Correct placement of the cannulae and fibers was verified by injections of fluorescent beads and post hoc analysis. Based on incorrect positioning, three rats (in which BL, as a consequence, did not decrease freezing responses) were excluded from further analysis. (Figure 5A, see also Experimental Procedures). To ensure basic activation of the amygdala during the behavioral Entinostat ic50 experiments, we trained all rats in a 2-day contextual fear-conditioning

protocol (see Figure 5B and Experimental Procedures) that resulted in similar freezing in the majority of animals (n = 25) after 2 days of conditioning (Figures 5C1 and 5D1). One animal was excluded from the experiment due to unusually low freezing levels. Hormonal cycle did not appear to affect these freezing levels (Figure S5). To assess acute effects of BL on freezing behavior, we placed rats on day 3 in the fear-conditioning box after optic fibers had been inserted through the guide cannulae to target the CeL. All rats exhibited maximal freezing upon and throughout exposure

to the context (Figure 5C1). After 10 min, 10 ms, 30 Hz BL pulses were given for either 20 or 120 s. As expected from the central role of the CeM in freezing behavior (Ciocchi et al., 2010 and Haubensak et al., 2010) and the inhibitory effects of BL on the CeM in vitro (Figure 4), BL efficiently decreased freezing (from 57.5 ± 0.9 to 32.1 ± 5.6 s/min, n = 6; one-way ANOVA, p < 0.05; Figure 5C1). The onset of see more this decrease (Figure 5C2; see also Movie S1) started in two rats as

fast as 2 s after BL onset and on average with a delay time of 21.5 ± 9.7 s across all animals (n = 6). Freezing returned after 70 ± 21 s upon termination of the 20 s BL stimulation and 108 ± 20 s after the 120 s BL exposure (n = 3 per group). The inhibiting effects of BL appeared specific to the fear-induced freezing response, because BL exposure in the same animals in a non-fear-conditioning context did not affect basic locomotor activity (Figure 5C3). To confirm involvement of endogenous OT release in these BL responses, we injected OTA on day 3 through the same guide cannulae through which the optic fibers were subsequently inserted and applied BL immediately very for 120 s before the rats were re-exposed (after removal of the optic fibers) to the fear-conditioning context. We thus measured the remaining block on the effects of BL by OTA, while at the same time providing more freedom of movement to the rats (now unobstructed by any attached optic fibers). We compared freezing behavior between four groups of rats, namely “Ctrl” (no BL, but optic fibers inserted prior to testing), “OTA” (OTA injected + optic fibers without BL), “BL” (BL application prior to exposure to context) and “OTA + BL” (injection of OTA followed by BL application prior to exposure to context). Ctrl or OTA-injected rats exhibited high freezing levels (Figure 5D2) comparable to those measured previously (Figure 5C1).