In vivo Microdialysis

Male adult Long-Evans rats (90–120 days of age) were included in the present experiments. Only one animal from each litter was used here while littermates were assigned to other experiments [as reported, in part, by McGaughy et al. (2014)]. For surgery, rats were anesthetized with 2% isoflurane with oxygen (0.6 L/min). Lidocaine with epinephrine was injected subcutaneously at the site of the incision. Surgeries were done under aseptic conditions. During surgery, guide cannulae (CMA 12, CMA/Microdialysis AB, Acton, MA, United States) were implanted bilaterally into the vmPFC such that the tip of the guide cannula was located at the coordinates of A 3.2 mm; L ± 0.8 mm; DV 2 mm with reference to bregma according to the atlas of Paxinos and Watson (2005). After the holes for the guide cannulae were drilled, the guide cannulae were slowly lowered into place over a 3-min period. Guide cannula were affixed to the skull using three stainless steel screws with dental acrylic covering the screws and the guides.

After 3 days of recovery following implantation of the guide cannulae, animals were placed in a large Plexiglas bowl with a collar attached by a guide wire to a suspension arm (CMA Microdialysis). All experiments took place between 0830 and 1200 h. On the day of the experiment, a 2 mm CMA-12 probe (CMA Microdialysis, Inc., N. Chelmsford, MA, United States) was placed into each cannula while the animal moved freely around the bowl. Artificial cerebrospinal fluid (artCSF; 147 mM NaCl, 1.26 mM CaCl₂, 2.5 mM KCl, and 1.18 mM MgCl in sterile water) was perfused through the probe using a CMA/Microdialysis Syringe pump and a 1.0 ml gastight Hamilton syringe at a rate of 1.0 μl/min. After allowing 3 h for equilibration of the probes, samples (20 μl) were collected every 20 min for another 3 h. Animals were only used in one microdialysis experiment.