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 solution21 (link). Drugs were injected into the brain of immobilized bees along the median ocellar nerve21 (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 way76 (link). Thirty min before the experiment, we injected: Epinastine hydrochloride, an OA receptor antagonist46 (link), cis-(Z)-flupentixol dihydrochloride, a DA receptor antagonist47 (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 application35 (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.
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