Transgenic ywhag mouse (CARD ID 1461) were generated by a group at Kumamoto University. In brief, exchangeable gene trap pU-21W vector was used for random gene trap mutagenesis. pU-21W is a promoter trap vector with three stop codons which were arranged in upstream of the ATG of the β-galactosidase (β-geo) in all three frames [30]. The strain name is depicted as B6;CB-YwhagGt (pU-21W)266Card, and the website address of the Database for the Exchange of Gene Trap Clones is http://egtc.jp/action/access/clone_detail?id=21-W266. Littermates including ywhag wild-type (WT), heterozygote knockout (Het), and homozygote knockout (KO) mice were used for this study. Mice were housed and maintained in standard laboratory conditions of 12:12 h light:dark cycle. Regular chow and water were provided ad libitum . All animal experiments were performed in accordance with the guidelines of Sejong University and Korea University Institutional Animal Care and Use Committee. Male mice were used for behavioral experiments.

ywhag KO (−/+ and −/−) mice were genotyped using polymerase chain reaction (PCR), and littermate ywhag (+/+) mice were used as a control. The following primers were used: ywhag common forward primer - 5′-TCATCAGCAGCATCGAGCAG-3′; ywhag WT reverse primer - 5′- ATGGCGTCGTCGAAGGC-3′ and ywhag KO reverse primer - 5′-AGGGGTCTCTTTGTCAGGGT-3′. The PCR protocol was 95℃ (10 min), then 35 cycles of 95℃ (30 s), 58℃ (30 s) 72℃ (30 s), and 72℃ (5 min). PCR products were examined using 1% agarose gel electrophoresis in TAE buffer.

The whole brain tissues obtained were lysed using RIPA buffer containing a protease inhibitor cocktail (Roche) and processed for western blotting using anti-14-3-3γ antibody (sc-398423, 1:1000, Santa Cruz Biotechnology) and mouse anti-actin (1:2000; Sigma-Aldrich). Signals were detected using enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA) following probing with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:3000, Jackson ImmunoResearch). Each experiment was performed with samples from three independent groups.

Animals were acclimatized to the behavior test room for a week before testing. Behavioral assays were commenced when the mice were aged 10 weeks. One cohort of mice were used for all behavioral assays (n=8 per genotype). The order of behavioral tests progresses from less stressful one (e.g. open field test) to more stressful one (e.g. forced swim test) and there were two or three day's interval between assays.

The open field test (OFT) was performed as described to measure locomotor activity [31]. The open field apparatus consisted of a square arena (40×40 cm) with 30 cm high walls that was illuminated using an electric bulb hanging 2.5 m above the floor. Open field behaviors of the mice were recorded and analyzed using the ANY-maze system (Stoelting, Wood Dale, IL). Animals were exposed to the open field for 10 minutes. Between subjects, the box was thoroughly cleaned with 70% ethanol and the ethanol was allowed to evaporate completely prior to testing mice.

The elevated plus maze (EPM) test was performed as described [32]. The apparatus consisted of two open arms and two enclosed arms arranged in a plus-sign orientation. The arms were elevated 50 cm above the floor with each 5-cm wide arm projecting 30 cm from the center. At the start of the test, each subject was placed in the center of the EPM facing a closed arm. Mice explored the maze for 5 minutes and exploratory activities in both the open and closed arms were recorded and analyzed using the ANY-maze system (Stoelting, Wood Dale, IL). Between subjects, the maze was thoroughly cleaned with 70% ethanol and the ethanol was allowed to evaporate completely prior to testing mice.

