Infusion Protocols for Hippocampal and VTA Manipulation

To help reduce any stress, all drugs were infused in the home cages. The stylets in the guide cannulae were replaced by a double infusion cannula (33 gauge, Plastics One) connected to two 5 μl microsyringes (WPI) in a microinfusion pump (Native Instruments) via flexible plastic tubing (C232CS, Plastics One) filled with Fluorinert (3M). The tips of infusion cannulae projected 0.5 mm below the tip of the guide cannulae. For intra-hippocampal microinjection, 0.5 μl of drug per cannula was infused at 0.2 μl min⁻¹ (2.5 min). Infusion cannulas were left in place for a further 2.5 min before being replaced with stylets to aid drug absorption. For intra-VTA microinjection, 0.3 μl was injected at a rate of 0.3 μl min⁻¹ (1 min) followed by 1-min of waiting. The mice were habituated to the experimental procedure of injection and to vehicle injection prior to the drug test in order to minimize novelty effect. Mice received drug injection 20 min (hippocampal microinfusions and i.p. injection of clonidine) or 3 min (VTA microinfusions) prior to the novelty exploration.