Lanteri et al.

Supplemental Figure 1. (A) Schematic representation of the experimental procedure. Mice were injected once daily for 4 days with either saline, MDMA or SR46349B + prazosin + MDMA. Then, both in vivo experiments (microdialysis analysis, locomotor activity monitoring, LORR test, hypothermia assay) as well as in vitro experiments (radiolabelling of a2A-adrenergic and 5-HT1A receptors, western blot and qRT-PCR analysis of Gai/o proteins and mRNAs expression in the LC and the DRN) were conducted 4 days or 1 month after this repeated treatment. (B) Schematic illustration of the localization of the dialysis probe in the mouse PFC, LC and DRN (adapted from the atlas by Paxinos and Franklin, 1997). (C) Schematic illustration of the microdissection of the LC and the DRN (adapted from the atlas by Paxinos and Franklin, 1997).

Supplemental Figure 2. Repeated MDMA fails to sensitize mesocortical dopaminergic neurons. Mice were repeatedly injected with either saline or MDMA (10 mg/kg, i.p.) and extracellular DA levels were monitored by in vivo microdialysis following amphetamine injection (2 mg/kg, i.p.) 4 days after withdrawal. Cortical extracellular DA levels are expressed as a percentage of the respective mean basal value ± SEM (absolute baseline values: repeated saline, 1.3 ± 0.18 pg DA/20 min; repeated MDMA, 0.92 ± 0.2 pg DA/20 min). n = 5-6 mice per group. There was no significant difference in cortical extracellular DA levels between the different conditions (p > 0.05).

Supplemental Figure 3. Repeated MDMA does not alter Gi proteins expression in the PFC. Mice were repeatedly injected with either saline or MDMA (10 mg/kg, i.p.). Four days or one month after the last injection, expression levels of Gai1, Gai2 and Gai3 were analyzed by western blot in the PFC. Experiments were performed 3 times with n = 8 mice per group. Data represent mean ± SEM. (p > 0.05) (unpaired Student’s t test).

Supplemental Figure 4. Pharmacological blockade of a2A-adrenergic and 5-HT1A receptors mimics neurochemical and behavioral sensitization. Mice were acutely injected with either saline, amphetamine (2 mg/kg, i.p.) or PCA (7 mg/kg, i.p.) after being pre-treated with either saline, efaroxan (2.5 mg/kg, i.p.) or WAY-100635 (1 mg/kg, i.p.) 30 min before. Cortical extracellular NE (A) and 5-HT (B) levels were monitored by in vivo microdialysis as well as the locomotor response induced by both compounds (C and D). Cortical extracellular monoamines levels are expressed as a percentage of the respective mean basal value ± SEM (absolute baseline values of NE: sal + sal, 0.55 ± 0.28 pg NE/20 min; efa + sal, 0.58 ± 0.32 pg NE/20 min; sal + amph, 0.59 ± 0.42 pg NE/20 min; efa + amph, 0.83 ± 0.59 pg NE/20 min. Absolute baseline values of 5-HT: sal + sal, 0.63 ± 0.51 pg 5-HT/20 min; WAY + sal, 0.95 ± 0.58 pg 5-HT/20 min; sal + PCA, 0.4 ± 0.12 pg 5-HT/20 min; WAY + PCA, 0.27 ± 0.09 pg 5-HT/20 min). There was no significant difference in basal cortical extracellular monoamines levels between the different conditions (p > 0.05). Locomotor activity is expressed as ¼ turn per 5 min ± SEM. Each group included at least 6 mice for microdialysis experiments and 8 mice for behavioral experiments.

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