S eticlopride hydrochloride

Cocaine hydrochloride was supplied by the National Institute on Drug Abuse (National Institutes of Health, Bethesda, MD). (S)-eticlopride hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO), SCH 23390 hydrochloride, from Tocris (Ellisville, MO). Cocaine and SCH 23390 were dissolved in 0.9% saline. Eticlopride was dissolved in ethanol and diluted to ≤1% ethanol in sterile water.

Primary antibodies used in this study include rabbit antibodies against phospho-S831 (pS831, PhosphoSolutions, Aurora, CO), phospho-S845 (pS845, PhosphoSolutions), GluA1 (Millipore), PKA catalytic subunit a (PKA Cα; Cell Signaling Technology, Danvers, MA), or PKA regulatory subunit IIβ (PKA RIIβ; Abcam, Cambridge, MA), or mouse antibodies against GluA2 (Millipore), GluA3 (Millipore), or tubulin (Millipore). Pharmacological agents, including (±)-6-chloro-PB hydrobromide (SKF81297), (-)-quinpirole hydrochloride, R(+)-SCH23390 hydrochloride, S-(-)-eticlopride hydrochloride and (-)-scopolamine hydrobromide, were purchased from Sigma-Aldrich. VU0152100 [3-amino-N-(4-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide] was purchased from Axon Medchem (Reston, VA). All agents were freshly prepared at the day of experiments. VU0152100 was dissolved in 10% Tween 80 and dH₂O with the pH adjusted to approximately 7.0 using 1 N NaOH. Other agents were dissolved in physiological saline.

S-(−)-eticlopride hydrochloride was purchased from Sigma. 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2) was purchased from Tocris (Bristol, UK). All agents were freshly prepared at the day of experiments. Eticlopride was dissolved in physiological saline. PP2 was dissolved in dimethyl sulfoxide (DMSO). In experiments with PP2, DMSO served as a vehicle control.

Primary antibodies used in this study include rabbit polyclonal antibodies against Src (Cell Signaling Technology, Danvers, MA), Fyn (Santa Cruz Biotechnology, Santa Cruz, CA), GluN1 (Millipore), GluN2A (Millipore), GluN2B (Millipore), phosphorylated GluN2B at tyrosine 1472 (pY1472; PhosphoSolutions), or actin (Sigma-Aldrich, St. Louis, MO), or mouse antibodies against Src (Cell Signaling), Fyn (Santa Cruz), or tubulin (Millipore). The rabbit antibody against pan pY416 was purchased from Cell Signaling. This antibody reacts with the Src family members when phosphorylated at the activation residue: Y416 (chicken Src), Y419 (rat Src), and Y420 (rat Fyn). Pharmacological agents, including (±)-6-chloro-PB hydrobromide (SKF81297), (-)-quinpirole hydrochloride, R(+)-SCH23390 hydrochloride, and S-(-)-eticlopride hydrochloride, were purchased from Sigma. VU0152100 [3-amino-N-(4-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide] was purchased from Axon Medchem (Reston, VA). All agents were freshly prepared at the day of experiments. VU0152100 was dissolved in 10% Tween 80 and dH₂O with the pH adjusted to approximately 7.0 using 1 N NaOH. Other agents were dissolved in physiological saline.

Primary antibodies used in the current study include rabbit antibodies against phosphorylated ERK1/2 (pERK1/2) at Thr202/Tyr204 (Cell Signaling Technology, Beverly, MA), ERK1/2 (Cell Signaling), or β-actin (Sigma-Aldrich, St. Louis, MO). Pharmacological agents, including (−)-scopolamine hydrobromide, R(+)-SCH23390 hydrochloride and S-(−)-eticlopride hydrochloride, were purchased from Sigma-Aldrich (St. Louis, MO). Scopolamine, SCH23390 and eticlopride were dissolved in physiological saline. All agents were freshly prepared at the day of experiments.

To examine effects of inhibiting DA D2Rs on cotinine-related behaviors, S-(−)-eticlopride hydrochloride, a selective D2R antagonist (Sigma, St. Louis, MO, USA) was injected subcutaneously into separated groups of rats self-administering cotinine (n = 8) and undergoing cue-induced reinstatement of cotinine-seeking behavior (n = 7). Eticlopride (0, 5, and 10 μg/kg) was administered approximately 30 min prior to operant sessions using a within-subject design with different doses administered in a random order. Non-treatment sessions were included to allow responses to return to basal levels between treatments. These doses of eticlopride have been shown to attenuate nicotine-related relapse behaviors in rats (Liu et al 2010 (link)).