Figure 3. Differential roles of dSub projections in hippocampal memory retrieval and retrieval-induced stress hormone responses.

(A, B) FN1-Cre mice were injected with a Cre-dependent virus expressing eArch3.0-eYFP into dSub. Optogenetic inhibition of dSub neurons during contextual fear conditioning (CFC) training had no effect on long-term memory (n = 12 mice per group; A). Inhibition of dSub neurons during CFC recall impaired behavioral performance (n = 12 mice per group; B). A two-way ANOVA followed by Bonferroni post-hoc tests revealed a behavioral epoch-by-eArch interaction and significant eArch-mediated attenuation of freezing (A–B: F_1,44 = 5.70, P < 0.05, recall). For dSub optogenetic manipulation experiments, injections were targeted to dSub cell bodies and the extent of virus expression is shown in Figures 1E–1K.

(C) Terminal inhibition of dSub projections to EC5 (bottom left), but not MB (bottom right), disrupted CFC memory recall (n = 11 mice per group). A two-way ANOVA followed by Bonferroni post-hoc tests revealed a dSub terminal-by-eArch interaction and significant eArch-mediated attenuation of freezing (F_1,40 = 7.63, P < 0.01, dSub→EC5 terminals).

(D) FN1-Cre mice were injected with a Cre-dependent virus expressing ChR2-eYFP into dSub. Optogenetic activation of dSub→EC5 terminals during CFC memory recall increased freezing levels (left), which was not observed in a neutral context (middle) or using no shock mice (right, n = 10 mice per group).

(E) Inhibition of dSub→EC5 terminals during trace fear conditioning (TFC) recall decreased tone (Tn)-induced freezing levels (n = 12 mice). A two-way ANOVA followed by Bonferroni post-hoc tests revealed a behavioral epoch-by-eArch interaction and significant eArch-mediated attenuation of freezing (E and Figure S4A: F_1,44 = 7.11, P < 0.05, recall). Pre-tone baseline freezing (Pre). Recall-induced freezing levels during individual tone presentations (left panel), averaged freezing levels during the two light-off tones and the two light-on tones (right panel).

(F) Inhibition of dSub→EC5 terminals during cocaine-induced conditioned place preference (CPP) recall impaired behavioral performance (n = 14 mice per group). Behavioral schedule (left, top part). Average heat maps showing exploration time during pre-exposure and recall trials (left, bottom part). Dashed white lines demarcate individual zones in the CPP apparatus. Pre-exposure preference duration (right, top graph) and recall preference duration (right, bottom graph). Saline (S or Sal), cocaine (C or Coc). A two-way ANOVA followed by Bonferroni post-hoc tests revealed a drug group-by-eArch interaction and significant eArch-mediated attenuation of preference duration (F_1,52 = 5.16, P < 0.05, cocaine). For CPP training inhibition, see Figure S4F. NS, not significant.

(G) Stress hormone: Terminal inhibition of dSub projections to MB, but not EC5, following CFC memory recall tests decreased stress responses as measured by corticosterone (CORT) levels. Optogenetic activation of dSub→MB terminals following CFC memory recall increased CORT levels (n = 10 mice per group). Context (ctx). CORT levels in CPP paradigm are shown in Figure S4H.

Unless specified, statistical comparisons are performed using unpaired t tests; *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM.