Brain region-specific gene expression activation required for reconsolidation and extinction of contextual fear memory

Abstract

During fear conditioning, animals learn an association between a previously neutral or conditioned stimulus (CS) and an aversive or unconditioned stimulus (US). Subsequent reexposure to the CS alone triggers two competing processes. Brief reexposure to the CS initiates reconsolidation processes that serve to stabilize or maintain the original CS-US memory. In contrast, more prolonged reexposure to the CS leads to the formation of an inhibitory extinction (CS-no US) memory. Previous studies have established that both reconsolidation and extinction require gene expression. Consistent with this, here we first show that genetic disruption of cAMP-responsive element-binding protein (CREB)-mediated transcription blocks both reconsolidation and long-term extinction of contextual fear memory. We next asked whether reconsolidation and extinction engage CREB-mediated transcription in distinct brain regions. Accordingly, we used immunohistochemical approaches to characterize the activation of the transcription factor CREB [as well as the expression of the CREB-dependent gene Arc (activity-regulated cytoskeleton-associated protein)] after brief versus prolonged reexposure to a previously conditioned context. After brief reexposure, we observed significant activation of CREB-mediated gene expression in the hippocampus and amygdala. In contrast, after the prolonged reexposure, we observed significant activation of CREB-mediated gene expression in the amygdala and prefrontal cortex. Finally, we showed that blocking protein synthesis in either the hippocampus or the amygdala blocked reconsolidation of contextual fear memory, whereas similar blockade in the amygdala and prefrontal cortex prevented the formation of extinction memory. These experiments establish that reactivated contextual fear memories undergo CREB-dependent reconsolidation or extinction in distinct brain regions.

Figure 1.

Effects of disrupting CREB function on memory reconsolidation and extinction. A, C, Experimental design used with data presented below. A, B, Effects of disrupting CREB function on reconsolidation. A, Freezing score during 3 min reexposure (WT/VEH, n = 13; CREB^IR/VEH, n = 11; WT/TAM, n = 13; CREB^IR/TAM, n = 11). B, Freezing score during test session. C, D, Effects of disrupting CREB function on extinction. C, Freezing score in 5 min blocks during 30 min reexposure (WT/VEH, n = 12; CREB^IR/VEH, n = 15; WT/TAM, n = 14; CREB^IR/TAM, n = 15). D, Freezing score during test session were shown. E, Summary of the relationship between the duration of reexposure and freezing score at test session (B, D). Error bars are SEM. *p < 0.05, versus WT/VEH, WT/TAM, and CREB^IR/VEH.

Figure 2.

Experimental design. The experimental design to investigate changes in CREB activation in mPFC, hippocampus, and amygdala induced by reconsolidation or extinction phases is shown.

Figure 3.

Effects of the reexposure duration on CREB activation in the mPFC. A, Representative PL pCREB-positive cell immunohistochemistry obtained from indicated mice. Scare bar, 100 μm. B, C, Effects of the 3 min reexposure duration on CREB activation in PL (B) and IL (C) regions of mPFC (Recon-33, n = 11; no US-33, n = 10; no association-33, n = 9; Recon-60, n = 9). D, E, Effects of the 30 min reexposure duration on CREB activation in PL (D) and IL (E) regions of mPFC (Ext-33, n = 10; no US-60, n = 15; no association-60, n = 14; Ext-60, n = 17). *p < 0.05, compared with the other groups. F, G, Comparison of CREB activation after 3 and 30 min reexposure in PL (F) and IL (G) regions of mPFC in the conditioned group. Error bars are SEM. CREB activation (relative pCREB/CREB levels) was calculated by normalizing the number of pCREB-positive cells to the total CREB positive cells. Data of CREB activation for each group were expressed as the percentage of the averaged values in the no US-33 control group.

Figure 4.

