Gbr12909

The functional integrity and synaptic function of DA neurons were evaluated by the ability of these neurons to accumulate [³H]-DA by active transport (50 nM; 40 Ci/mmol; PerkinElmer, Courtaboeuf, France), as previously described [43 (link)]. GBR-12909 (0.2 μM; Sigma Aldrich, L’Isles d’Abeau Chesnes, France) was used to obtain blanks that were subtracted from raw values.

Brain sections for DAT binding were pre-incubated in 50 mM Tris-HCl buffer containing 120 mM NaCl and 0.1% bovine serum albumin (pH 7.4) for 20 min at 4 °C. Sections were then incubated for 2 h in 15 nM [³H]WIN35428 (specific activity, 85.9 Ci/mmol; PerkinElmer, Waltham, MA, USA). Non-specific binding was determined by the addition of 10 µM GBR12909 (Sigma-Aldrich, Castle Hill, NSW, Australia) to subsequent sections. Sections were then washed twice for 1 min in ice-cold buffer, dipped in ice-cold distilled water and then air dried [40 (link),85 (link),88 (link)].

HEK 293T cells were from the American Type Culture Collection. Anti-FLAG M2 agarose gel, monoclonal anti-FLAG antibody, 3 x FLAG peptide, 4-[4-(dimethylamino) phenyl]-1-methylpyridinium (APP⁺), 4-[4-(dimethylamino) styryl]-N-methylpyridinium (ASP⁺), fluoxetine, GBR12909, and desipramine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sulfosuccinimidyl 2-(biotinamido) ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin), streptavidin agarose gel, and Super Signal West Pico were from Thermo Fisher Scientific (Waltham, MA, USA). All other reagents were of analytical grade.

The drug MDMA was dissolved in physiological saline (in a volume of 2 ml/kg) and administered at doses of 0.5, 2.5, and 5 mg/kg. Morphine (ICN Hungary Plc.) was also dissolved in physiological saline (in a volume of 2 ml/kg) and administered at a dose of 1 mg/kg 30 min before the social interaction assay. GBR‐12909 (Sigma) was dissolved in physiological saline (in a volume of 5 ml/kg) and administered at doses of 1, 3, and 10 mg/kg. All the drugs were administered by subcutaneous (s.c.) injection in the flank region 30 min prior to the behavioral observations. In the coadministration experiment, MDMA and GBR‐12909 were administered to the rats in the two flank regions within a short period of time 30 min prior to the observations. DA‐3,4‐[7,8‐3H(N)], with a specific activity of 60 Ci/mmol, and choline chloride [methyl‐3H], with a specific activity of 80 Ci/mmol, were purchased from American Radiolabeled Chemicals (ARC). β‐PEA was obtained from Sigma. All the other chemicals were obtained from Sigma.

D‐Amphetamine (D‐Amph) was sourced from Tocris (UK), paroxetine from Santa Cruz (USA), p‐chloroamphetamine (pCA) and GBR12909 were from Sigma‐Aldrich (USA). Hellmanex was from Hellma France S.A.R.L. (Paris, France). Radiochemicals are [³H] 1‐methyl‐4‐phenylpyridinium ([³H]MPP+; 80–85 μCi × mmol⁻¹) from American Radiolabeled Chemicals (St. Louis, MO, USA) and [³H]5‐HT (28.3 μCi x mmol⁻¹) from PerkinElmer (Boston, MA, USA). Other chemicals and cell culture supplies were obtained from Sigma‐Aldrich (St. Louis, MO, USA), and cell culture dishes were from Sarstedt (Nuembrecht, Germany).

Neurotransmitter release was evoked by either bath application of a high concentration of potassium or electrical stimulation. Potassium-evoked release was performed by perfusing tissue with high K⁺ (100 mM, replacing an equimolar amount of NaCl) aCSF for 1 min once a stable 30 s baseline recording had been obtained. Electrically-evoked release was performed with a bipolar stimulating electrode placed close to the carbon fiber recording electrode within the dorsal telencephalon. Current pulses were generated by the acquisition software and applied via a stimulus isolator (Iso-Flex; AMP Instruments). The tissue was allowed to recover for a minimum of either 5 min (electrical stimulation) or 30 min (for high K⁺ stimulation) between stimulations. In some experiments we added drugs targeting neurotransmitter systems to the aCSF: 10 μM GBR 12909 (selective DA reuptake inhibitor; Sigma Aldrich D052); or 10 μM cocaine hydrochloride (DA, NA and 5-HT reuptake inhibitor; Sigma Aldrich C5776). GBR 12909 was first dissolved in DMSO with gentle warming before being directly added to the aCSF. Cocaine was made into a stock solution in water before being added to aCSF. Drugs were not perfused onto tissue until at least 3 stable baseline recordings had been obtained.