Figure 4.

BLA Neuronal Downregulation Induces Passive Defensive Reactions upon Imminent Threat

(A) Virus injection site and expression of hM4D revealed by mCherry immunohistochemistry. Scale bar, 200 μM.

(B) (Left) Example traces of action potentials fired during 400-ms incremental current injections (from −100 pA to 300 pA; red trace representing 200 pA; see inset) before (top) and during CNO treatment (bottom). (Right) Mean action potential frequency as function of current injected before CNO (black) and during CNO (purple) is shown.

(C) Experimental design for the TET and ASR assessment: day 1, TET conditioning; day 2, TET testing with vehicle (n = 6, gray) or CNO (n = 7, purple) intraperitoneal (i.p.) injected in the BLA 30 min prior to (1) exposure in threat and escape task (TET) to distant (4 kHz, yellow), imminent (12 kHz, orange), or inescapable (12 kHz, red; in separate experiment) threat. (i) “No escape” illustrates shuttling only upon foot shock exposure; (ii) “escape” illustrates avoidance of foot shock by shuttling before end of tone. (2) Acoustic startle response measured after exposure to 4- or 12-kHz tones followed by white noise burst on day 8 (ASR habituation), followed on day 9 by TET recall, and on day 10 by ASR 30 min after vehicle (gray) or CNO (purple) i.p. injection.

(D and E) CNO (purple) as compared to vehicle (gray) i.p. injections in hM4D-infected rats that were exposed to imminent threat (D) reduced escape responses (two-way ANOVA: treatment × threat imminence; F_{(1, 22) =} 19.48; p < 0.001) and (E) increased freezing levels (two-way ANOVA: treatment effect; F_{(1, 22) =} 16.69; p < 0.001). Imminent threat induces significantly more freezing compared to distant threat in rats injected with CNO compared to Veh (imminence effect F_{(1, 22)} = 5.06; p < 0.05). CNO has no effects on freezing after inescapable threats.

(F) CNO enhanced the potentiation of the startle reflex (ASR; two-way ANOVA: treatment × threat imminence; F_{(1, 20)} = 10.21; p < 0.01; n = 6 each group) that occurs upon exposure to imminent, but not to distant or inescapable, threats. Data from TET test conducted under naive conditions and vehicle treatments were pooled and converted to percent escape behavior and were normally distributed (D’Agostino and Pearson normality test; for distant threat, K₂ = 5.40, p = 0.06; for imminent threat, K₂ = 1.11, p = 0.57). ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Error bars represent standard error of the mean. See also Figures S2 and S3.