The total duration click here of each training trial was 500 s. A training trial started with placing the mouse in a square chamber with grid floor (Coulbourn Instruments; H10-11RTC, 120W × 100D × 120H). At 198 s, 278 s, 358 s, and 438 s, a foot shock was delivered (2 s, 0.70 mA). On days 2 and 3, FC+EXT mice were subjected to four extinction trials per day (day 2: E1 to E4; day 3: E5 to E8). Each extinction trial lasted 1,800 s with a trial interval of 2 hr. For each extinction trial, mice were placed in the same box used for

fear conditioning without receiving foot shocks. On day 4, FC and FC+EXT mice were tested over 500 s during a single retrieval test. For the retrieval test, mice were placed in the same box used for fear conditioning without receiving foot shocks. The design of experiment 2 is summarized in Figure 5A. The FC group in experiment 2 was subjected to the same protocol as the FC group in experiment 1. The HC group consisted of mice that stayed in their home cage during the entire experiment. HC mice were perfused at the same time as the FC mice. Freezing behavior was measured using a digital camera connected to a computer with Actimetrics FreezeFrame software. The bout length was 1 s and the threshold for freezing behavior was determined by an experimenter blind to

experimental conditions and animal group. Freezing scores were obtained by averaging freezing during minutes 2 and 3 of each trial. Ninety minutes after retrieval testing, mice were deeply anesthetized with ketamine/xylazine Birinapant molecular weight and transcardially perfused with 0.1 M phosphate buffer (PB) followed by 4% paraformaldehyde (PFA 4%) dissolved in 0.1 M PB. Brains were extracted and postfixed in PFA 4% for 24 hr. Brains were transferred to 30% sucrose for 48–72 hr before slicing 20 μm coronal sections of the entire brain using a cryostat. Sections were stored

in cryoprotectant at −20°C until use. Free-floating sections were rinsed extensively in PBS with 0.25% Triton X-100 (PBS-T). Sections Terminal deoxynucleotidyl transferase were blocked for 1 hr at room temperature in PBS-T with 10% normal goat serum (or 3% donkey serum for CB1R). Sections were incubated in rabbit anti-Zif268 (Santa-Cruz; polyclonal; 1:3,000) with either mouse anti-GAD67 (Millipore; monoclonal; 1:10,000), mouse anti-PV (Millipore; monoclonal; 1:2,000), mouse anti-CCK/Gastrin (Center for Ulcer Research and Education UCLA; monoclonal; 1:1,000), or goat anti-CB1 (kind gift of Dr. K. Mackie; polyclonal; 1:2,000) (Harkany et al., 2005). Additional primary antibodies used were rabbit anti-CamKII (kind gift of Dr. M. Jacob; polyclonal; 1:2,000) and rabbit anti-Rab3b (kind gift of Dr. T. Südhof; polyclonal; 1:4,000). Primary antibodies were diluted in the blocking solution, incubated at 4°C for 72 hr, and rinsed three times for 15 min in PBS-T.