1xpbs

Basic procedures for stereotaxic surgery and implanting guide cannulae in mice were similar to that previously described (Griffin et al., 2014 (link); Griffin et al., 2009b (link)). Under isoflurane anesthesia, mice had the bilateral guide cannulae (Plastics One, Inc) implanted targeting the mPFC (AP: +2 mm, ML: ±0.4 mm, DV: −1.2 mm, Bregma was used as a reference point) (Franklin and Paxinos, 2008 ). Bilateral microinjections were delivered using dual syringe Model 11 Pumps (Harvard Apparatus) in a volume of 0.25 μl/side administered over 2 min, with the injector left in place for an additional 2 min to allow diffusion of the injected agent. Recombinant human BDNF (rhBDNF, R&D Systems) was prepared in 1xPBS (Boston Bioproducts) immediately prior to microinjection. For BDNF overexpression studies, 0.25 μl of either AAV- BDNF or AAV-GFP was bilaterally infused into the mPFC (AP: +2 mm, ML: ±0.4 mm, DV: −1.7 mm) using a 0.5 μl, 33-gauge Hamilton Neuros syringe (Reno, NV) at a rate of 0.05 μl per minute. After a 5 min diffusion period, the syringe was slowly retracted over an additional 5 min period. All animals undergoing surgery were given 1–2 weeks recovery time before experiments commenced.

At the end of the study, mice were sacrificed with deep anesthesia (3 g/kg urethane) followed by transcardiac perfusion using approximately 10 ml ice-cold 1X PBS (diluted from 10X stock Boston Bioproducts, Inc) followed by approximately 12 ml ice-cold 4% paraformaldehyde (PFA). The brain was post-fixed overnight in 4% PFA and then placed in 30% sucrose until sectioning. Brains were sectioned (40 microns) using a cryostat and floated in 1X PBS prior to immunostaining. All solutions were diluted in PBS with 0.3% Triton-X (PBST), and sections were rinsed in PBS 3 times between each step. Sections were first incubated in a 1% hydrogen peroxide solution for 60 min followed by overnight incubation in primary antibody (1:20,000 rat anti-mCherry, Life Technologies #M11217) diluted in blocking solution (5% normal goat serum in PBST). Sections were then placed in secondary antibody (1:1000 biotinylated goat anti-rat, Jackson Immuno #112–066-003) for 30 min and 1:1000 ABC (Vector Elite Kit, Vector Labs) for 60 min. All rinses and incubations occurred on a rocker at room temperature. Finally, sections were incubated in a solution of 0.05% 3,3’-diaminobenzidine and 0.015% hydrogen peroxide for 10 min.