Tcs ox2 29

After 3-wk of LDA, mice began to be handled for habituation and systemic injections. The selective OX1R antagonist, SB-334867 (SB, Tocris), was dissolved in 2% DMSO in 25% (2-Hydroxypropyl)-β-cyclodextrin (Sigma) and 0.9% saline; the OX2R antagonist TCS-OX2-29 (TCS, Tocris) (Huang et al., 2010 (link); Plaza-Zabala et al., 2013 (link)) was dissolved in 0.9% saline. Animals were injected, intraperitoneally (i.p.), 30-min prior to the drinking sessions. Subjects received either 0, 0.3, 1, 3 or 10-mg/kg-body-weight of SB, or 3 or 10-mg/kg-body-weight of TCS at a volume of 10-ml/kg (similar to previously used volumes, e.g. Olney et al., 2015 (link)). The selected SB doses were lower than what was previously used because we hypothesized mice drinking alcohol-quinine would be more sensitive to the effects of OX1R inhibition. Each given dose was injected twice (on different test days) and averaged per mouse; injections were counter-balanced with their vehicle across test days and mice using a Latin Squares design. There was at least one drinking session day between drug treatments to limit the impact of carryover effects after drug treatment, and drinking levels returned to baseline on these untreated days (not shown). Also, no more than two injections were performed in a given week (Fig. 1). The SacQ group was treated with either 0- or 3-mg/kg-body-weight of SB.

To determine if the effects from OX agonists might be due to endogenous OX activity and also which receptor might participate in these effects, Set 6 (N = 13) was initially treated like Set 4, with n = 6 cannulated in the aPVT and n = 7 in the pPVT. They were injected with the OX2R antagonist TCS OX2 29 (10 nmol; Tocris Bioscience, Minneapolis, MN, USA) (Hirose et al., 2003 (link)) or vehicle, counterbalanced in a within-subject design across two ethanol days, and then, after one week of recovery, were injected with the OX1R antagonist SB 334867 (10 nmol; Tocris) (Smart et al., 2001 (link)) or vehicle. The OX2R antagonist was dissolved in 0.9% NaCl (Hospira), at a dose that produced behavioral effects with pPVT injection (Li et al., 2011b (link)), while SB 334867 was dissolved in dimethyl sulfoxide (Sigma–Aldrich, St. Louis, MO, USA).

Orexin A, AL-orexin B, SB 334867 and TCS-OX2-29 were purchased from Tocris Bioscience (Bristol, UK). Naloxone, naltrexone, WIN 55,212-2 and AM 251 were bought from Sigma-Aldrich (St. Louis, MO). Morphine sulfate was purchased from Division of Controlled Drugs, Food and Drug Administration, Department of Health, Excutive Yuan, Taiwan. Orexin A, AL-orexin B, morphine, TCS-OX2-29, Naloxone and naltrexone were dissolved in normal saline. For i.pag. microinjection, SB 334867, WIN 55,212-2 and AM 251 were dissolved in dimethylsulfoxide (DMSO). For i.p. injections, SB334867 and AM 251 were dissolved in a water solution containing 10% (w/v) encapsin and 2% (v/v) DMSO. All drugs were prepared at the working concentrations for either i.pag. microinjections or i.p. injections. The i.p. injection volume was 10 ml/kg.