Biotinylated Dextran Amine (BDA) Injections and Immunohistochemistry

Rats (4 rats for each sample) were anesthetized with isofluorane (4% for induction and 1–1.5% for maintenance) and stereotaxically injected with 1 μL of 10% BDA 10 kDa (Thermo Fisher) at a rate of 0.1 μL/30 s with a 33-G Hamilton syringe in the BLA (AP = 2.8 mm, ML = 4.8 mm, and DV = 8.2 mm from Bregma). Seven days after the surgery, the animals were decapitated to prepare synaptosomes from the PFC or perfused for immunohistochemistry. Animals were perfused intracardially with 4% paraformaldehyde (PFA), and brains were postfixed in 4% PFA overnight and then kept in 20% sucrose for 48 hours. Brains were sliced in 30-μm coronal sections with a cryostat (Leica CM 1510, Wetzlar, Germany). BDA immunohistochemistry was performed as described (55). Coronal sections were mounted on gelatin-coated slides and coverslip using Entellan (Merck), and the images were captured in an epifluorescence microscope (Nikon, FLV1000). In the case of PFC synaptosomes prepared from BDA-injected animals, they were processed for immunofluorescence using the secondary antibody Streptavidin-AlexaFluor⁶⁴⁷ (1:200; Thermo Fisher).