Extracellular signal-regulated kinase 2 (ERK2) knockdown mice show deficits in long-term memory; ERK2 has a specific function in learning and memory

Abstract

The extracellular signal-regulated kinase (ERK) 1 and 2 are important signaling components implicated in learning and memory. These isoforms display a high degree of sequence homology and share a similar substrate profile. However, recent findings suggest that these isoforms may have distinct roles: whereas ERK1 seems to be not so important for associative learning, ERK2 might be critically involved in learning and memory. Thus, the individual role of ERK2 has received considerable attention, although it is yet to be understood. Here, we have generated a series of mice in which ERK2 expression decreased in an allele dose-dependent manner. Null ERK2 knock-out mice were embryonic lethal, and the heterozygous mice were anatomically impaired. To gain a better understanding of the influence of ERK2 on learning and memory, we also generated knockdown mice in which ERK2 expression was partially (20-40%) reduced. These mutant mice were viable and fertile with normal appearance. The mutant mice showed a deficit in long-term memory in classical fear conditioning, whereas short-term memory was normal. The mice also showed learning deficit in the water maze and the eight-arm radial maze. The ERK1 expression level of the knockdown mice was comparable with the wild-type control. Together, our results indicate a noncompensable role of ERK2-dependent signal transduction in learning and memory.

Figure 1.

Schematic diagram of targeted knockdown of the mouse Erk2 gene. A, The targeting vector (top), wild-type Erk2 allele (middle), and Erk2(floxN) allele (bottom). White boxes represent Erk2 exons (Ex), and black boxes represent the Pgk–neo cassette. A KpnI restriction site was generated in intron 3 (black bar). The 5′ outer probes used for Southern blot analysis are shown as gray boxes. The primers used for PCR are shown as arrows. B, Southern blot analysis of wild-type and heterozygous adult mice genomic DNA. DNA samples were digested with KpnI and hybridized with the 5′ outer probe. The positions and sizes of wild-type and mutant fragments are indicated. C, PCR genotyping of wild-type and homozygous mice. The positions and sizes of PCR fragments for wild-type and mutant mice are indicated.

Figure 2.

Western blot analysis of Erk2^+/+, Erk2^floxN/+, and Erk2^floxN/floxN mice. A, B, Expressions of ERK2, ERK1, phospho-ERK2, and β-actin in an extract obtained from the cerebrum, hippocampus, and cerebellum from 1-month-old (A) or 12-month-old (B) mice. The gels shown are representative examples. β-Actin served as controls for protein loading. C–E, The graphs depict the averaged results from eight (1 month of age) or six (12 months of age) experiments. *p < 0.05; **p < 0.01.

Figure 3.

ERK2 knockdown mice showed normal histology in the brain. A–D, Nissl staining of hippocampus (A), pyramidal layer of hippocampus (B), cerebellum (C), and Purkinje layer of cerebellum (D). E, Synaptic density in ERK2 knockdown mice was not altered. Representative dendritic segments of dentate gyrus neurons from Erk2^+/+ (left) and Erk2^floxN/floxN (right) mice. Scale bars: A, C, 500 μm; B, D, 50 μm; E, 10 μm.

Figure 4.

ERK2 knockdown mice showed normal behavior in the open-field, elevated plus maze, Y-maze, and wheel-running tests. A, Open-field test (total distance traveled in 10 min for 3 consecutive days, Erk2^+/+, n = 12; Erk2^floxN/+, n = 11; Erk2^floxN/floxN, n = 12). B, Elevated plus maze test (percentage time spent in open arms, Erk2^+/+, n = 12; Erk2^floxN/+, n = 16; Erk2^floxN/floxN, n = 12). C, Y-maze test (percentage correct alternation response). No significant differences were observed between wild-type and ERK2 knockdown mice in these tests (Erk2^+/+, n = 12; Erk2^floxN/+, n = 11; Erk2^floxN/floxN, n = 12). D, E, Representative actograms in wheel-running test for Erk2^+/+ (D) and Erk2^floxN/floxN (E) mice in double-plotted format. Each horizontal line represents 48 h. Vertical bars represent periods of wheel-running activity. Animals were initially housed in 12 h LD cycle for 14 d and then were transferred to DD. The LD cycle is indicated by the bar above the top records. Numbers on the left indicate days of study.

Figure 5.

ERK2 knockdown mice showed impaired memory performance in both contextual and cued tests compared with wild-type littermates. A, Freezing response was measured in the context before shock (basal freezing) and in the conditioning chamber (contextual fear response) 2 h after conditioning (Erk2^+/+, n = 12; Erk2^floxN/floxN, n = 12). B, Freezing response was measured in the context before shock (basal freezing) and in the conditioning chamber (contextual fear response) 2 d after conditioning (Erk2^+/+, n = 12; Erk2^floxN/floxN, n = 12). C, Freezing response (for the same set of mice) was measured in an alternative context without auditory cue (basal freezing) or with a cue 2 d after conditioning. D, Representative Western blots of ERK2 phosphorylation levels. E, Densitometric analysis for ERK2 phosphorylation 1 h after training (Erk2^+/+, n = 5; Erk2^floxN/floxN, n = 5). *p < 0.05; ***p < 0.001.

Figure 6.

ERK2 knockdown mice showed partially impaired learning in the Morris water maze compared with wild-type littermates. A, ERK2 knockdown mice (filled triangles) showed longer escape latency on days 2–7 and day 9, but the difference diminished after day 10 compared with wild-type littermates (filled circles). Erk2^+/+, n = 12; Erk2^floxN/floxN, n = 12. *p < 0.05; **p < 0.01. B, C, Representative traces of an ERK2 knockdown (B) or a wild-type (C) mouse on day 5. Black circles represent the platform.

Figure 7.

ERK2 knockdown mice were partially impaired in the 8-8 version (8 arms fully baited) eight-arm radial maze task. A–C, Mice were trained in one trial per day for 12 consecutive days and 12 trials. Learning performance is expressed as the mean latency (A), the mean number of correct (distinct) arm choices during the first eight trials (B), and total number of revisiting errors (C). ERK2 knockdown mice required a longer time to complete a trial on days 3 and 4, but, on day 5, statistical differences between genotypes were no longer seen and both genotypes apparently reached a learning plateau (A). ERK2 knockdown mice showed no significant genotype effect in correct choices (during the first 8 trials) (B) and made higher error scores only from days 2 to 4 (C) (Erk2^+/+, n = 14; Erk2^floxN/floxN, n = 14). *p < 0.05; ***p < 0.001.

Figure 8.

ERK2 knockdown mice showed impaired reference memory in learning in the 4-8 version (only 4 arms baited) of the eight-arm radial maze task. Mice were trained for four trials per day for 16 consecutive days and 64 trials. A, ERK2 knockdown mice required a longer time and did not reach the wild-type curve. B, C, As a measure of reference memory, learning performance is expressed as the mean number of correct (baited) arm choices during the first four trials (B) and total number of visiting errors of never baited arms (C). ERK2 knockdown mice showed fewer reference memory correct choices (B) and higher error scores (C) throughout the experiment; however, in the same task, working memory error was not critically impaired. D, As a measure of working memory, correct (distinct) choices (during the first 4 choices) were almost the same as the control. E, F, Total number of revisiting errors for baited arms (E) or revisiting errors for unbaited arms (F) shows that the curve of ERK2 knockdown mice reached that of wild-type mice (after days 6 and 9, respectively) although the mutant mice had significantly higher scores in reference memory errors (Erk2^+/+, n = 12; Erk2^floxN/floxN, n = 12). *p < 0.05; **p < 0.01; ***p < 0.001.