Figure 4. Optogenetic stimulation of CeACAM-LPBN projections in behaving rats reduces nocifensive responses and hampers aversive learning involving a noxious unconditioned stimulus. A, Experimental design for examining nocifensive responses to mechanical (von Frey) and thermal (Hargreaves) stimulation of the hindpaws. Rats received bilateral intra-CeA injections of either AAV-ChR2 or the control vector AAV-mCherry, and optic fibers were implanted above the LPBN. In each session, each paw was stimulated with laser stimulation off or on (intermingled trials). On the last day of testing (day 0), the test session was preceded by a capsaicin injection into the left hindpaw. B, C, Time course of von Frey experiment for mechanical sensitivity, for the AAV-ChR2 group (B) and for the AAV-mCherry group (C). Measurements were taken over 3 d. The red arrowhead indicates capsaicin injection into the left hindpaw. D, The mean 50% paw withdrawal threshold was significantly higher in the laser on condition as compared with the laser off condition in the AAV-ChR2 group (***p < 0.0001), but not in the AAV-mCherry group (p = 0.13). E, F, Time course of Hargreaves experiment for heat sensitivity in the AAV-ChR2 group (E) and in the AAV-mCherry control group (F). Measurements were taken over 3 d. The red arrowhead indicates capsaicin injection into the left hindpaw. G, On average, paw withdrawal latencies were significantly higher in the laser on condition as compared with the laser off condition in the AAV-ChR2 group (***p = 0.0004), but not in the AAV-mCherry group (p = 0.35). H, Experimental design for examining the effect of CeACAM-LPBN stimulation on unconditioned and conditioned freezing in a threat conditioning paradigm. In the conditioning session, a tone-CS (2 kHz at 70 dB, for 10 s) was paired with a co-terminating electrical shock-US (1 mA for 0.5 s; 2 pairings, 5-min intertrial interval). To inhibit shock-evoked LPBN responses in the AAV-ChR2 group, laser stimulation was delivered to the LPBN for 2 s in each trial, beginning 1 s before shock onset. On the next day, animals underwent a “cue test” session, in which they were placed in a novel chamber, and the tone-CS was presented for three consecutive minutes. The % of time rats spent freezing following shocks was used as a measure of shock aversiveness, while the % of time spent freezing during tone presentation was used as a measure of the previously-acquired aversive learning. I, Both groups showed similar amount of freezing following shock presentations (conditioning trials 1 and 2; p = 0.86 and p = 0.3, respectively). During the cue test, rats in the AAV-ChR2 group showed significantly less conditioned freezing compared with the AAV-mCherry group (**p = 0.009; n = 15 rats per group). D, G, I, n.s. = non significant.