Microinjection of Neuromodulators in Honeybees

Drugs were dissolved in honeybee Ringer [NaCl 130 mM, KCl 6 mM, MgCl₂ 4 mM, CaCl₂ 5 mM, HEPES 10 mM, glucose 25 mM, sucrose 160 mM]. Drugs or Ringer alone (control) were injected into the brain through the median ocellar tract. In addition to normal preparation, the head of each harnessed bee was fastened to the holder with a small drop of wax to avoid movements. A Harvard GC 100-10 microelectrode filled with the drug to be injected was connected to an IM 300 Narishige microinjector and used to deliver 10×20 nl into the brain. Volumes injected were calibrated before and after injection by means of a Malassez cell. Injections were performed 30 min before conditioning, as earlier experiments (reviewed in ref. 42) showed that pharmacological injections of catecholamines and their inhibitors are effective approximately 30 min after drug application. Octopaminergic (mianserine, epinastine) and dopaminergic receptor antagonists (fluphenazine, flupentixol, SCH23390, spiperone) (Sigma Aldrich, Deisenhofen, Germany) were used at µm and mM doses (Fig. 6). Low-dose experiments (except for flupenthixol 0.2 µm) and their respective Ringer control were performed in parallel (yellow rows in Fig. 6). High-dose experiments (and flupentixol 0.2 µm) were also performed in parallel with their respective Ringer control (pink rows in Fig. 6). The group injected with epinastine received one dose and had a separate Ringer control (blue row in Fig. 6).