Associative Fear Conditioning and Extinction. All mice were handled once per day for two days and then pre-exposed once to the test xxxxxxxx (Med Associates Inc., St Albans, VT) the day prior to training. Fear conditioning and extinction experiments were performed in different contexts, where light, odor, and tactile cues were shifted. FreezeFrame and FreezeView software (Xxxxxxxxx Instruments, #ACT-100, Allentown, PA) were used to examine percent time spent freezing during tone presentations as a measure of fear behavior. For the Cnr1 antagonist (SR141716A) and fatty acid amide (FAAH) inhibitor (URB597) experiments, C57BL/6J mice received two days of 10 paired conditioned stimulus (CS) tones (30 s, 6 kHz, 75-80 dB) which co-terminated with the unconditioned stimulus (US) shock (500 ms,
Associative Fear Conditioning and Extinction. All mice were handled once per day for two days and then pre-exposed once to the test xxxxxxxx (Med Associates Inc., St Albans, VT) the day prior to training. Fear conditioning and extinction experiments were performed in different contexts, where light, odor, and tactile cues were shifted. FreezeFrame and FreezeView software (Xxxxxxxxx Instruments, #ACT-100, Allentown, PA) were used to examine percent time spent freezing during tone presentations as a measure of fear behavior. For the experiments shown in Figure 4.4-2, 4.4-3, and 4.4A-B, the behavioral protocol of Mariscano et al. was replicated (Xxxxxxxxx, Wotjak, et al. 2002). In Figure 4.4-2 and Figure 4.4-3, Cnr1 wild-type and knockout littermates received one paired conditioned stimulus (CS) tone (180 sec, 9 kHz, 75-80 dB) which co-terminated with an unconditioned stimulus (US) shock (1 sec, 0.7 mA). Three days after fear conditioning, subjects were exposed to one CS trials (180 sec tone) to assess cued fear expression/extinction. One, two, three, and four days after fear expression/extinction training, subjects were again tested to one CS trial (180 sec tone) to assess extinction retention. For the Cnr1 antagonist experiment depicted in Figure 4.4-4A-B, subjects received one day of one paired conditioned stimulus (CS) tone (180 sec, 9 kHz, 75-80 dB) which co-terminated with the unconditioned stimulus (US) shock (1 s, 0.7 mA). Three days after fear conditioning, C57BL/6J mice were injected with vehicle or 3 mg/kg SR141716A, a potent Cnr1 antagonist, 20 minutes prior to cued fear expression/extinction training (one CS trial, 180 s). Subjects were assessed for extinction retention (one CS trial, 180 sec) 24 hours after cued fear expression/extinction training. For experiments shown in Figure 4.4-4C-D, the same behavioral protocol was replicated as in the prior experiments, however the intensity and length of US was reduced to 0.4 mA, 0.5 sec. CS tone (180 sec) was averaged according to 30 second bins for analysis.
