ERK2 contributes to the control of social behaviors in mice

Abstract

Signaling through extracellular signal-regulated kinase (ERK) is important in multiple signal transduction networks in the CNS. However, the specific role of ERK2 in in vivo brain functions is not fully understood. Here we show that ERK2 play a critical role in regulating social behaviors as well as cognitive and emotional behaviors in mice. To study the brain function of ERK2, we used a conditional, region-specific, genetic approach to target Erk2 using the Cre/loxP strategy with a nestin promoter-driven cre transgenic mouse line to induce recombination in the CNS. The resulting Erk2 conditional knock-out (CKO) mice, in which Erk2 was abrogated specifically in the CNS, were viable and fertile with a normal appearance. These mice, however, exhibited marked anomalies in multiple aspects of social behaviors related to facets of autism-spectrum disorders: elevated aggressive behaviors, deficits in maternal nurturing, poor nest-building, and lower levels of social familiarity and social interaction. Erk2 CKO mice also exhibited decreased anxiety-related behaviors and impaired long-term memory. Pharmacological inhibition of ERK1 phosphorylation in Erk2 CKO mice did not affect the impairments in social behaviors and learning disabilities, indicating that ERK2, but not ERK1 plays a critical role in these behaviors. Our findings suggest that ERK2 has complex and multiple roles in the CNS, with important implications for human psychiatric disorders characterized by deficits in social behaviors.

Figure 1.

Generation of Erk2 CKO mice. A, Schematic diagram of targeted knock-out of the mouse Erk2 gene. The Erk2(flox(Neo+)) allele is converted to the Erk2(flox(Neo−)) allele by in vivo cre-mediated partial recombination using EIIA-cre mice. White boxes represent Erk2 exons and black boxes represent the Pgk-neo cassette. The 5′ and 3′ outer probes used for Southern blotting are shown as gray boxes. The locations of the primers used for genotyping are indicated underneath each scheme. B, C, Southern blot analysis of wild-type and mutant mouse genomic DNA. DNA samples are digested with KpnI and hybridized with the 5′ (B) or 3′ (C) outer probe. The positions and sizes of the wild-type and mutant fragments are indicated. D, PCR genotyping of wild-type and mutant mice. The positions and sizes of PCR fragments for wild-type and mutant mice are indicated. E–G, Expression profile in adult Erk2 CKO mice. E, Expression of ERK1 and 2 in extracts of the hippocampus, cortex and cerebellum. F, Phosphorylation status of ERK1 and 2 in basal conditions. In E and F, β-actin serves as the control for protein loading. G, Loss of ERK2 protein in the nervous system in Erk2 CKO mice. Immunohistochemical analysis of the hippocampus (bottom) and hippocampal pyramidal cells (top) show the distribution of ERK2 in neuronal cells from nestin-cre−; Erk2^flox/flox (control) mice, and that ERK2 is absent in nestin-cre+; Erk2^flox/flox (Erk2 CKO) mice. Slides are counterstained with hematoxylin. Scale bars: top, 10 μm; bottom, 500 μm.

Figure 2.

ERK2 protein is abrogated in neuronal and glial cells in Erk2 CKO mice. A–C, ERK2 is expressed in neuronal cells in control mice at 12 weeks of age. Double staining for ERK2 (A) and the postmitotic neuronal marker NeuN (B) in the neocortex show that ERK2 is expressed in neurons, as indicated by colocalization (C). D–F, ERK2 is abrogated in neuronal cells in Erk2 CKO mice. Double staining for ERK2 (D) and NeuN (E) with a merged image (F) show that ERK2 is not detectable in neuronal cells in Erk2 CKO mice. G–I, ERK2 is expressed weakly in astrocytes in control mice. Double staining for ERK2 (G) and the astrocyte marker GFAP (H) in the neocortex with a merged image (I) show partial colocalization of ERK2 and GFAP, indicating that ERK2 is expressed weakly in some astrocytes (arrows) although not in other astrocytes. Arrowhead indicates probable expression of ERK2 in neurons. J–L, ERK2 is abrogated in astrocytes in Erk2 CKO mice. Double staining for ERK2 (J) and GFAP (K) with a merged image (L) show that ERK2 is not detectable in astrocytes in Erk2 CKO mice. Scale bars, 20 μm.

Figure 3.

Neuronal number is not altered in Erk2 CKO mice. A, B, Cortical coronal sections are immunostained for the neuronal marker NeuN. No significant difference is detected in the number of NeuN⁺ cells between controls and Erk2 CKO mice at 13 (A) and 7 (B) weeks of age. C, The fold differences in the numbers of NeuN⁺ cells at 13 weeks of age are calculated (CKO vs control, n = 5 mice for each, t = 0.33, t test, p > 0.05). Scale bars, 100 μm.

Figure 4.

Inactivation of ERK2 results in more astrocytes within the cerebral cortex. Immunohistochemistry with GFAP, a marker for astrocyte, reveals that GFAP immunoreactivity is increased in the cortices of Erk2 CKO mice compared with controls at 13 weeks of age. Note that GFAP⁺ cells seem to be more abundant in outer layers than inner layers of cortices in Erk2 CKO mice. Scale bar, 100 μm.

Figure 5.

Synaptic density is not altered in Erk2 CKO mice. A, Representative confocal images of DiI-impregnated dendritic segments of layer II/III neurons from control and Erk2 CKO mice. No prominent difference is observed in dendritic spines between the genotypes. B, Representative electron micrographs of layer II/III spines in the temporal cortex from control and Erk2 CKO mice. The postsynaptic density is clearly visible as a dark band located right beneath the postsynaptic membrane in the spine head (arrows). Scale bars: A, 10 μm; B, 1 μm. C, The length of postsynaptic density from layer II/III in the temporal cortex is not significantly different in Erk2 CKO mice compared with controls. D, The number of spines per 100 μm² segments of layer II/III in the temporal cortex is not significantly different between the groups. E, The percentage of perforated spines in apical dendrites of layer II/III in the temporal cortex is not different between the groups. F, The length of postsynaptic density from the hippocampus is not significantly different in Erk2 CKO mice compared with controls. G, The number of spines per 100 μm² segments of the hippocampus is not significantly different between the groups. H, The percentage of perforated spines in apical dendrites of the hippocampus is not different between the groups.

Figure 6.

Erk2 CKO mice are aggressive and impaired in maternal behaviors. A–C, Maternal nurturing in newly postpartum Erk2 CKO mice. A, In nurturing analysis of postpartum females, a mother is deprived of her first litter of pups for 10 min and then rechallenged with three pups placed individually in the corners of her cage. The typical behavior of a control mother is shown, approaching one pup immediately and collecting all three pups into one corner within a short latency (left), then crouching over them. The Erk2 CKO mother typically ignores the pups and does not retrieve them immediately (right). B, Time spent crouching over the pups in the nest of control (n = 10) and Erk2 CKO (n = 10) mothers. C, The percentage of scattered pups from the same set of mothers as in B. D–F, Aggressive behaviors of male Erk2 CKO mice as measured by the resident-intruder test (control, n = 7; Erk2 CKO, n = 7). **p < 0.01, ***p < 0.001.

Figure 7.

Erk2 CKO mice are impaired in social behaviors. A–D, Abnormal behaviors in social interaction tests. A, Olfactory investigations in Erk2 CKO mice are used for the social recognition test. Social memory by male mice is measured as the difference in investigation time (control, n = 12; Erk2 CKO, n = 12). The data depict the amount of time spent investigating the same female during each of four successive 1 min trials. A fifth trial depicts the response to a new female. B, When exposed to caged social and inanimate targets in the open field, Erk2 CKO mice show a decreased duration of interaction with the social target, and a similar duration of interaction with the inanimate object (control, n = 29; Erk2 CKO, n = 23). Percentage time is depicted in B–D. C, In the sociability test in a three-room chamber, Erk2 CKO mice spend less time than controls with the social target (control, n = 11; Erk2 CKO, n = 11). D, In the social novelty test, controls show a preference for social novelty, while Erk2 CKO mice show no preference between the novel and familiar targets. Erk2 CKO mice spend significantly less time than controls interacting with the novel target. The same set of mice is used as in C. E, Erk2 CKO mice are not significantly different from controls in the latency to find a buried treat following overnight food deprivation. The same set of mice is used as in B. F, Erk2 CKO mice show a significant decrease in interaction with a novel object in their home cages. The same set of mice is used as in B. G, Erk2 CKO mice show significant deficits in nest formation (control, 6 cages, n = 4 mice per cage; Erk2 CKO, 6 cages, n = 4 mice per cage). H, I, Representative photographs of control (H) and Erk2 CKO (I) cages, 45 min after the introduction of cotton nesting materials into each cage. Note the fluffy nest built in the control cage and the huddling of mice in this nest, in contrast to the poorly formed nests in the Erk2 CKO cage. **p < 0.01, ***p < 0.001. N.S., Not significant.

Figure 8.

Erk2 CKO mice exhibit normal locomotor activity but decreased anxiety-related behaviors. A–D, Open field test (control, n = 19; Erk2 CKO, n = 20). A, The average speed without resting time is not different between control and Erk2 CKO mice. B, The 10 min total path-length traveled is not significantly different between control and Erk2 CKO mice. C, Erk2 CKO mice exhibit reduced anxiety-like behavior, because they spend significantly more time in the central zone of the open field apparatus. D, Representative tracks of control and Erk2 CKO mice in the open field chamber over 10 min. E, Erk2 CKO mice exhibit reduced anxiety-like behavior in the elevated plus-maze test (the same set of mice as in A). ***p < 0.001.

