Extinction of remotely acquired fear depends on an inhibitory NR2B/PKA pathway in the retrosplenial cortex

Abstract

As memories age, their processing increasingly relies upon cortical rather than hippocampal circuits, but the adaptive significance and mechanisms of this shift are not fully understood. Here we investigated the behavioral features and cortical mechanisms underlying extinction of remotely versus recently acquired context fear in mice. Behaviorally, extinction and reinstatement were similar, but re-extinction of remote fear was significantly faster, suggesting time-dependent engagement of mechanisms specific for processing remote memory. Using pharmacological manipulations of NMDA receptors and associated signaling pathways in the in the retrosplenial cortex, we demonstrated that extinction of remote fear uniquely required NR2B-mediated downregulation of the cAMP-dependent protein kinase (PKA)/cAMP response element-binding protein pathway. Interestingly, NR2B/PKA interactions weakened independently of the age of the memory, but the functional significance of this molecular change was evident only as memory retrieval became PKA-dependent over time. Thus, cortical PKA signaling may provide a molecular signature of when a memory has become "remote," and inhibition of this pathway may open the door for modulation of remote memories.

Figure 1.

Facilitated re-extinction of remote context fear. A, Design of the experiment in B. Extinction of recent and remote context fear began 1 d (recent) or 35 days (remote) postconditioning. B, Extinction of remote, but not recent, context fear is resistant to fear relapse. Mice in the recent and remote groups extinguished fear to the conditioning context at similar rates and to a similar extent. After receiving a strong footshock (arrow), both groups demonstrated similar levels of fear reinstatement (R). After reinstatement, however, re-extinction was facilitated in the remote group; *p < 0.01.

Figure 2.

NR2B subunit-containing NMDAR in RSC mediate remote context fear extinction. A, Extinction of recent context fear was unaffected by infusion of either the NR2A-preferring antagonist NVP or NR2B-specific antagonist Ro (left). In contrast, Ro, but not NVP, blocked extinction of remote fear (right). B, Cannula placements in RSC. The photomicrograph shows a cresyl violet-stained coronal section with representative bilateral cannula placements in RSC. Illustrated below that are the locations of individual cannula placements. Atlas templates adapted from Paxinos and Franklin (2001). C, Recent fear extinction was prevented by inactivation of RSC via the GABAa receptor agonist MUS. D, Extinction of remotely acquired fear was unaffected by infusion of the AMPAR antagonist CNQX. All arrows indicate post-extinction infusions; *p < 0.05 from the Veh group.

Figure 3.

NR2B blockade during remote extinction leads to increased pCREB. A, After 3 d of extinction training, we found robust immunostaining for NR2B and pCREB, but not pERK, in RSC. pERK was highly expressed in ACC in the same brain. Arrowheads point to regions of dense immunostaining. Scale bar, 250 μm. B, Consistent with our previous results, infusion of Ro into RSC blocked remote fear extinction. Ro also led to an increase in pCREB+ cells relative to vehicle controls. Arrows indicate post-extinction infusions; *p < 0.05.

Figure 4.

NR2B/PKA interactions in RSC are reduced after both recent and remote fear extinction. A, Coimmunoprecipitation of NR2B and PKA revealed decreased NR2B/PKA complexes after recent and remote extinction; *p < 0.01. B, Validation of the coimmunoprecipitation approach, demonstrating a strong PKA band after incubation of membrane lysates with NR2B antibody but not IgG. C, Representative immunoblots showing significant reductions in NR2B/PKA complexes after recent and remote fear extinction.

Figure 5.

Retrieval and extinction of recently acquired fear do not require changes in pCREB or PKA-dependent signaling. A, Top, Freezing levels during the final extinction session for each group. Bottom, Although freezing levels were much lower for the day 6 group, the number of pCREB+ cells was similar among naive, day 3, and day 6 groups; *p < 0.01. B, Representative sections showing immunostaining of pCREB+ cells in layer 2/3 of RSC. Scale bar, 125 μm. C, Inhibition of PKA via Rp-cAMPs had no effect on retrieval of recent fear memory. D, PKA inhibition had no effect on extinction of recently acquired fear. Arrows indicate post-extinction infusions.

Figure 6.

Retrieval and extinction of remotely acquired fear are mediated by increased and decreased PKA activity and CREB phosphorylation, respectively. A, Top, Freezing levels during the final extinction session for each group. Bottom, the number of pCREB+ cells in RSC was increased relative to naive mice after 3 d, and returned to baseline levels after 6 d, of extinction training, mirroring the differences in freezing between the day 3 and day 6 groups. B, Representative sections showing immunostaining of pCREB+ cells in layer 2/3 of RSC. Scale bar, 125 μm. C, Inhibition of PKA via Rp-cAMPS blocked retrieval of remote fear memory. D, Inhibition of PKA facilitated extinction of remotely acquired fear. E, Activation of PKA via 8-Br-cAMPs blocked remote extinction. F, Remote extinction was unaffected by infusions of the β-receptor antagonist nadolol or the D1 receptor antagonist SCH23390. Arrows indicate post-extinction infusions; *p < 0.05.