The similarities and diversities of signal pathways leading to consolidation of conditioning and consolidation of extinction of fear memory

Abstract

It is generally believed that consolidation of long-term memory requires activation of protein kinases, transcription of genes, and new protein synthesis. However, little is known about the signal cascades involved in the extinction of memory, which occurs when the conditioned stimulus is no longer followed by the unconditioned stimulus. Here, we show for the first time that an intra-amygdala injection of transcription inhibitor actinomycin D at the dose that blocked acquisition failed to affect extinction of a learned response. Conversely, protein synthesis inhibitor anisomycin blocked both acquisition and extinction. Extinction training-induced expression of calcineurin was blocked by anisomycin but not by actinomycin D. NMDA receptor antagonist, phosphatidylinositol 3-kinase (PI-3 kinase), and MAP kinase inhibitors that blocked the acquisition also blocked the extinction of conditioned fear. Likewise, PI-3 kinase inhibitor blocked fear training-induced cAMP response element-binding protein (CREB) phosphorylation as well as extinction training-induced decrease in CREB phosphorylation, the latter of which was associated with calcineurin expression and could be reversed by a specific calcineurin inhibitor. Thus, molecular processes that underlie long-term behavioral changes after acquisition and extinction share some common mechanisms and also display different characteristics.

Figure 1.

Intra-amygdala administration of anisomycin, but not actinomycin D, blocks extinction of fear memory. A, Behavioral procedure used in experiment B. B, Bilateral amygdala infusion of anisomycin (125 μg dissolved in 1.6 μl of vehicle; 0.8 μl per side) before light-alone trials blocked extinction of fear memory, whereas actinomycin D (10 μg dissolved in 1.6 μl of vehicle; 0.8 μl per side) was without effect. ^*p < 0.001 versus preextinction tests. C, Cannula tip placements from paired rats infused with vehicle (open circle), anisomycin (open triangle), or actinomycin D (filled diamond). Filled stars represent the tip locations in the unpaired rats given anisomycin.

Figure 2.

Extinction training-induced calcineurin expression is blocked by anisomycin but not by actinomycin D. A, Behavioral procedure used in experiment B. Extinction-trained rats were killed by decapitation immediately after light-alone trials. The LA and BLA were microdissected, and cytosolic calcineurin levels were measured as described in Materials and Methods. B, Calcineurin levels in the extinction-trained rats given drugs or vehicle were normalized to those of unpaired controls. Light-alone trials induced an increased expression of calcineurin in cytosolic (α-actin as internal control) fraction that could be blocked by anisomycin (125 μg dissolved in 1.6 μl of vehicle; 0.8 μl per side). In contrast, actinomycin D (10 μg dissolved in 1.6 μl of vehicle; 0.8 μl per side) was without effect. ^*p < 0.01 versus vehicle. C, Cannula tip placements from paired rats infused with vehicle (open circle), anisomycin (filled circle), or actinomycin D (filled triangle).

Figure 3.

Effects of NMDA and non-NMDA receptors, PI-3 kinase, and MEK inhibitors on the extinction of fear startle. A, Percentage of startle potentiation before (pretest) and after (post-test) three sessions of extinction training in rats given various pharmacological agents before light-alone trials. Extinction of fear memory was blocked by intra-amygdala infusion of d-APV (25 nmol dissolved in 1.6 μl of artificial CSF; 0.8 μl per side), wortmannin (5 μg dissolved in 1.6 μl of DMSO; 0.8 μl per side), or U0126 (2 μg dissolved in 1.6 μl of DMSO; 0.8 μl per side), whereas CNQX (25 nmol dissolved in 1.6 μl of DMSO; 0.8 μl per side) was without effect (n = 6 rats in each group). In addition, a control experiment was conducted in which U0126 was injected into the amygdala of the unpaired rats, and the result showed that this drug did not affect the startle response. ^*p < 0.001 versus pretest. B, Cannula tip placements from paired rats infused with vehicle (open circle), d-APV (filled square), CNQX (filled star), wortmannin (filled circle), or U0126 (filled triangle). Filled diamonds represent the tip locations in the unpaired rats given U0126.

Figure 4.

Effect of extinction training on CREB phosphorylation. A, Representative blots and mean ± SE percentage of pCREB immunoreactivity from unpaired control rats (lane 1), conditioned rats (lane 2), and conditioned rats given light-alone training (lane 3). Fear conditioning resulted in an increase in pCREB, whereas extinction training caused a decrease. Bilateral infusion of FK-506 (10 μg dissolved in 1.6 μl of DMSO; 0.8 μl per side) before extinction trials blocked extinction-induced decrease in pCREB (lane 4). ^*p < 0.01 versus conditioned. B, Time course of calcineurin expression induced by light-alone trials in nuclear fraction. Figure shows the representative blots and mean ± SE of calcineurin immunoreactivities from rats decapitated at various time points (n = 6 rats in each time point) after extinction training (histone used as internal control). ^*p < 0.001 versus conditioned.

Figure 5.

Block of conditioning-induced CREB phosphorylation and extinction-induced dephosphorylation of pCREB by PI-3 kinase inhibitors. A, Behavioral procedure used in experiment. B, Rats were killed 1 hr after preextinction tests, and the nuclear extracts of the amygdala were analyzed. Conditioned rats treated with vehicle exhibited a greater degree of CREB phosphorylation (lane 2) compared with unpaired controls (lane 1). Bilateral infusion of wortmannin (lane 3) or LY 294002 (lane 4) 30 min before training blocked CREB phosphorylation. ^**p < 0.01 versus control. C, Behavioral procedure used in experiment D. D, Extinction-induced dephosphorylation of pCREB is blocked by PI-3 kinase inhibitor. Rats were given an intra-amygdala injection of wortmannin (5 μg dissolved in 1.6 μl of DMSO; 0.8 μl per side) or vehicle before receiving extinction trials. Immunoblotting for pCREB from nuclear extract was performed 20 min after extinction training. In contrast to those of the control and vehicle groups, extinction trials did not cause CREB dephosphorylation in wortmannin-treated animals (n = 4 rats in each group). ^*p < 0.05 versus vehicle.

Figure 6.

A model of consolidation of conditioning and consolidation of extinction of memory in the amygdala. Both acquisition and extinction trigger calcium influx through either NMDA receptors or L-type calcium channels in the amygdala. The increase in intracellular Ca²⁺ leads to the activation of protein kinases (e.g., PKA, PI-3 kinase, and MAPK). The activated kinases then translocate into the nucleus in which they phosphorylate CREB to initiate gene transcription and translation. Extinction training not only reactivates original memory this way but also promotes calcineurin synthesis via the existed mRNA. Calcineurin then exerts a negative feedback effect to dephosphorylate kinases and weakens the original memory. Thus, the outcome of the effect of protein synthesis inhibition on the reconsolidation-extinction may depend on a competition between these two processes.