Effects of the reexposure duration on CREB activation in the hippocampus. A, Representative CA1 pCREB-positive cell immunohistochemistry obtained from indicated mice. Scare bar, 100 μm. A–D, Effects of the 3 min reexposure duration on CREB activation in CA1 (B), CA3 (C) and DG (D) regions of hippocampus (Recon-33, n = 11; no US-33, n = 10; no association-33, n = 9; Recon-60, n = 9). *p < 0.05, compared with the unconditioned groups (no US-33 and no association-33). E–G, Effects of the 30 min reexposure duration on CREB activation in CA1 (E), CA3 (F) and DG (G) regions of hippocampus (Ext-33, n = 10; no US-60, n = 15; no association-60, n = 14; Ext-60, n = 17). H–J, Comparison of CREB activation after 3 and 30 min reexposure in CA1 (H), CA3 (I), and DG (J) regions of hippocampus in the conditioned group. Error bars are SEM. CREB activation was calculated by normalizing the number of pCREB-positive cells to the total CREB-positive cells. Data of CREB activation for each group were expressed as the percentage of the averaged values in the no US-33 control group.

Figure 5.

Effects of the reexposure duration on CREB activation in the amygdala. A, Representative BLA pCREB-positive cell immunohistochemistry obtained from indicated mice. Scare bar, 100 μm. B–D, Effects of the 3 min reexposure duration on CREB activation in LA (B), BLA (C), and Ce (D) regions of amygdala (Recon-33, n = 11; no US-33, n = 10; no association-33, n = 9; Recon-60, n = 9). *p < 0.05, compared with the unconditioned groups (no US-33 and no association-33). E–G, Effects of the 30 min reexposure duration on CREB activation in LA (E), BLA (F) and Ce (G) regions of amygdala (Ext-33, n = 10; no US-60, n = 15; no association-60, n = 14; Ext-60, n = 17). *p < 0.05, compared with the other groups. H–J, Comparison of CREB activation after 3 and 30 min reexposure in LA (H), BLA (I), and Ce (J) regions of amygdala in the conditioned group. Error bars are SEM. CREB activation was calculated by normalizing the number of pCREB-positive cells to the total CREB-positive cells. Data of CREB activation for each group were expressed as the percentage of the averaged values in the no US-33 control group.

Figure 6.

Effects of the duration of reexposure on Arc expression in the mPFC, amygdala, and hippocampus. A, B, Experimental design used with data presented below. A, Effects of the 3 min reexposure duration on Arc expression in the mPFC, amygdala, and hippocampus (reconsolidation, n = 10; control, n = 9). *p < 0.05, compared with the control group. B, Effects of the 30 min reexposure duration on Arc expression in mPFC, amygdala, and hippocampus (extinction, n = 11; control, n = 11). *p < 0.05, compared with the control group. Error bars are SEM. Data of Arc expression for each group were expressed as the percentage of the averaged values in the control group. Scale bar, 100 μm.

Figure 7.

Effects of protein synthesis inhibition in the mPFC, hippocampus, or amygdala on reconsolidation and extinction. A, Experimental design used with data. Mice were infused VEH or ANI into the mPFC, hippocampus, or amygdala immediately after 3 or 30 min reexposure. B, Effects of protein synthesis inhibition in mPFC, hippocampus, or amygdala on reconsolidation. Freezing scores during the test session are shown (mPFC: VEH, n = 10; ANI, n = 11; hippocampus: VEH, n = 12; ANI, n = 12; amygdala: VEH, n = 9; ANI, n = 9). C, Effects of protein synthesis inhibition in mPFC, hippocampus, or amygdala on extinction. Freezing scores during test session are shown (mPFC: VEH, n = 9; ANI, n = 9; hippocampus: VEH, n = 10; ANI, n = 10; amygdala: VEH, n = 8; ANI, n = 9). During reexposure, freezing scores in 5 min blocks are presented. Error bars are SEM. *p < 0.05 versus VEH-treated groups.