-MDMA (2 mg/kg), revealed a main effect of pre-treatment [F(1,415) = 49.36, p < 0.00001], area [F(1,415) =14.02, p < 0.0002], and treatment [F(1,415) = 29.80, p < 0.008] and a significant pre-treatment × treatment [F(1,415) = 43.52, p < 0.00001] interaction. Tukey’s post hoc test (p < 0.05) showed that R,S(±)-MDMA increased pERK density/neuron in the NAc shell and core with respect to saline and that SCH 39166 pre-treatment prevented it. Three-way anova, of the effects of raclopride and R,S(±)-MDMA (2 mg/kg), revealed a main effect of treatment [F(1,413) = 32.18, p < 0.00001], and area [F(1,413) = 4.44, p < 0.03] and a significant treatment × area [F(1,413) = 10.17, p < 0.001] interaction. Tukey’s post hoc test (p < 0.05) showed that R,S(±)-MDMA increased pERK density/neuron in the NAc shell and core with respect to saline and that raclopride pre-treatment failed to prevent it.

pERK-positive neurons/area

Two-way anova revealed a main effect of dose [F(2,58) =48.84, p < 0.00001] but not of area [F(1,58) = 0.02, NS] nor a significant dose × area interaction [F(2,58) = 0.018, NS]. Tukey’s post hoc test (p < 0.05) showed that R,S(±)-MDMA (1 and 2 mg/kg) increased the number of pERK-positive neurons in the shell and in the core with respect to saline and that the effect of R,S(±)-MDMA (2 mg/kg) was greater than R,S(±)-MDMA (1 mg/kg) in the NAc shell and core. Three-way anova, of the results of SCH 39166 and R,S(±)-MDMA (2 mg/kg), revealed a main effect of pre-treatment [F(1,36) = 9.9, p < 0.003], treatment [F(1,36) = 36.44, p < 0.00001], and a significant pre-treatment × treatment interaction [F(1,36) = 17.22, p < 0.0001], but not a significant effect of area [F(1,36) = 0.83, NS] nor significant treatment × area [F(2,36) = 0.27, NS] and pre-treatment × treatment × area interactions [F(1,36) = 0.003, NS]. Tukey’s post hoc test (p < 0.05) showed that R,S(±)-MDMA increased the number of pERK-positive neurons in the shell and in the core and that SCH 39166 pre-treatment prevented it. Three-way anova, of the effects of raclopride and R,S(±)-MDMA (2 mg/kg), revealed a main effect of treatment [F(1,56) = 52.746, p < 0.0001], area [F(1,56) = 4.06, p < 0.04], and a significant pre-treatment × treatment interaction [F(1,56) = 21.52, p < 0.0001] but not significant pre-treatment × area [F(1,56) = 3.9, NS], treatment × area [F(1,56) = 2.7, NS] nor pre-treatment × treatment × area interactions [F(1,56) = 2.92, NS]. Tukey’s post hoc test (p < 0.05) showed that R,S(±)-MDMA increased the number of pERK-positive neurons in the shell and in the core and that raclopride pre-treatment failed to prevent this increase.

Effects of S(+)-MDMA on pERK immunoreactivity

Figure 5 shows the effect of S(+)-MDMA (0.64 and 1 mg/kg) on pERK density/neuron (top panel) and on pERK-positive neurons/area (bottom panel) in the NAc shell and core.

Figure 5 also shows the effects of pre-treatment with the DA D1 receptor antagonist, SCH 39166 (50 µg/kg s.c.), on S(+)-MDMA (1 mg/kg) induced increases of pERK density/neuron (top panel) and of pERK-positive neurons/area (bottom panel), in the NAc shell and core.

pERK density/neuron

Two-way anova revealed a main effect of dose [F(2,324) = 68.50, p < 0.0001], area [F(1,324) = 8.9, p < 0.003], and a significant dose × area interaction [F(5,413) = 5.62, p < 0.0008]. Tukey’s post hoc test (p < 0.05) showed that S(+)-MDMA increased significantly pERK density/neuron in the NAc shell and core with respect to saline at the dose of 1 mg/kg but not 0.64 mg/kg, and that the increase in pERK density/neuron was greater in the NAc shell than NAc core. Three-way anova, of the results of SCH 39166 and S(+)-MDMA (1 mg/kg), revealed a main effect of pre-treatment [F(1,428) = 43.07, p < 0.0001], treatment [F(1,428) = 23.08, p < 0.0001], area [F(1,428) = 6.86, p < 0.009], and significant pre-treatment × treatment [F(1,428) = 36.98, p < 0.0001] and treatment × area [F(1,428) = 18.75, p < 0.0001] interactions. Tukey’s post hoc test (p < 0.05) revealed that S(+)-MDMA increased pERK density/neuron in the NAc shell and core with respect to saline and that SCH 39166 pre-treatment prevented it.