This test was performed as described [33]. The apparatus consisted of two (light and dark) chambers (34×24×24 cm) joined together. There was an aperture (8×8 cm) between the two chambers that the mice could use to move between chambers. The chambers were made of white and black opaque Plexiglas. The black chamber was covered with a black lid (the dark box), while the white chamber was covered with a transparent lid and illuminated using ambient room lighting to ~450 lux (the light box). During the test, a subject was placed in the center of the light box facing away from the aperture connecting the two chambers and allowed to freely explore both chambers for 5 minutes. Between animals, the box was cleaned with 70% ethanol and allowed to completely dry. All sessions were recorded and analyzed using the ANY-maze system (Stoelting, Wood Dale, IL).

Mice were tested in a Plexiglas cage divided into three chambers comprising two equal-sized end areas (31.5×25.5 cm each) and a smaller neutral section between them (10.5×25.5 cm). During the habituation phase, the end areas contained an empty “holding cell” (10.16 cm in diameter and 13.97 cm tall). Each mouse was placed in the center of the box and allowed to explore the entire box for 5 minutes. The subject was then returned to the cage while another adult male (C57BL/6 WT mice) was placed under the holding cell (on one randomly selected side) and the empty cell was placed on the opposite side. During the test phase, the subjects were placed in the center and allowed to investigate the entire box for 5 minutes. The ANY-maze system recorded and scored the number of entrances into each side and the time spent investigating the novel mouse. Investigation was defined as the test mouse's nose touching the novel mouse through the bars or sniffing within 1 cm. The test arena was carefully cleaned with 70% ethanol between subjects and allowed to completely dry.

The rotarod test was performed to evaluate the basic mobility of animals in each group as described [31]. Mice were placed on the stationary cylinder of the rotarod apparatus and trained on the apparatus for at least four consecutive trials in which the rod was kept at a constant speed (4 rpm) and returned to their cages for 1 min between trials. Once the animals were able to stay on the rod rotating at 4 rpm for at least 60 s, they were subjected to the rotarod test. Mice were placed on the rod rotating at an accelerating speed from 4 to 40 rpm over 300 s. The time before animals fell off the rod was recorded with a maximum cut-off of 300 s. Mice were tested for eight consecutive trials with at least 5-min intervals. The data from the last four trials were averaged as the latency to fall. Each mouse was returned to its home cage and remained there for 5 min before the next trial. If the mouse remained on the rod in 5 minutes, latency to fall was recorded as 300 s. Between subjects, the rotarod apparatus was thoroughly cleaned with 70% ethanol and allowed to completely dry.

Mice were subjected to a schedule of restraint stress before each behavior test to induce behavioral and physiological phenotypes. Each mouse was restrained using a 50 ml conical tube with an open end, exposing the nose for breathing. Acute stress was applied for one hour and each mouse was returned to its home cage immediately after restraining stress.

All statistical analyses were carried out using GraphPad Prism version 5.00 for Windows. Numerical data are presented as means±standard error of the mean (SEM). The error bars in graphs denote the SEM. The statistical significance of the data was assessed using unpaired or paired Student's t-tests, with the significance level denoted by asterisks (^*p<0.05, ^**p<0.01, or ^***p<0.001).

The genomic organization of the mouse 14-3-3γ gene (ywhag) and KO strategy using the gene trap system are illustrated (Fig. 1A and Fig. 2A) [30]. The sequence analysis of mRNA from 14-3-3γ homozygote pups showed that the exon 1, the partial exon 2 and the inserted sequence from the gene-trap pU-21W vector were expressed (Fig. 1B). This genetic insertion of foreign sequence results in truncated N-terminal 14-3-3γ peptide (71 amino acids of 14-3-3γ and 13 amino acid long peptide of gene-trap vector). This N-terminal fragment covers three initial domains (A~C) of 14-3-3 proteins (Fig. 1C).

