Flupentixol

A small hole was pricked into the cornea of the median ocellus to allow the insertion of a 10 µl-syringe (World Precision Instrument), which was used to inject 200 nl of each drug solution^{21 (link)}. Drugs were injected into the brain of immobilized bees along the median ocellar nerve^{21 (link)}. This method ensures that drugs migrate through the ocellar tract and distribute within the bee brain in a fast (less than 5 min) and homogenous way^{76 (link)}. Thirty min before the experiment, we injected: Epinastine hydrochloride, an OA receptor antagonist^{46 (link)}, cis-(Z)-flupentixol dihydrochloride, a DA receptor antagonist^{47 (link)}; or PBS (control). Epinastine and flupentixol were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France). PBS was obtained from EUROMEDEX (Strasbourg, France). Injection time was chosen based on experiments, which showed that the effects of aminergic blockers reach a stable level approximately 30 min after drug application^{35 (link),77 (link)–79 (link)}. One mg of each drug was diluted in 1 ml PBS. Final concentrations obtained were 3.5 mM of Epinastine and 1.94 mM of flupentixol. To test for dose-response effects, we prepared for each drug an additional dilution of 1:10000. In all cases, aliquots were kept in −20 °C until use. Each aliquot was used for one whole week and kept during this time in 4 °C.

The main chemicals used in this study, including FGAs (chlorpromazine, chlorprothixene, clothiapine, droperidol, flupentixol, fluphenazine, haloperidol, levomepromazine, loxapine, pimozide, prochlorperazine, sulpiride, thioridazine, and trifluoperazine) and SGAs (amisulpride, aripiprazole, brexpiprazole, clozapine, lurasidone, olanzapine, paliperidone, quetiapine, risperidone, ziprasidone, and zotepine) were acquired from Sigma-Aldrich (St. Louis, Missouri, USA) and were of highest purity grade. Before usage, the compounds were dissolved in dimethyl sulfoxide (DMSO) at the proper concentrations. Human hepatoma HepaRG™ cells were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). For 2 weeks, frozen cells were defrosted and kept alive in Williams’ E medium (Sigma-Aldrich, St. Louis, Missouri, USA) with 10% FetalClone™ II serum (HycloneTM, GE Healthcare, Chicago, Illinois, USA), 1 × L-glutamine, 5 μg/mL human insulin, and 50 μM hydrocortisone hemisuccinate without antibiotics as supplements. To produce differentiated hepatocyte-like features, the medium was then changed to the aforementioned medium plus 2% DMSO for an additional 2 weeks. Cells were grown at 37°C in a humidified 5% CO₂ environment. P-nitrophenylphosphate (PNPP) was used in an acid phosphatase (ACP) experiment to measure cell viability (29 (link)).