Epac activation initiates associative odor preference memories in the rat pup

Abstract

Here we examine the role of the exchange protein directly activated by cAMP (Epac) in β-adrenergic-dependent associative odor preference learning in rat pups. Bulbar Epac agonist (8-pCPT-2-O-Me-cAMP, or 8-pCPT) infusions, paired with odor, initiated preference learning, which was selective for the paired odor. Interestingly, pairing odor with Epac activation produced both short-term (STM) and long-term (LTM) odor preference memories. Training using β-adrenergic-activation paired with odor recruited rapid and transient ERK phosphorylation consistent with a role for Epac activation in normal learning. An ERK antagonist prevented intermediate-term memory (ITM) and LTM, but not STM. Epac agonist infusions induced ERK phosphorylation in the mitral cell layer, in the inner half of the dendritic external plexiform layer, in the glomeruli and, patchily, among granule cells. Increased CREB phosphorylation in the mitral and granule cell layers was also seen. Simultaneous blockade of both ERK and CREB pathways prevented any long-term β-adrenergic activated odor preference memory, while LTM deficits associated with blocking only one pathway were prevented by stronger β-adrenergic activation. These results suggest that Epac and PKA play parallel and independent, as well as likely synergistic, roles in creating cAMP-dependent associative memory in rat pups. They further implicate a novel ERK-independent pathway in the mediation of STM by Epac.

Figure 1.

Epac activation is a sufficient UCS to generate STM and LTM. (A) Olfactory bulb infusion of 8-pCPT (Epac agonist) 20 min before odor exposure generates 24-h LTM even in the absence of noradrenergic activation. No inverted U-curve response occurred with different concentrations of the drug, which was also seen with the activation of another cAMP target, PKA, as the UCS (Grimes et al. 2012). (B) Intrabulbar activation of Epac (5 µg 8-pCPT) is sufficient to produce 3-h STM. (C) Activation of Epac (5 µg 8-pCPT) 20 min before training resulted in odor-specific learning rather than generalized preference to a presented odor. That is, pups trained with one odor demonstrated the ability to discriminate between the learned and nonlearned odors 24 h later. Data are expressed as mean ± SEM. (**) P < 0.01, (*) P < 0.05. The odor preference test required a choice of either odor (peppermint- or orange-scented bedding) or no odor (normal-scented bedding). n values are shown within the column for each group.

Figure 2.

Immunohistochemistry was used to visualize pERK or pCREB expression in the olfactory bulb. Unilateral infusion of 8-pCPT (Epac agonist) into the olfactory bulb 20 min prior to odor training results in increased pERK in several layers (A). The external plexiform layer was measured and found to be significantly darker than vehicle side (B). Increased pCREB activation was also observed in several layers (C) and density measurements within the mitral cell layer was significant (D) compared with the vehicle treated olfactory bulb. Large arrows indicate sites of the cannula track. Data in B and D are expressed as mean ± SEM. Paired t-tests were used to evaluate differences in relative optical density staining between vehicle- and 8-pCPT-infused olfactory bulbs. (**) P < 0.01. (epl) External plexiform layer, (gcl) granule cell layer, (gl) glomerular layer, (mcl) mitral cell layer, (onl) olfactory nerve layer.

Figure 3.

Odor preference learning causes immediate activation of ERK that decays quickly, while nonlearning animals demonstrate two periods of ERK activation. (A,B) The phosphorylation of ERK1 increases immediately after learning and remains elevated for 10 min in learning animals (2 mg/kg Iso + odor), after which it returns to normal control levels. However, in nonlearning animals (6 mg/kg Iso + odor) the phosphorylation of ERK1 increases at two separate time periods. It increases immediately after learning and remains elevated for 10 min after which it returns to normal control levels, but then becomes elevated again 1 h after learning. (C,D) The phosphorylation of ERK2 increases immediately after learning and returns to normal control levels 10 min later in learning animals. However, in nonlearning animals the phosphorylation of ERK2 increases at two separate time periods. It increases immediately after learning and remains elevated for 10 min after which it returns to normal control levels, but then becomes elevated again 1 h after learning. (E,F) A representative immunoblot of phosphorylated ERK1/2 (1:1000, Cell Signaling) under learning (2 mg/kg Iso + odor) and nonlearning (6 mg/kg Iso + odor) conditions. (G,H) A representative immunoblot of total ERK1/2 (1:1000, Cell Signaling) under learning and nonlearning conditions demonstrates no change in total ERK between conditions and across all time points. n = 5 for all groups. Data are expressed as phosphorylated ERK normalized to total ERK and as percentage relative to saline nonlearning control ran in the same experiment. Error bars are SEM. (*) P < 0.05, (**) P < 0.01.

Figure 4.

The activation of bulbar ERK is required for 5-h ITM and 24-h LTM, but not 3-h STM. (A) Olfactory bulb infusion of the ERK inhibitor U0126 before learning does not affect 3-h STM formation. This ERK inhibition had no effect on learning (2 mg/kg Iso + odor) resulting in a normal inverted U-curve response to isoproterenol (noradrenergic) activation. (B) Olfactory bulb infusion of U0126 before learning causes the inhibition of 5-h ITM formation. This ERK inhibition disrupted normal learning (2 mg/kg Iso + odor) resulting in ITM being reduced to nonlearning control levels. (C) Olfactory bulb infusion of U0126 before learning also caused the inhibition of 24-h LTM formation. The inhibition of bulbar ERK before learning disrupts normal learning (2 mg/kg Iso + odor) and inhibits LTM formation. However, 24-h LTM is restored with increased β-adrenoceptor activation (6 mg/kg Iso) suggesting an alternate pathway is involved. (D) When both ERK and PKA pathways are blocked (using U0126 and Rp-cAMPs, respectively) before learning, 24-h memory is blocked with either normal (2 mg/kg Iso) or supraoptimal (6 mg/kg Iso) noradrenergic activation at training. This suggests that when alternate pathways are blocked, learning is difficult or not possible to achieve. Data are expressed as mean ± SEM. (**) P < 0.01. n values are shown within the columns.