Figure 6. CGRP^SPFp→LA and CGRP^PBel→CeA circuits relay unconditioned stimulus information and are crucial for aversive memory formation during Pavlovian threat conditioning.

(A) Heatmap of Z-scored CGRP^SPFp single-cell calcium activity during threat conditioning. n = 81 (habituation), n = 90 (conditioning), and n = 93 cells (cue test) from three mice. Each row represents the averaged value during three to six stimulus presentations in a single cell. White vertical lines indicate the onsets of tone or foot shock used for (B).

(B) Left: averaged peri-stimulus activity of all recorded CGRP^SPFp neurons. Stimulus onset is indicated by white vertical lines in (A). Right: area under the curve (AUC) of all recorded CGRP^SPFp neurons. AUCs were calculated from 0 to 5 s after stimulus onset. CGRP^SPFp neurons were activated by the unconditioned stimulus (foot shock) and not by the conditioned stimulus (non-aversive tone) during habituation or cue tests. n = 81 (habituation), n = 90 (conditioning), and n = 93 cells (cue test) from three mice.

(C and D) During the optogenetic threat-conditioning test wherein CGRP^SPFp→LA circuit photostimulation was used as an unconditioned stimulus (US), the ChR2 group displayed higher freezing levels during both the context (C) and cue (D) retrieval tests than EYFP controls. n = 13 (EYFP), n = 10 (ChR2) mice. “BL” in the cue test indicates freezing during baseline before the first tone was delivered, and “CS+” is the average freezing during the three tones.

(E and F) Inhibiting the CGRP^SPFp→LA circuit with PPO during threat conditioning reduced freezing level in the context (E) and cue retrieval tests (F). n = 8 (EYFP), n = 7 (PPO) mice.

(G) Heatmap of Z-scored CGRP^PBel single-cell calcium activity during threat conditioning. n = 56 (habituation), n = 50 (conditioning), and n = 59 cells (cue test) from three mice. White vertical lines indicate the onsets of tone or foot shock used for (H).

(H) Same design as in (B). CGRP^PBel neurons were activated by the unconditioned stimulus (foot shock), but not by the cues during either habituation or cue tests. n = 56 (habituation), n = 50 (conditioning), and n = 59 cells (cue test) from three mice.

(I and J) During the optogenetic threat-conditioning test wherein CGRP^PBel→CeA circuit photostimulation was used as a US, the ChR2 group displayed higher freezing levels during both the context (I) and cue (J) retrieval tests than EYFP controls. n = 5 (EYFP), n = 6 (ChR2) mice.

(K and L) Inhibiting the CGRP^PBel→CeA circuit with ArchT during threat conditioning reduced freezing behavior in the cue retrieval test (L) but not the context test (K). n = 6 (EYFP), n = 6 (ArchT) mice.

Data are presented as mean ± SEM. Statistical analyses were performed using Kruskal-Wallis one-way ANOVA on ranks with Dunn’s multiple comparisons (B and H), two-tailed unpaired t tests (C, E, I, and K), or repeated-measures two-way ANOVA with post hoc Sidak’s multiple comparison (D, F, J, and L); see also Table S5 for statistical details. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S7-S9.