Flupenthixol

Tetrodotoxin (TTX) and flupenthixol were purchased from Tocris Bioscience (Bristol, UK), flavopiridol was from Selleckchem (Houston, TX, USA), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MI, USA). Peptides were synthesized by Biomatik (Wilmington, DE, USA). DA was made fresh every 30 min to minimize oxidation. Antagonists were administered 10 min before DA application. Concentrations of flavopiridol (100 nM) and 5,6-dichloro-1-β-D-ribobenzimidazole (DRB, 100 μM) were chosen based on previously demonstrated effective dosages (Chao and Price, 2001 (link); Bensaude, 2011 (link); Yuan and Burrell, 2013 (link); Krenz et al., 2014 (link)).

Three weeks following virus infection, mice labeled with Mor-Ens or Sal-Ens were given six daily intracranial self-stimulation (ICSS) training sessions. Behavioral training and testing were performed in a mouse-operant apparatus (Med Associates) interfaced with optogenetic stimulation equipment (Newdoon Inc and Inper Tech, Hangzhou, Zhejiang, China). Each operant behavior chamber was equipped with a dim-light source and a nose-poke hole equipped with infrared photobeams connected to a computer. A 1-h fixed-ratio one (FR1) schedule was performed every training day. A nose poke in the target hole produced 2 s of 473-nm light (5 ms, 20 Hz, 10 mW). For drug-infusion studies, mice received injections of either flupenthixol (0.5 mg/kg, Tocris, Bristol, UK) or saline vehicle intraperitoneally 30 min prior to ICSS sessions. For negative-reinforcement procedures [68 (link)], mice were placed into the chamber and delivered continuous 473-nm (5 ms, 20 Hz, 10 mW) optical stimulation with an interstimulus interval of 1 s. The mice were trained on a FR1 training schedule, in which each active nose poke produced a 20-s laser shut-off and the terminals were not optogenetically activated.

Fluoxetine (#H6995, Bosche Scientific), citalopram (#C505000, Toronto Research Chemicals), paroxetine (#2141, Tocris), fluvoxamine (#1033), venlafaxine (#2917, Tocris), reboxetine (#1982, Tocris), imipramine (#I7379-5G, Sigma–Aldrich), clomipramine (#C7291, Sigma–Aldrich), desipramine (#3067, Tocris), phenelzine (#P6777, Sigma–Aldrich), rolipram (#R6520, Sigma–Aldrich), ketamine (#3131, Tocris), 2R, 6R-Hydroxynorketamine and 2S, 6S-Hydroxynorketamine (#6094 and #6095, respectively, Tocris), carbamazepine (#4098, Tocris), chlorpromazine (#C8138, Sigma–Aldrich), flupenthixol (#4057, Tocris), and pimozide (#0937, Tocris) were investigated in this study. The compounds were dissolved in dimethyl sulfoxide (DMSO) that was also used as a vehicle in all the experiments. Concentrations tested (Table 1) were selected based on earlier in vitro studies from our research group (Fred et al., 2019 (link); Casarotto et al., 2021 (link)) and other laboratories (Johansen et al., 2015 (link)).