ENU-mutagenesis mice with a non-synonymous mutation in Grin1 exhibit abnormal anxiety-like behaviors, impaired fear memory, and decreased acoustic startle response

General health, neurological reflex, and motor coordination/learning in Grin1 ^Rgsc174 /Grin1 ⁺ mice

No abnormal neurological features, such as whisker twitch or righting reflex, were found in Grin1^Rgsc174/Grin1⁺ mice (Table 1). Body weight was significantly reduced (Figure 1A; F_1,19 = 11.940, p = 0.0026) and latency to fall was significantly decreased in the wire hang test (Figure 1E; F_1,19=4.956, p=0.0383) in these mutant mice compared to wild-type mice. There were no significant differences in body temperature between the genotypes (Figure 1B; F_1,19 = 0.188, p = 0.6693), grip strength (Figure 1C; F_1,19 = 1.552, p = 0.228) or latency to the first hind-paw response in the hot plate test (Figure 1E; F_1,19 = 0.286, p = 0.5992). Grin1^Rgsc174/Grin1⁺ mice showed a significantly longer latency to fall in the accelerating rotarod test compared to wild-type mice (Figure 1F; F_1,19=11.554, p = 0.030). Previous studies revealed that body weight is negatively correlated with rotarod performance [42, 43]. A similar negative correlation was observed in Grin1^Rgsc174/Grin1⁺ mice (Figure 1G; body weight vs. average latency (1st day); r = −0.566, p = 0.0065). Analysis of covariance (ANCOVA) with body weight as a covariate found no significant effect of genotype on latency to fall (F_1,19 = 0.026, p = 0.8730), indicating that the seemingly improved rotarod performance may reflect the reduced body weight of Grin1^Rgsc174/Grin1⁺ mice. In the gait analysis, Grin1^Rgsc174/Grin1⁺ mice exhibited a significant narrowed stance width of the hind paws (width between hind limbs) compared with wild-type mice (Figure 1O, F_1,15 = 8.135, p= 0.0121). A previous study reported that the stance width was widened in rats with injured spinal cords and became narrowed during injury recovery after a locomotor training paradigm [44]; therefore, the narrowed stance width in Grin1^Rgsc174/Grin1⁺ mice may represent improved motor coordination. No significant differences were detected between genotypes in any other indices of the gait analyses (Figure 1G-R; Figure 1O, F_1,15 = 8.135, p= 0.0121). These observations indicate that there are no clear deficits in nociception, neuromuscular strength, or motor coordination/learning in Grin1^Rgsc174/Grin1⁺ mice.

	Wild-type	Grin1Rgsc174/Grin1+
Coat state (% with normal coat state)	100	100
Ear twitch (% with quick response)	100	100
Whisker (% with)	100	100
Whisker twitch (% with normal response)	100	100
Righting reflex (% with normal response)	100	100
Key jangling (% with normal response)	100	100
Reaching (% with normal response)	100	100

Wild-type mice, n = 10; Grin1^Rgsc174/Grin1⁺ mice, n = 11.

Increased locomotor activity in Grin1^Rgsc174/Grin1⁺ mice

In the open field test, the total distance traveled was significantly increased (Figure 2A; F_1,19 = 31.867, p < 0.0001; genotype × time interaction, F_23,437 = 4.700, p < 0.0001), while no significant increase in time spent in the center area was detected in Grin1^Rgsc174/Grin1⁺ mice compared to wild-type mice (Figure 2B; F_1,19 = 0.461, p = 0.5053). These observations are consistent with a previous study [31]. There was no significant effect of the genotype in vertical activity for 120 min (Figure 2C; F_1,19 = 0.501, p = 0.4876), although there was a significant genotype × time interaction between genotypes (Figure 2C; F_23,437 = 1.882, p =0.0085). Stereotypic behavior was significantly increased in Grin1^Rgsc174/Grin1⁺ mice compared with wild-type mice (Figure 2D; F_1,19 = 9.015, p = 0.0073). Increased locomotor activity of Grin1^Rgsc174/Grin1⁺ mice was detected in other behavioral measures: total distance traveled in a light chamber in the light/dark test (Figure 3D; F_1,19=4.942, p=0.0385), the social interaction test in a novel environment (Figure 4D; F_1,38=8.141, p=0.0214), and the Crawley’s sociability (Figure 4I; F_1,19 = 12.224, p =0.0024) and social novelty preference tests (Figure 4L; F_1,19 = 61.313, p < 0.0001).

