MicroRNA-182 regulates amygdala-dependent memory formation

Abstract

De novo protein synthesis supports long-lasting functional and structural plasticity and is a molecular requirement for new memory formation. Recent evidence has suggested that microRNAs may be involved in regulating the molecular mechanisms underlying neural plasticity. MicroRNAs are endogenous, noncoding RNAs capable of post-transcriptional repression of their mRNA targets. To explore the potential for microRNA-mediated regulation of amygdala-dependent memory formation, we performed expression profiling of microRNAs in the lateral amygdala of rats 1 h after auditory fear conditioning. Microarray analysis revealed that over half of all known microRNAs are endogenously expressed in the lateral amygdala, with 7 microRNAs upregulated and 32 downregulated by auditory fear training. Bioinformatic analysis identified several of the downregulated microRNAs as potential repressors of actin-regulating proteins known to be involved in plasticity and memory. Downregulation of one of these microRNAs by auditory fear conditioning, miR-182, was confirmed by quantitative real-time PCR. Overexpression of miR-182 within the lateral amygdala resulted in decreased expression of the protein but not mRNA of two synapse-enriched regulators of actin known to modulate structural plasticity, cortactin and Rac1. The overexpression of miR-182 also disrupted long-term but not short-term auditory fear memory. These data indicate that learning-induced suppression of miR-182, a microRNA previously uncharacterized in the brain, supports long-term memory formation in the amygdala and suggests it does so, at least in part, through the derepression of key actin-regulating proteins. These findings further indicate that microRNAs may represent a previously underappreciated mechanism for regulating protein synthesis during memory consolidation.

Figure 1.

Auditory fear conditioning downregulates miR-182 in the LA. A, Schematic of experimental design. B, Representation of the miRNAs on the GeneArray Chip that are basally expressed in the LA of rats. C, Differential expression of the 206 miRNAs expressed in the LA 1 h after training, relative to Naive and Unpaired controls. D, 1 h post-training expression of miRNAs specifically predicted to have ARP targets. Color scale represents miRNA expression levels normalized to Naive. Gray shading represents miRNAs below detection in the LA. E, Predicted ARP targets of each miRNA depicted in D.

Figure 2.

Auditory fear conditioning decreases miR-182 at 1 h and 24 h post-training A, qRT-PCR validation of microarray results for miR-30c and miR-182 expression 1 h post-training, normalized to U6. Difference between Paired and Naive (#) or Unpaired (*) animals is shown, p < 0.05. B, qRT-PCR quantification of miR-182 levels 24 h post-training, normalized to U6. Difference between Paired and Naive (#) or Unpaired (*) animals, p < 0.05. C, Representative qRT-PCR trace of miR-30c (a) and miR-182 (b) expression levels from same Naive LA sample. Error bars represent ± SEM.

Figure 3.

Overexpression of miR-182 in the LA represses cortactin and Rac1 expression following auditory fear conditioning A, Relative change in miR-182 levels 48 h after intra-LA infusion of rno-miR-182OE in Naive animals, normalized to miR-182 levels in cel-miR-67OE control. B, Schematic of experimental design for effect of rno-miR-182OE on ARPs. C, Western blot analysis of miR-182-predicted ARPs normalized to GAPDH and intra-LA cel-miR-67OE controls (Cortactin, *p < 0.0005; Rac1, ^#p < 0.01). D, qRT-PCR analysis of Cortactin and Rac1 mRNA levels, normalized to GAPDH and intra-LA cel-miR-67OE controls. Error bars represent ± SEM.

Figure 4.

Overexpression of miR-182 in the LA disrupts long-term memory formation. A, Schematic of LTM experimental design. B, C, Intra-LA rno-miR-182OE disrupts long-term memory formation (*p < 0.005) (B) without affecting immediate learning (C). D, Schematic of STM experimental design. E, Intra-LA rno-miR-182OE does not affect short-term memory formation. F, Location of needle tips for intra-LA infusions. Because of overlap, not all needle tip locations are resolvable in the figure. Error bars represent ± SEM.

Figure 5.

Proposed model of miR-182's role within the amygdala during the formation of auditory fear memory.