To verify the genetic deletion of ywhag in mice, we performed PCR-based genotyping of tail samples of pups born from 14-3-3γ Het breeding pairs at P0 (Fig. 2B). We also examined the expression level of 14-3-3γ using a specific anti-14-3-3γ antibody with brain homogenates prepared from pups at P0 (Fig. 2C). It is noted that the 14-3-3γ signal from homozygote KO mice was absent, and the level of 14-3-3γ from heterozygote (Het) mice was significantly reduced compared to that in littermate control mice. These data indicate that ywhag gene was successfully disrupted in these 14-3-3γ KO mice. Beta-gal staining of adult Het mice showed the broad expression of 14-3-3γ in the brain (Fig. 2D). Since β-geo expression in 14-3-3γ Het mice is controlled by the endogenous promoter of the ywhag gene [30], β-geo expression in our 14-3-3γ Het mice represents the endogenous expression pattern of 14-3-3γ. The β-geo expression clearly showed in the pyramidal layer of the hippocampus, and its expression pattern is consistent with the mRNA expression pattern of 14-3-3γ in the Allen brain atlas database (http://mouse.brain-map.org/experiment/show?id=1124).

Interestingly, at weaning (P21), we found that there were no surviving homozygote KO mice among the 242 mice born from 15 breeding pairs (36 litters of pups) (Fig. 3A). This is clearly distinct from the other two homozygote KO mice of 14-3-3 isoform genes (ywhae and ywhaz ) that survive long enough for behavioral experiments [10,11]. In addition, we also noticed that the body size and weight of 14-3-3γ Het mice was significantly less than those of littermate controls (Fig. 3B and 3C). Based on these data, we concluded that 14-3-3γ is critically important for the maturation and survival of mice in contrast to a previous report [29]. We also examined the expression level of 14-3-3γ using brain homogenates prepared from adult 14-3-3γ WT and Het mice and found that the reduced expression of 14-3-3γ in Het mice was maintained (Fig. 3D). For behavioral experiments, we selected Het mice as similar as possible in size and body weight to WT littermate controls to see whether differences in behavioral phenotypes were directly related to the genetic disruption of 14-3-3γ. We also measured the body weights of the two genotypes throughout adulthood until we finished the behavioral experiments (Fig. 3E).

Since 14-3-3γ is abundantly expressed in the brain, to investigate its role in the expression of behavioral characteristics, we performed an OFT to assess basal locomotor activity. Intriguingly, 14-3-3γ Het mice traveled significantly more distance in the center zone than WT mice (Fig. 4A), which may be due to hyperactivity and not related to less anxiety, because the total distance traveled and mean speed of 14-3-3γ Het mice were significantly longer and faster than those in WT mice, respectively (Fig. 4B and 4C). In addition, the Het mice also showed significant longer margin distance compared to WT mice (data not shown). Because there was no significant difference between WT and Het mice in the rotarod test, any change in muscle strength or coordination of Het mice was not related (Fig. 4D).

Since the OFT suggested that the reduced level of 14-3-3γ in Het mice results in hyperactive behavior, to determine whether the 14-3-3γ haploinsufficiency may affect mood-related behaviors, we conducted the forced swim test (FST) and three-chamber social interaction test (3CT). Interestingly, Het mice displayed significantly longer immobile time in the FST than WT mice (Fig. 5A), which suggests that their depressive-like phenotype was induced by 14-3-3γ haploinsufficiency, while there was no deficit in the social interaction of Het mice in the 3CT (Fig. 5B).

Hyperactive and depressive symptoms are common in patients with several neuropsychiatric diseases. Since these patients often have a deficit in the appropriate response to acute stress [34], we subjected Het mice to restraint stress and performed several behavioral tests including the OFT, light and dark box (LDB) test, and EPM test. In the OFT, the hyperactivity of Het mice was significantly decreased, as seen in the total distance traveled and mean speed (Fig. 6A and 6B), while WT littermate control mice did not show any significant change. In the LDB test, total distance traveled in the light zone and number of transitions were significantly increased in Het mice in response to acute stress (Fig. 6C and 6D). Similarly, Het mice showed significantly increased time spent in open ends and total distance traveled in open arms in response to acute stress in the EPM test (Fig. 6E and 6F).