Abnormal anxiety-like behaviors in Grin1^Rgsc174/Grin1⁺ mice

In the light/dark transition test, the number of transitions between chambers was significantly decreased in Grin1^Rgsc174/Grin1⁺ mice (Figure 3A; F_1,19=11.418, p=0.0032). There were no significant differences between genotypes in the time they remained in a light chamber (Figure 3B; F_1,19=3.554, p=0.0748) or in the first latency to enter the light chamber (Figure 3C; F_1,19=0.979, p=0.3348). Grin1^Rgsc174/Grin1⁺ mice traveled significantly longer distances in the light chamber (Figure 2D; F_1,19=4.942, p=0.0385), but not in the dark one (Figure 2D; F_1,19=1.776, p=0.1983). In the elevated plus maze test, the percentage of time spent in the open arms and the percentage of entries into the open arms by Grin1^Rgsc174/Grin1⁺ mice were significantly higher than those by wild-type mice (Figure 2E; time spent in the open arms, F_1,18=54.874, p<0.0001; Figure 2F; entries into the open arms, F_1,18=55.392, p < 0.0001). There were no significant differences between the genotypes in the total number of entries into the arms (Figure 2G; F_1,18=1.814, p = 0.1948) or in the distance traveled (Figure 2H; F_1,18 = 1.985, p = 0.1759). The number of transitions between the chambers decreased in the light/dark transition test, suggesting increased anxiety-like behavior in Grin1^Rgsc174/Grin1⁺ mice. On the other hand, the mutant mice displayed a higher percentage of time spent in open arms and a higher percentage of entries into the open arms in the elevated plus maze test, which are generally interpreted as decreased anxiety-like behaviors. Thus, apparently opposite anxiety-like behaviors were observed between the light/dark transition test and elevated plus maze test. For a more detailed interpretation of these results, see the Discussion section.

No abnormalities in the Porsolt forced swim or tail suspension tests in Grin1^Rgsc174/Grin1⁺ mice

There were no significant differences between the Grin1^Rgsc174/Grin1⁺ and wild-type mice in immobility on day 1 (Figure 5A; genotype effect, F_1,19 = 2.949 × 10^-5, p = 0.9957; genotype × time interaction, F_9,171 = 1.099, p = 0.3659) or day 2 (Figure 5B; genotype effect, F_1,19 = 0.033, p = 0.8585; genotype × time interaction, F_9,171 = 0.180, p = 0.9959), or in distance traveled on day 1 (Figure 5C; genotype effect, F_1,19 = 1.642, p = 0.2154) or day 2 (Figure 5D; genotype effect, F_1,19 = 1.062, p = 0.3156) in the Porsolt forced swim test. In the tail suspension test, there was no significant difference in immobility between the genotypes (Table 2).

