The experiment was concluded by euthanizing the animals, microinjection of Thionin dye, and rapid freezing of the brains. Animals were euthanized with inhaled isofluorane in enclosed Plexiglas® boxes. Microinjections with 0.25% thionin were performed the same way as microinjections during the experiment, and served the purpose of providing a marker for microinjection location. After microinjection, brains were extracted and rapidly frozen in chilled isopentane solution. The frozen brains were then stored at -80°C. Brains were sliced in 40 micron slices on a cryostat which had an internal temperature of -20°C. Placement of the microinjections was confirmed through the atlas of Paxinos and Watson (1998) using conventional light microscopy at 4-20× (Fig 1). An example photomicrograph showing the LDTg infusion site after multiple infusions at the end of the experiment is shown in Fig 1.