Figure 9.

Erk2 CKO mice show impaired memory performance in both contextual and cued tests. A, The freezing response is measured in the context before shock (basal freezing) and in the conditioning chamber (contextual fear response) 24 h after conditioning (control, n = 12; Erk2 CKO, n = 12). B, The freezing response (the same set of mice as in A) is measured in an alternative context either without an auditory cue (basal freezing after conditioning) or with a cue 48 h after conditioning. **p < 0.01, ***p < 0.001.

Figure 10.

Plasma Oxt levels are not altered in Erk2 CKO mice. A, B, Levels of plasma Oxt in male (control, n = 10; Erk2 CKO, n = 10) (A), and female (control, n = 8; Erk2 CKO, n = 8) mice (B). The levels in Erk2 CKO mice are not different from those in controls.

Figure 11.

Subcutaneous injection of Oxt (10 ng/kg body weight) significantly increases phosphorylated ERK levels in the hippocampus. A, Hippocampus homogenates from vehicle-treated or Oxt-treated control (n = 4 for each) and Erk2 CKO littermates (n = 4 for each) are analyzed simultaneously for phospho-ERK1/2 (pERK1/2) and ERK1/2 by quantitative Western blotting. In control mice, ERK1 phosphorylation level is significantly elevated 10 min after Oxt injection. Similarly, ERK1 phosphorylation is significantly elevated 10 min after Oxt injection in Erk2 CKO mice. There is no difference in total ERK1 expression level among all groups. The ERK2 phosphorylation level is also elevated in control mice after Oxt injection without change of total ERK2 expression level. β-Actin serves as the control for protein loading. B–D, To evaluate phosphorylation, the intensities of the phospho-ERK1 and phospho-ERK2 bands are divided by their corresponding loading control (β-actin). Then, relative levels of ERK1 (B), ERK2 (C), and total ERK (D) phosphorylation are normalized to the mean control with vehicle injection. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 12.

Pharmacological blockade of ERK1 phosphorylation in Erk2 CKO mice does not additionally affect long-term memory. A, ERK1 and 2 phosphorylation levels in the hippocampus, cortex, and cerebellum are significantly inhibited 60 min after injection of 30 mg/kg MEK inhibitor SL327 in the hippocampus, cortex and cerebellum. There is no concurrent decrease in the total expression level of either isoform. B, The phosphorylation levels of ERK1 and 2 in the hippocampus as analyzed by band density (control, n = 5; Erk2 CKO, n = 5). β-Actin serves as the control for protein loading. C, In control mice, SL327 attenuates freezing in the contextual and cued fear conditioning after receiving one pairing of a tone and shock, compared with vehicle (vehicle, n = 10; SL327, n = 10). However, no significant difference is observed between animals treated with SL327 or vehicle for Erk2 CKO mice (vehicle, n = 10; SL327, n = 10). D, In control mice, SL327 attenuates freezing in contextual fear conditioning after receiving three pairings of a tone and shock compared with vehicle, but the additional pairings eliminated the effect of SL327 on freezing in response to the cue (vehicle, n = 10; SL327, n = 10). There is no significant difference in freezing in response to context and cue between Erk2 CKO mice treated with SL327 or vehicle (vehicle, n = 10; SL327, n = 10). *p < 0.05, **p < 0.01. N.S., Not significant.

Figure 13.

Pharmacological blockade of ERK1 phosphorylation in Erk2 CKO mice does not additionally affect social behaviors. A–C, Treatment with the MEK inhibitor SL327 (30 mg/kg) reduces social behaviors in control mice, but does not additionally affect impaired social behaviors in Erk2 CKO mice. A, When SL327 in control mice are exposed to caged social and inanimate targets in the open field, the duration of interaction with the social target is significantly decreased compared with vehicle (n = 14 mice for each). On the other hand, there is no significant difference in the spent with the social target between SL327- and vehicle-treated Erk2 CKO mice (n = 14 mice for each). Percentage time is depicted in A–C. B, In the sociability test in the three-room chamber, SL327 reduces the time spent with the social target compared with vehicle in control mice. On the other hand, there is no significant difference in the time spent with the social target between SL327- and vehicle-treated Erk2 CKO mice. The same set of mice is used as in A. C, In the social novelty test, vehicle-treated controls show preference for the novel target, while SL327-treated control mice does not. Furthermore, SL327 in control mice reduces the time spent with the social target compared with vehicle. On the other hand, there is no significant difference in the time spent with the novel target between SL327- and vehicle-treated Erk2 CKO mice. The same set of mice is used as in A and B. *p < 0.05, **p < 0.01, ***p < 0.001 compared with vehicle-injected control mice; ^#p < 0.05; ^###p < 0.001. N.S., Not significant.