No obvious deficit in the social interactions of Grin1^Rgsc174/Grin1⁺ mice

In the social interaction test conducted in a novel environment, there were no significant differences between the Grin1^Rgsc174/Grin1⁺ and wild-type mice in the number of contacts (Figure 4A; F_1,8 = 2.364, p = 0.1627), mean duration per contact (Figure 4B; F_1,8 = 2.017, p = 0.1934), or total duration of contact (Figure 4C; F_1,8 = 0.646, p = 0.4447). These results are inconsistent with the previous results indicating that the total interaction time in Grin1^Rgsc174/Grin1⁺ mice had significantly decreased compared to that in wild-type mice in an open field during a social interaction test [31]. In the test of social behaviors in home cage, no significant difference was detected in social interaction between Grin1^Rgsc174/Grin1⁺ and wild-type mice (Figure 4E; whole period, F_1,6 = 0.076, p = 0.7924; light period, F_1,6 = 0.089, p=0.7752; dark period, F_1,6 = 0.278, p=0.6170). There was no significant effect of genotype on locomotor activity, which was quantified as the number of pixels changed between each pair of successive frames in home cage (Figure 4F; whole period, F_1,6 = 2.101, p = 0.1974; day period, F_1,6 = 0.005, p = 0.9480; night period, F_1,6 = 3.843, p=0.0976), although there was a significant genotype × time interaction between the genotypes (Figure 2C; F_23,437 = 1.882, p =0.0085). Given that the mutant mice showed significantly increased locomotor activity in their home cages in the previous observations [31] and in one-sided testing of the present study (p= 0.0488), the non-significant genotype effect was most likely caused by the small number of pairs (Wild-type, N=5; Mutant mice, N=3). In fact, Grin1^Rgsc174/Grin1⁺ mice displayed apparently increased locomotor activity throughout the night period, and this activity was significantly increased during the time period from 5:00 a.m. to 6:00 a.m. (Figure 4F; F_1,6 = 7.297, p=0.0355) in their home cage. In the Crawley’s three-chamber social approach test, there was no significant difference in preference between the empty cage and the cage with a stranger mouse, either in wild-type or Grin1^Rgsc174/Grin1⁺ mice (Figure 4G; time spent around the cages; stranger cage vs. empty cage; paired t-test; wild-type mice, t₁₀= 1.774, p= 0.1065; Grin1^Rgsc174/Grin1⁺ mice, t₉ = 1.005, p = 0.3410). No significant difference between genotypes was detected in the total time spent around the empty chamber or the chambers of the stranger mouse (Figure 4H; genotype, F_1,19 = 1.788, p = 0.1970). In the social novelty preference test, Grin1^Rgsc174/Grin1⁺ mice spent more time in the cage with a novel (stranger) mouse compared to the time spent in the cage with the familiar mouse (the first, already-investigated mouse), whereas wild-type mice did not show a preference between the familiar mouse and a stranger mouse (Figure 4J; time spent in cages (familiar vs. stranger); paired t-test; Grin1^Rgsc174/Grin1⁺ mice, t₉ = 3.877, p = 0.0038; wild-type mice, t₁₀ = −0.706, p = 0.4963). Similar results were obtained in the time spent around the chambers (familiar side vs. stranger side, paired t-test; wild-type mice, t₁₀ =−0.451, p=0.6616; Grin1^Rgsc174/Grin1⁺ mice, t₉ =3.721, p=0.0048). In comparison with wild-type mice, Grin1^Rgsc174/Grin1⁺ mice spent significantly less time in the cage with the familiar mouse (Figure 4J; genotype, F_1,19 = 11.847, p = 0.0027), while no significant difference between the genotypes was detected in time spent in the cage with a novel mouse (Figure 4J; genotype, F_1,19 = 0.711, p = 0.4096). These observations suggest that social novelty preference is increased in Grin1^Rgsc174/Grin1⁺ mice. There was no significant difference between the genotypes in the total time spent around both the cages of the familiar and of the stranger mice (Figure 4K; genotype, F_1,19 = 3.180, p = 0.0905). There were no obvious impairments in the Grin1^Rgsc174/Grin1⁺ mice in three different tests for social behavior.

Severely impaired fear memory in Grin1^Rgsc174/Grin1⁺ mice

In the contextual and cued fear conditioning test, there was no significant difference in freezing between genotypes in the conditioning phase (Figure 6A; genotype effect, F_1,15 = 0.879, p = 0.3633; genotype × time interaction, F_7,105 = 1.701, p = 0.1165). Contextual freezing at 1 day after training was significantly decreased in Grin1^Rgsc174/Grin1⁺ mice compared to wild-type mice (Figure 6B; genotype effect, F_1,15=8.848, p = 0.0095). In the altered context, although there was no significant difference in freezing during the pre-tone period between genotypes (Figure 6C; genotype effect, F_1,15 = 0.078, p = 0.7839), freezing during the tone period was significantly lower than in wild-type mice (Figure 6C; genotype effect, F_1,15 = 7.206, p = 0.017). Grin1^Rgsc174/Grin1⁺ mice showed a significant increase in distance traveled immediately after the first footshock compared to wild-type mice, and no significant differences were detected in these measurements after the second or third footshocks (Figure 6D; Footshock 1, F_1,15 = 6.270, p = 0.0243; Footshock 2, F_1,15 = 0.566, p = 0.4635; Footshock 3, F_1,15 = 0.221, p = 0.6449). These findings demonstrate that both contextual and cued fear memory are impaired in Grin1^Rgsc174/Grin1⁺ mice.

Moderately impaired spatial working memory in Grin1^Rgsc174/Grin1⁺ mice

In the eight-arm radial maze test, there were no significant differences in the number of revisiting errors during the trials without delays (1–19 blocks, Figure 6E; genotype effect, F_{1, 15} =1.112, p = 0.3083; genotype × trials interaction, F_18,270 = 0.647, p = 0.8602) or during the trials with delay (20–22 blocks Figure 6H; genotype effect, F_1,15 =0.072, p = 0.7926; genotype × trials interaction, F_2,30 = 0.398, p = 0.6753) between the Grin1^Rgsc174/Grin1⁺ and wild-type mice. No significant difference was detected in the number of different arm choices during the first eight entries in the trials without delays (1–19 blocks, Figure 6F; genotype effect, F_1,15= 0.724, p = 0.4082; genotype × trials interaction, F_18,270 = 0.837, p = 0.6562). To increase the difficulty of the task, a delay period (30 sec, 2 min, and 5 min) was provided after four pellets had been taken by confining the mice to the center platform in the 20th, 21st, and 22nd blocks of the trials. Grin1^Rgsc174/Grin1⁺ mice showed a significantly lower number of different arm choices during the first 8 entries in the trials with delay (Figure 6I; genotype effect, F_1,15 =5.431, p=0.0341; genotype × trials with delay interaction, F_2,30 = 0.251, p=0.7792). No significant difference was detected in the latency to obtain all pellets between Grin1^Rgsc174/Grin1⁺ and wild-type mice (Figure 6G; without delay, genotype effect, F_1,15 = 1.833, p = 0.1945; genotype × trials interaction, F_18,270 = 1.143, p = 0.2611; Figure 6J; with delay, genotype effect, F_1,15= 0.370, p = 0.8678; genotype × trials interaction, F_2,30 = 0.370, p = 0.8678). The results of the eight-arm radial maze test suggest moderately impaired spatial working memory in Grin1^Rgsc174/Grin1⁺ mice. However, it is possible that the increased locomotor activity causes the mild performance deficit to become a confounding factor.

Decreased startle response of Grin1^Rgsc174/Grin1⁺mice

Grin1^Rgsc174/Grin1⁺ mice had markedly lower startle amplitude than wild-type mice at both 110 dB and 120 dB (Figure 7A; genotype effect, 110 dB: F_1,18 = 57.464, p < 0.0001, 120: F_1,18=83.542, p < 0.0001). We did not detect significant differences in PPI between the genotypes (Table 2).

Expression pattern of maturation markers of DG neurons in Grin1^Rgsc174/Grin1⁺ mice

To address whether Grin1^Rgsc174/Grin1⁺ mice show maturation abnormalities in dentate granule cells, the expression patterns of markers for “immature dentate gyrus (iDG)”, upregulation of dopamine receptor D1A (Drd1a) and downregulation of desmoplakin (Dsp), tryptophan 2,3-dioxygenase (Tdo2), and Calbindin-28k (Calb1), were assessed by quantitative RT-PCR using total RNA extracted from the hippocampus. The expression of Tdo2 and Calb1 was slightly but significantly reduced in the mutant mice compared to wild-type mice (Figure 8; Tdo2, p = 0.006; Calb1, p = 0.002). There were no significant differences between Grin1^Rgsc174/Grin1⁺ and wild-type mice in the expressions of Dsp or Drd1a (Figure 8; Dsp, p = 0.197; Drd1a, p=0.652). In Grin1^Rgsc174/Grin1⁺ mice, two out of the four marker genes showed iDG-specific gene expression patterns, suggesting that they may partially display the iDG phenotype.

Animals and experimental design

Neurological screen

The neurological screen was performed as previously described [35]. The righting, whisker touch, and ear twitch reflexes were evaluated. A number of physical features, including the presence of whiskers or bald hair patches, were also recorded.

Neuromuscular strength

Neuromuscular strength was evaluated with the grip strength test and wire hang test. A grip strength meter (O'Hara & Co., Tokyo, Japan) was used to assess forelimb grip strength. Mice were lifted and held by their tail so that their forepaws could grasp a wire grid. The mice were then gently pulled backward by the tail with their posture parallel to the surface of the table until they released the grid. The peak force applied by the forelimbs of the mouse was recorded in Newtons (N). Each mouse was tested three times, and the greatest value measured was used for the statistical analysis. In the wire hang test, the mouse was placed on a wire mesh that was then inverted and waved gently, so that the mouse gripped the wire. Latency to fall was recorded, with a 60 sec cut-off time.

Hot plate test

The hot plate test was used to evaluate sensitivity to a painful stimulus. Mice were placed on a 55.0 (± 0.3)°C hot plate (Columbus Instruments International, Columbus, OH), and latency to the first hind-paw response was recorded. The hind-paw response was defined as either a foot shake or a paw lick.

Gait analysis (front and hind paws)

The gait of adult mice during spontaneous walk/trot locomotion was analyzed using the DigiGait™ Imaging System (Mouse Specifics Inc, Watertown, MA). In this system, mice walking on a motorized transparent treadmill belt are recorded on video, and the software automatically identifies the stance and swing components of the stride, and calculates stance width, stride length, step angle, and paw angle. Equivalent stride times for the fore and hind paws were composed of a shorter stance and a longer swing time. Peak vertical reaction force increased with decreasing stance time, and the results of the forelimbs were approximately 5% greater than those of the hind paws over the whole stance time range studied.

Light/dark transition test

A light/dark transition test was conducted as previously described [80]. The apparatus used for the light/dark transition test was composed of a cage (21 × 42 × 25 cm) divided into two sections of equal size by a partition with a door (O'Hara & Co.). One chamber was brightly illuminated (390 lux), whereas the other chamber was dark (2 lux). Mice were placed into the dark side and allowed to move freely between the two chambers through an open door for 10 min. The total number of transitions, latency to first enter the light chamber, distance traveled, and time spent in each chamber were recorded by ImageLD software (see 'Data analysis').

Elevated plus maze test

An elevated plus-maze test was conducted as previously described [81]. The elevated plus-maze consisted of two open arms (25 × 5 cm) and two enclosed arms of the same size with 15-cm high transparent walls. The arms and central square were made of white plastic plates and elevated 55 cm above the floor. To minimize the likelihood of animals falling from the apparatus, 3-mm-high Plexiglas walls surrounded the sides of the open arms. Arms of the same type were located opposite from each other. Each mouse was placed in the central square of the maze (5 × 5 cm), facing one of the closed arms. Mouse behavior was recorded during a 10-min test period. The number of entries into an arm and the time spent in the open and enclosed arms were recorded. The percentage of entries into open arms, the time spent in open arm (s), the number of total entries, and the total distance traveled (cm) were analyzed. Data acquisition and analysis were performed automatically using Image EP software (see 'Data analysis').

Open field test

Locomotor activity was measured using an open field test. Each mouse was placed in the corner of the open field apparatus (40 × 40 × 30 cm; Accuscan Instruments, Columbus, OH). The chamber of the test was illuminated at 100 lux. The total distance traveled (in cm), vertical activity (rearing measured by counting the number of photobeam interruptions), and time spent in the center area (20 × 20 cm), and beam-break counts for stereotyped behaviors were recorded. If the animal broke the same beam (or set of beams) three times, then the monitor considers the animal to have exhibited stereotypic activity, including grooming and head bobbing. The stereotypy count is the number of beam breaks that occur during this period of stereotypic activity. The data were collected for 120 min.

Social interaction test in a novel environment

In the social interaction test, two mice of identical genotypes that were previously housed in different cages were placed in a box together (40 × 40 × 30 cm) and allowed to explore freely for 10 min [82]. Because a pair of mice was used as a sample in the test, the number of samples is half. Social behavior was monitored with a CCD camera connected to a Macintosh computer. Analysis was performed automatically using ImageSI software (see 'Data analysis'). The total number of contacts, total duration of active contacts, total contact duration, mean duration per contact, and total distance traveled were measured. The active contact was defined as follows. Images were captured at 3 frames per second, and distance traveled between two successive frames was calculated for each mouse. If the two mice made contact and if the distance traveled by either mouse was longer than 4 cm, the behavior was considered as an “active contact”.

Social interaction test in home cage

Social interaction monitoring in home cage was conducted as previously described [54]. The system comprised the home cage (29 × 18 × 12 cm) and a filtered cage top, separated by a 13-cm high metal stand containing an infrared video camera attached at the top of the stand. Two mice of the same genotype that had been housed separately were placed together in a home cage. Their social behavior was then monitored for 1 week. Output from the video camera was fed into a Macintosh computer. Images from each cage were captured at a rate of one frame per second. Social interaction was measured by counting the number of particles detected in each frame; two particles indicated that the mice were not in contact with each other, while one particle (i.e., the tracking software could not distinguish two separate bodies) indicated contact between the two mice. We also measured locomotor activity during these experiments by quantifying the number of pixels that changed between each pair of successive frames. Analysis was performed automatically using ImageHA software (see 'Data analysis').

Crawley’s sociability and preference for social novelty test

The test for sociability and preference for social novelty is a well-designed method to investigate the complex genetics of social behaviors [83]. The apparatus comprised a rectangular, three-chambered box and a lid containing an infrared video camera (O'Hara & Co.). Each chamber was 20 × 40 × 22 cm and the dividing walls were made from clear Plexiglass, with small square openings (5 × 3 cm) allowing access into each chamber. An unfamiliar C57BL/6J male (stranger 1) that had no prior contact with the subject mouse was placed in one of the side chambers. The placement of stranger 1 in the left or right side chambers was systematically alternated between trials. The stranger mouse was enclosed in a small, circular wire cage, which allowed nose contact between the bars but prevented fighting. The cage was 11-cm high, with a bottom diameter of 9 cm and bars spaced at 0.5 cm intervals. The subject mouse was first placed in the middle chamber and allowed to explore the entire social test box for 10 min. The amount of time spent within a 5-cm distance of the wire cage in each chamber and the time spent in each chamber was measured with the aid of a camera fitted on top of the box. After the first 10 min, each mouse was tested in a second 10-min session to quantify social preference for a new stranger. A second, unfamiliar mouse was placed in the chamber that had been empty during the first 10-min session. This second stranger was enclosed in an identical small wire cage. The test mouse had a choice between the first, already-investigated unfamiliar mouse (stranger 1), and the novel unfamiliar mouse (stranger 2). As described above, the amount of time spent within a 5-cm distance of each wire cage and in each chamber during the second 10-min session was recorded. The stranger mice used in this experiment were 8- to 12-week-old C57BL/6J male mice that were not littermates. Analysis was performed automatically using ImageCSI software (see 'Data analysis').

Contextual and cued fear conditioning test

Each mouse was placed in a test chamber (26 × 34 × 29 cm) inside a sound-attenuated chamber and allowed to explore freely for 2 min. A 60 dB white noise, which served as the conditioned stimulus (CS), was presented for 30 sec, followed by a mild footshock (2 sec, 0.5 mA) serving as the unconditioned stimulus (US). Two more CS-US pairings were presented with a 2-min inter-stimulus interval. Context testing was conducted 24 h after conditioning in the same chamber. Cued testing with altered context was performed after conditioning using a triangular box (35 × 35 × 40 cm) made of white opaque Plexiglas, which was located in a different room. The chamber of the test was illuminated at 100 lux. Data acquisition, control of stimuli (i.e., tones and shocks), and data analysis were performed automatically using ImageFZ software (see 'Data analysis'). Images were captured at 1 frame per second. For each pair of successive frames, the amount of area (pixels) that the mouse moved was measured. When this area was below a certain threshold (i.e., 20 pixels), the behavior was judged as ‘freezing.’ When the amount of area equaled or exceeded the threshold, the behavior was considered as ‘non-freezing.’ The optimal threshold (amount of pixels) to judge freezing was determined by adjusting it to the amount of freezing measured by human observation. ‘Freezing’ that lasted less than the defined time threshold (i.e., 2 sec) was not included in the analysis. The parameters were constant for all mice assessed.

Eight-arm radial maze

The eight-arm radial maze test was performed using fully automated eight-arm radial maze apparatuses [35] (O'Hara & Co.). The floor of the maze was made of white plastic, and the wall (25-cm high) consisted of transparent plastic. Each arm (9 × 40 cm) radiated from an octagonal central starting platform (perimeter 12 × 8 cm) like the spokes of a wheel. Identical food wells (1.4-cm deep and 1.4-cm in diameter) with pellet sensors were placed at the distal end of each arm. The pellet sensors were able to automatically record the pellet intake of the mice. The maze was elevated 75 cm above the floor and placed in a dimly-lighted room with several extra-maze cues. During the experiment, the maze was maintained in a constant orientation. One week before pre-training, the animals were deprived of food until their body weight was reduced to 80% to 85% of their initial levels. In the pre-training, each mouse was placed in the central starting platform and allowed to explore and consume food pellets scattered over the whole maze for a 30-min period (one session per mouse). After completion of the initial pre-training, the mice received another pre-training to retrieve a food pellet from each food well after it had been placed at the distal end of each arm. A trial was finished when the mouse consumed the pellet. This procedure was repeated eight times, using eight different arms, for each mouse. After these pre-training trials, the actual maze acquisition trials were performed. In the spatial working memory task of the eight-arm radial maze, all eight arms were baited with food pellets. Mice were placed on the central platform and allowed to obtain all eight pellets within 25 min. A trial was terminated immediately after all eight pellets were consumed or 25 min had elapsed. An 'arm visit' was defined as travelling more than 5 cm from the central platform. The mice were confined at the central platform for 5 sec after each arm choice (without delay). The animals went through one trial per day (22 trials total). For each trial, arm choice, latency to obtain all pellets, distance traveled, number of different arms chosen within the first eight choices, the number of arm revisited, and omission errors were automatically recorded. The number of different arms chosen during the first eight choices is considered a measure of working memory, and to be relatively independent of locomotor activity levels [84–86]. To increase the difficulty of the task, in the 20th block of trials, a 30-sec delay was provided after four pellets had been taken by confining the mice in the center platform. During the 21st and 22nd blocks of the trial, the delay period was extended to 2 min and 5 min, respectively. A trial was terminated immediately after all of the pellets were consumed or 25 min had elapsed. After each trial, the maze was cleaned with water. The locations of the maze arms were randomly relocated after each session to prevent animals from using intra-maze cues. Data acquisition, control of guillotine doors, and data analysis were performed by ImageRM software (see 'Data analysis').

Startle response prepulse inhibition tests

A startle reflex measurement system was used (O'Hara & Co.) to measure the startle response and prepulse inhibition. A test session began by placing a mouse in a plastic cylinder where it was left undisturbed for 10 min. White noise (40 msec) was used as the startle stimulus for all trial types. The startle response was recorded for 140 msec (measuring the response every 1 msec) starting from the onset of the prepulse stimulus. The background noise level in each chamber was 70 dB. The peak startle amplitude recorded during the 140-msec sampling window was used as the dependent variable. A test session consisted of six trial types (i.e., two types for startle stimulus only trials, and four types for prepulse inhibition trials). The intensity of the startle stimulus was 110 or 120 dB. The prepulse sound was presented 100 msec before the startle stimulus, and its intensity was 74 or 78 dB. Four combinations of prepulse and startle stimuli were used (74–110, 78–110, 74–120, and 78–120). Six blocks of the six trial types were presented in pseudorandom order such that each trial type was presented once within a block. The average inter-trial interval was 15 sec (range: 10–20 sec).

Porsolt forced swim test

The Porsolt forced swim test apparatus consisted of four Plexiglass cylinders (20-cm high × 10-cm diameter). A nontransparent panel separated the cylinders to prevent the mice from seeing each other (O'Hara & Co.). The cylinders were filled with water (23°C) up to a height of 7.5 cm. Mice were placed into the cylinders, and the immobility and distance traveled were recorded over a 10-min test period. Images were captured at one frame per second. For each pair of successive frames, the amount of area (pixels) that the mouse moved in was measured. When the amount of area was below a certain threshold, mouse behavior was judged as “immobile.” When the amount of area equaled or exceeded the threshold, the mouse was considered as “moving.” The optimal threshold used for judging was determined through adjustments it to the amount of immobility measured by human observation. Immobility lasting for less than 2 sec was not included in the analysis. Retention tests were administered 24 h after training. Data acquisition and analysis were performed automatically using ImageTS software (see 'Data analysis').

Rotarod test

Motor coordination and balance were tested with the rotarod test. The rotarod test, using an accelerating rotarod (UGO Basile North America Inc., Collegeville, PA), was performed by placing mice on rotating drums (3-cm diameter) and measuring the time each animal was able to maintain its balance on the rod. The speed of the rotarod accelerated from 4 to 40 rpm over a 5-min period. The animals went through three trials per day on two consecutive days. The trials were separated by more than 1-hr intertrial intervals.

Quantitative RT-PCR

Quantitative RT-PCR analysis was conducted as previously described [35]. Total RNA was isolated from the hippocampi of 27- to 29-wk-old Grin1^Rgsc174/Grin1⁺ mice and wild-type mice. First-strand cDNA was synthesized from 1 μg of DNase I-treated total RNA using the Superscript® VILO™ cDNA synthesis kit (Life Technologies, Grand Island, NY). The expression of related genes was quantified using SYBR GreenER qPCR SuperMix for ABI PRISM (Life Technologies) following the instructions of the manufacturer. Quantitative PCR was performed using ABI PRISM7700 (Life Technologies) with the following conditions: 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 sec at 94°C and 1 min at 60°C. β-actin was amplified from all samples to normalize expression. The following primer sequences were obtained from the Primer Bank [87–89] (http://pga.mgh.harvard.edu/primerbank/index.html): calbindin-28K (56–185); desmoplakin (7–113); tryptophan 2,3-dioxygenase (1–105); Drd1a (133–251); and β-actin (851–962). The Ct values used were the mean values of triplicates.

Data analysis

The applications used for the behavioral studies (ImageLD, ImageSI, ImageTS, ImageCSI, ImageRM, ImageFZ) were developed by Dr. Tsuyoshi Miyakawa (available through O’Hara & Co.) based on the NIH Image program (NIH, Bethesda, MD, available at http://rsb.info.nih.gov/nih-image/) and ImageJ software (Imagejdev.Org, available at http://imagejdev.org/). Statistical analysis was conducted using StatView software (SAS Institute, Cary, NC). Data were analyzed by one-way analysis of variance (ANOVA), two-way repeated measures ANOVA, analysis of covariance (ANCOVA), the paired t-test, or Pearson’s correlation coefficient. Values in graphs are expressed as the mean ± SEM.

Availability of supporting data

Dataset, such as experimental date, age, raw data, and summary data (mean ± SEM), of the behavioral tests are available in the mouse phenotype database repository, http://www.mouse-phenotype.org/.