Anisomycin

Brains of 9–12-day-old flies were dissected in cold Haemolymph-Like saline solution 3 (HL3) (NaCl 70 mM, KCl 5 mM, MgCl₂ 4 mM, trehalose 5 mM, sucrose 115 mM, HEPES 5 mM, NaHCO₃ 10 mM, pH 7.2–7.3) and transferred into Nunc Lab-Tek II Chamber Slide (Thermofisher, #154526) with either 500 µL of HL3 or HL3 supplemented with neurotransmitter for 30 min at 25°C. Brains were protected from light during incubations. Neurotransmitters were used at the following final concentrations: Acetylcholine (Sigma, # A6625): 10 mM; Tyramine (Sigma, # T2879): 10 mM; Dopamine (Sigma, # H8502): 10 mM; Octopamine (Sigma, # O0250): 10 mM. For treatment with the translational inhibitor anisomycin (sigma, #A9789), anisomycin was added 20 min prior to Tyramine at a final concentration of 40 µM and maintained throughout Tyramine treatment. After treatment, brains were collected, fixed with 4% formaldehyde in HL3 for 25 min, washed thrice with phosphate-buffered saline supplemented with 0.5% Triton-X (PBT) and either directly mounted in vectashield (Vector Laboratories) to image endogenous fluorescence or further immuno-stained.

All chemicals were obtained from Sigma-Aldrich unless stated otherwise. All chemicals were dissolved in DM at a concentration such that the added volume into CM was less than 1% of total CM volume. In the experiments with the protein synthesis blocker anisomycin (12.5 ug/mL; A9789; Sigma-Aldrich) the cells were pre-incubated with anisomycin in DM prior to CM addition. Cadmium was used at a final concentration of 100 μM and nifedipine at 10 μM.

The general procedures for intra-hippocampal infusions were as described in previous reports (Quevedo et al., 1999 (link); Luft et al., 2006 (link); Roesler et al., 2006 (link)). At the time of infusion, a 30-gauge infusion needle was fitted into the guide cannula. The tip of the infusion needle protruded 1.0 mm beyond the guide cannula and was aimed at the CA1 area of the dorsal hippocampus. The animals received, via the infusion cannula, a bilateral 0.8 μl infusion of vehicle (20% dimethylsulfoxide, DMSO, in saline), the PDE4 inhibitor rolipram (7.5 μg /side dissolved in vehicle; Sigma-Aldrich, St. Louis, USA), the protein synthesis inhibitor anisomycin (80.0 μg/side dissolved in vehicle; Sigma-Aldrich, St. Louis, USA), or rolipram combined with anisomycin at the doses described above. Drug doses were chosen on the basis of previous studies (Quevedo et al., 1999 (link); Vianna et al., 2001 (link); Luft et al., 2006 (link); Werenicz et al., 2012 (link)). Drug or vehicle was infused over a 30-s period. Solutions were freshly prepared before each experiment.
In different experiments, intra-hippocampal infusions were given immediately after the first retrieval session (which also served as extinction training), 1 h after retrieval (delayed infusion controls), 24 h after training in the absence of retrieval (no retrieval controls), or immediately after training.