cAMP signaling in the DH is sufficient to rescue retrieval in NE/E-deficient Dbh^−/− mice. The protocol used is that described for Fig. 1, except Dbh^−/− mice were used. These mice exhibit impaired intermediate-term (day 0–day 4 after training) contextual fear memory retrieval (20). (A) The PDE-resistant cAMP analog Sp-2′-O-monobutyryl-cAMPS (a prodrug for Sp-cAMPS) or Sp-8-Br-cAMPS was infused before testing contextual fear memory. The analogs significantly enhanced freezing in the Dbh^−/− mice (n = 6, 5, 6, 5, 4). (B) Sp-MB (2 μg) was infused before testing cued fear memory and was without effect. To generate cued freezing comparable with that observed for contextual freezing in Dbh^−/− mice, a 0.4-mA (instead of 1-mA) shock was used during training (n = 4 per group). (C) Sp-MB (2 μg) was infused before testing contextual fear memory on day 1. The same mice were tested on day 2 without infusion. Sp-MB produced a transient enhancement in freezing on day 1 (n = 5 per group). Values are mean ± SEM; *, P < 0.05; ⋀, P < 0.01.

PKA signaling in the DH is required for intermediate-term contextual fear memory retrieval. The protocol used is that described for Fig. 1, except that CMIQ infusions were performed 15 min before testing, and 10% dimethylformamide in saline (Sal) was used as vehicle for CMIQ. (A) As an antagonist of the ATP-binding site of PKA, CMIQ was infused before testing contextual fear. CMIQ significantly reduced freezing (n = 5 per group). (B) A myristoylated, amidated peptide fragment [14–22] from the PKI protein (myr-PKI[14–22]amide or PKI*) was infused before testing contextual fear. PKI* significantly reduced freezing at 2 and 5 μg (n = 7, 4, 5, 5, 5). (C) PKI* (2 μg) was infused before testing cued fear and was without effect (n = 7, 5). (D) PKI* (2 μg) was infused before testing contextual fear on day 1. The same mice were tested on day 2 without infusion. PKI* produced a transient impairment in freezing on day 1 (n = 5 per group). (E) PKI* (2 μg) was infused before testing contextual fear in separate groups of mice on day 1–day 5 (n = 5, 8, 8, 5, 5). Significant reductions in freezing occurred on day 1–day 3, a nonsignificant reduction in freezing occurred on day 4, whereas day 5 was unaffected, relative to Sal on day 1 (n = 5). Values are mean ± SEM; *, P < 0.05; ⋀, P < 0.01; #, P < 0.001.

Combined treatment with PKA- and Epac-selective agonists in the DH is required to rescue retrieval in NE/E-deficient Dbh^−/− mice. The protocol used is that described for Fig. 1, except Dbh^−/− mice were used. (A) The PDE-resistant PKA-selective agonist Sp-6-Phe-cAMPS (Sp-“PKA”), the PDE-resistant Epac-selective agonist Sp-8-pCPT-2′-O-Me-cAMPS (Sp-“Epac”), or the combination was infused before testing contextual fear. The combination significantly enhanced freezing in the Dbh^−/− mice at doses that had no effect on their own (n = 6, 7, 4, 8, 5, 5, 4, 6, 6, 7, 7). The highest dose (2 μg) of Sp-Epac also significantly enhanced freezing; however, this effect was blocked by the PKA-selective antagonist PKI* (see legend for B). (B) The Epac-selective agonist 8-pCPT-2′-O-Me-cAMP (Epac) was used to replace Sp-Epac from A. Sp-PKA + Epac (0.5 μg each) significantly enhanced freezing in the Dbh^−/− mice, whereas 2 μg of Epac alone had no effect (n = 5, 4, 5). (C) To better detect potential enhancements or reductions in retrieval, a 0.75-mA shock was used during conditioning. The cAMP analogs Sp-PKA, Epac, or the combination (all at 0.5 μg) were infused into the DH of wild-type mice 30 min before testing retrieval. Relative to saline (Sal), none of the infusions had a significant effect on retrieval (n = 5 per group), although the individual agonists tended to enhance freezing modestly. Values are mean ± SEM; *, P < 0.05; ⋀, P < 0.01; #, P < 0.001

cAMP signaling in the DH is required for intermediate-term contextual fear memory retrieval. Dbh^+/− mice, which possess normal tissue levels of NE/E, were used to examine the requirement for cAMP signaling in retrieval. DH infusions were made 1–5 days after training through chronically implanted cannulae 30 min before testing, with isotonic saline (Sal) as vehicle. Freezing was quantitated as an indicator of fear memory. (A) The PDE-resistant cAMP analog Rp-cAMPS, Rp-2′-O-monobutyryl-cAMPS (Rp-2′-O-MB-cAMPS or Rp-MB, a prodrug for Rp-cAMPS), or Rp-8-Br-cAMPS was infused before testing contextual fear memory. The latter two analogs significantly reduced freezing (n = 7, 5, 5, 8, 6, 6, 7 mice for each group, left to right). (B) Rp-MB (5 μg) was infused before testing cued fear memory and was without effect (n = 7 per group). (C) Rp-MB (5 μg) was infused before testing contextual fear memory 1 day after training (D1). The same mice were tested on day 2 without infusion. Rp-MB produced a transient impairment in freezing on day 1, consistent with a block of retrieval (n = 5 for Sal, 6 for Rp-MB). (D) Rp-MB (5 μg) was infused before testing contextual fear memory in separate groups of mice on day 1–day 5 (n = 6, 8, 5, 7). Significant reductions in freezing occurred on day 1–day 3, whereas day 5 was unaffected, relative to Sal on day 1 (n = 5). Sal on day 1 was used as reference because previous studies indicated that freezing is stable across days for Sal-infused mice (20, 24). Values are mean ± SEM; *, P < 0.05; #, P < 0.001.