pERK-positive neurons/area

Two-way anova revealed a main effect of dose [F(2,86) = 12.39, p < 0.0001], but not of area [F(1,86) = 0.15, NS] nor a significant dose × area interaction [F(2,86) = 0.06, NS]. Tukey’s post hoc test (p < 0.05) showed that 1 mg/kg of S(+)-MDMA but not 0.64 mg/kg increased the number of pERK-positive neurons in the shell and in the core with respect to saline. Three-way anova, of the results of SCH 39166 and S(+)-MDMA (1 mg/kg), revealed a main effect of pre-treatment [F(1,92) = 6.53, p < 0.012], a significant pre-treatment × treatment interaction [F(1,92) = 8.56, p < 0.004], but not a significant effect of area [F(1,92) = 0.04, NS] nor significant treatment × area [F(2,36) = 0.27, NS] and pre-treatment × treatment × area interactions [F(1,92) = 0.01, NS]. Tukey’s post hoc test (p < 0.05) showed that S(+)-MDMA increased the number of pERK-positive neurons in the shell and in the core and that SCH 39166 pre-treatment prevented this increase.

Effects of R(−)-MDMA on pERK immunoreactivity

Figure 5 shows the effect of the administration of R(−)-MDMA (1 and 2 mg/kg) on pERK density/neuron (top panel) and on pERK-positive neurons/area (bottom panel) in the NAc shell and core.

pERK density/neuron

Two-way anova revealed a main effect of dose [F(2,318) = 21.79, p < 0.00001] but not of area [F(1,18] = 1.10, NS] nor a significant area × dose interaction [F(2,318) = 0.75, NS]. Tukey’s post hoc test (p < 0.05) showed that at the dose of 1 mg/kg R(−)-MDMA increased and at the dose of 2 mg/kg decreased pERK density/neuron with respect to saline in the NAc shell. Tukey’s post hoc test also showed that R(−)-MDMA failed to affect pERK density/neuron at the dose of 1 mg/kg while, at the dose of 2 mg/kg, decreased it in the NAc core.

pERK-positive neurons/area

Two-way anova failed to reveal main effects of dose [F(2,22) = 0.09, NS], area [F(1,22) = 1.84, NS], and a significant dose × area interaction [F(2,22) = 0.49, NS].

Effect of time on R,S(±)-MDMA-induced pERK immunoreactivity

Table 2 shows the effect of the administration of R,S(±)-MDMA (2 mg/kg i.v.) on pERK density/neuron and on pERK-positive neurons/area in the NAc shell and core after three different time intervals (2.5, 5, and 10 min) from R,S(±)-MDMA administration.

Table 2.   Effect of time (2.5, 5, and 10 min) since saline or R,S(±)-MDMA (2 mg/kg i.v.) administration on pERK activation in the shell and core of the NAc
 % density/neuron ± SEM% pERK-positive neurons/area ± SEM
ShellCoreShellCore
  1. Values represent the average ± SEM. Effect of saline or R,S(±)-MDMA (2 mg/kg i.v.) administration on ERK density/neuron and on % ERK-positive neurons/area in the shell and core of the NAc. Values represent the average ± SEM.

  2. *indicates p < 0.05 with respect to sal/sal, same area. NSindicates not significantly different versus sal/sal.

  3. MDMA, 3,4-methylenedioxymethamphetamine; pERK, phosphorylated extracellular signal regulated kinase; NAc, nucleus accumbens.

pERKSal/Sal100 ± 2100 ± 2100 ± 13100 ± 11
Sal/(±)MDMA 2 mg/kg @ 2.5′122* ± 4109NS ± 2259* ± 23447* ± 48
Sal/(±)MDMA 2 mg/kg @ 5′121* ± 3108NS ± 2289* ± 24364* ± 49
Sal/(±)MDMA 2 mg/kg @ 10′123* ± 5107NS ± 2308* ± 35469* ± 29
ERKSal/Sal100 ± 2100 ± 2100 ± 13100 ± 6
Sal/(±)MDMA 2 mg/kg96NS ± 192NS ± 296NS ± 1589NS ± 9
pERK density/neuron

Three-way anova, of the results of R,S(±)-MDMA administration on pERK density/neuron, revealed a main effect of treatment [F(1,510) = 57.78, p < 0.0001], area [F(1,510) =8.55, p < 0.003], and significant treatment × area interaction [F(1,510) = 21.81, p < 0.0001], but not a significant effect of time [F(2,510) = 1.08, N.S.]. Newman–Keuls post hoc test (p < 0.05) revealed that R,S(±)-MDMA increased pERK density/neuron in the NAc shell with respect to saline at 2.5, 5, and 10 min from R,S(±)-MDMA administration.

pERK-positive neurons/area

Two-way anova revealed a main effect of area [F(1,28) = 21.52, p < 0.0001] but failed to reveal main effects of time [F(2,28) = 1.37, NS] and significant time × area interaction [F(2,28) = 1.31, NS].

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The present study shows that R,S(±)-MDMA and S(+)-MDMA but not R(−)-MDMA increased dialysate DA and ERK phosphorylation in the NAc. These effects were obtained at i.v. doses of MDMA corresponding to those previously reported to be self-administered by rats (Ratzenboeck et al. 2001) and to affect dialysate DA (Cadoni et al. 2005) and acetylcholine (Acquas et al. 2001). In agreement with Cadoni et al. (2005), R,S(±)-MDMA and S(+)-MDMA increased DA transmission preferentially in the shell of the NAc. The S(+)-isomer of MDMA, given at doses of 0.64 and 1 mg/kg, resulted approximately twice as potent as racemic MDMA, suggesting that this isomer fully accounted for the DA stimulant properties of racemic MDMA (Table 1). On the other hand, the property of preferentially stimulating DA transmission in the NAc shell adds MDMA to the list of drugs with abuse potential that share this property (Pontieri et al. 1995, 1996; Tanda et al. 1997; Lecca et al. 2006a,b).

In spite of the inability to release DA in the NAc shell, R(−)-MDMA stimulated behavior as indicated by the increased time spent as active and still-aroused during the first 20 min after 0.64 mg/kg and during the first 30 min after 1 mg/kg of R(−)-MDMA. The circumstance that R(−)-MDMA failed to increase DA in the NAc but stimulated behavior would suggest that R(−)-MDMA has DA-independent psychostimulant properties. This possibility is consistent with the observation of Fantegrossi et al. (2002) that R(−)-MDMA is self-administered by monkeys and this property is almost completely prevented by MDL 100 907, an antagonist of 5HT2 receptors.

R,S(±)-MDMA and S(+)-MDMA increased pERK in the NAc, while R(−)-MDMA was ineffective. This effect was specifically dependent upon DA D1 receptors as it was blocked by a D1 receptor antagonist (SCH 39166) but not by a D2/3 antagonist (raclopride). These observations would be consistent with the notion that ERK activation is the post-synaptic correlate of the increase of dialysate DA.

In order to test the specificity for the phosphorylated isoforms of ERK in our experimental setup, we also studied in parallel experiments the effects of racemic MDMA on pERK and total ERK immunostaining by determining the optical density/neuron and the number of pERK-positive neurons/area in the NAc shell and core (Table 2). These results indicate that in the same rats this treatment determines an increase of the immunostaining for the active, dually phosphorylated form but not for the inactive, non-phosphorylated form of the kinases (Table 2).

Besides counting pERK-positive neurons as in the studies by Caboche and colleagues (Valjent et al. 2000, 2001a, 2004), we analyzed at the highest magnification (100×) the optical density of pERK-positive neurons of the NAc. Using this measure, R,S(±)-MDMA and S(+)-MDMA but not R(−)-MDMA, preferentially increased pERK density/neuron in the shell with respect to the core. This effect is parallel to that on dialysate DA in the NAc. However, the changes in pERK density/neuron do not fully match the changes in the number of pERK-positive neurons/area as this measure fails to detect the preferential increase of pERK in the shell as compared with the core shown by density/neuron measurements (bottom panel of Fig. 5). The reason for this discrepancy might be related to the differences in the degree of maximal activation of DA transmission in these two areas. If this reasoning is correct, measures of pERK density/neuron would be a more faithful index of post-synaptic activation in response to activation of pre-synaptic DA transmission than number of pERK-positive neurons/area.

Our findings appear in general agreement with the study by Salzmann et al. (2003), indicating a role for the activation of ERK’s pathway in the motivational effects of racemic MDMA in mice and with the findings by Valjent and colleagues (Valjent et al. 2000, 2001a, 2004) who reported that cocaine and Δ9-THC did increase the number of pERK-positive neurons in the NAc, although this effect was of a similar magnitude in the shell and core. The preferential increase of pERK density/neuron in the shell versus the core has also been observed by us following acute administration of cocaine and morphine (Acquas E. et al., manuscript in preparation). Therefore, this effect might represent a common modality by which extracellular DA increased by drugs of abuse acts post-synaptically to produce behavioral and biochemical consequences (Valjent et al. 2001b). In agreement with Valjent et al. (2000, 2001a), we found that blockade of DA D1 receptors by SCH 39166 (McQuade et al. 1991), a selective antagonist with no affinity for 5HT2 receptors, prevented the increase in pERK (density/neuron and number of pERK-positive neurons) elicited by racemic MDMA. This finding, in keeping with the notion that many of the behavioral consequences of increased DA transmission in the shell of the NAc are mediated by the D1 receptor subtype, supports the view that pERK represents a post-synaptic correlate of increased pre-synaptic DA transmission.

In further agreement with the observations by Valjent et al. (2000, 2001a) on cocaine and Δ9-THC, raclopride failed to affect the increase of pERK induced by MDMA in the NAc shell and core indicating that pERK activation is specifically D1-dependent. Nonetheless, raclopride by itself significantly increased pERK in the NAc core. This effect might result from combined blockade of D2 receptors and stimulation of DA release onto D1 receptors by raclopride. Stimulation of D1 receptors activates the phosphorylation of ERKs via the activation of adenylate cyclase-protein kinase A pathway, whereas stimulation of D2 receptors may exert an opposite action on ERKs’ phosphorylation: (i) by activating via βγ-subunits of the Gi protein and (ii) by reducing adenylate cyclase activity and therefore reducing the contribution of protein kinase A. Blockade of D1 receptors in the presence of R,S(±)-MDMA-induced increased extracellular concentrations of DA can reduce the activation of ERKs directly (blockade of Gs protein-coupled receptors) and indirectly via D2 receptors stimulation (stimulation of Gi protein-coupled receptors). These changes might take place on two different neuronal populations of the NAc core that, as in the dorsal striatum, differentially express D1 and D2 receptors.

The administration of R(−)-MDMA, which failed to affect dialysate DA in the shell and core of the NAc, slightly activated ERKs (density/neuron and number of neurons/area) in the shell at the dose of 1 mg/kg but failed to affect these measures in the core at 1 mg/kg and decreased them at 2 mg/kg. These results suggest that the failure to increase pERKs may reside in the lack of stimulation of DA transmission in the NAc, as indicated by microdialysis (Fig. 3). Although unable per se to activate pERK, R(−)-MDMA might potentiate D1-dependent stimulation of pERK by S(+)-MDMA. This would explain the observation that a dose of 1 mg/kg of S(+)-MDMA, while more potent than S,R(±)-MDMA in releasing DA in the NAc, is similarly potent in stimulating pERK in the NAc.

In conclusion, our study, while providing a pharmacological characterization of the effects of racemic MDMA and its stereoisomers on DA transmission in the shell and core of the NAc, confirms the notion that drugs of abuse preferentially increase DA transmission in the shell compared with the core (Di Chiara 1999, 2002) and suggests that activation of pERKs via a D1-dependent mechanism represents a post-synaptic correlate of drug-induced increases of DA transmission.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study was supported by funds from Ministero dell’Istruzione, Università e Ricerca, MIUR (Centre of Excellence for Studies on the Neurobiology of Addiction, and FIRB) to G. Di Chiara and PRIN, Regione Autonoma della Sardegna (RAS), Fondazione Banco di Sardegna (Sassari, Italy) and from the University of Cagliari to G. Di Chiara and E. Acquas. G. Zernig’s contribution was supported by FWF grant P16394-B05. A. Pisanu was supported by a PhD fellowship from Fondazione Banco di Sardegna (Sassari, Italy). The excellent technical assistance of Elena Sias (Department of Toxicology), Renato Mascia (Dipartimento Farmaco Chimico Tecnologico), and Mauro Argiolas (Department of Animal Biology and Ecology) is gratefully acknowledged.

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  6. Acknowledgements
  7. References
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