All experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Stanford University and were performed based on the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Behavioral testing was conducted on a total of 97 Npas4 mice. This line has been described previously 
. Male Npas4 wild-type (WT), heterozygotes (HET), and knockout (KO) mice were obtained by HETxHET breeding at the Stanford Behavioral and Functional Neuroscience core facility (USA). Mice were housed in groups (2–6 mice per cage) and maintained on a 12-h reverse light-dark cycle with access to food and water ad libitum
. All mice were at least 7 weeks old at the beginning of experiments, and behavioral procedures were conducted during the dark phase of the cycle. The battery of behavioral tests was performed over several weeks. KO mice were found to die at a much younger age (around 3 months of age) than WT and HET mice, which explains the absence of KO mice tested in the elevated-zero maze and the low number of KO mice tested in the novel object recognition test.
Npas4 expression in the brain after a social encounter was tested by immunohistochemistry. This experiment was conducted on male C57Bl/6j mice (n
8) from Jackson Laboratory (Bar Harbor, ME, USA). Mice were group-housed on a 12-h reverse light-dark cycle with access to food and water ad libitum
. They were tested at 12 weeks of age.
All experiments were conducted blind to the genotype of the mice.
- Test of anxiety and locomotor activity
a. Elevated zero-maze.
Animals (WT n
15; HET n
17) were tested in 8-min trials of elevated zero-maze to assess anxiety. The maze was elevated 40 cm from the ground and featured two opposite open arms and two opposite closed arms. The closed arms were characterized by 40 cm high walls. Mice were individually placed in an open arm, facing a closed arm. Their behavior was recorded via an overhead camera and the automated tracking system Ethovision. At the end of the test, the maze was cleaned with 70% ethanol. Time spent in the open arms was recorded and analyzed as an indicator of anxiety.
b. Open-field test. Twenty-two WT, 22 HET, and 8 KO were placed in the center of an empty novel arena (76×76 cm) with opaque walls, surrounded by privacy blinds. For 10 minutes, each animal was allowed to explore the entire arena while being tracked by Ethovision (Noldus Information Technology, Wageningen, the Netherlands). At the end of each trial, the animal was placed back in its home cage and the arena was cleaned with 10% ethanol. The total distance moved and the percent of time spent in the center of the arena (53.5×53.5 cm) were analyzed.
- Tests of social behavior and social memory
a. 6-trial social test.
The 6-trial social test was used to assess social behavior and social discrimination. It consists of presenting ovariectomized females (OEF) to each subject mouse (WT n
10, HET n
13, KO n
9) over six 1-min trials separated by a 10-min inter-trial interval (ITI). During trials 1 to 4, subject mice were exposed to the same never-before-met OEF. In the 5th
trial, subject mice were exposed to a new never-before met OEF to test for social discrimination. During the 6th
trial, subject mice were exposed to the same OEF presented during trials 1 to 4. Each trial was videotaped and videos were analyzed offline by a trained experimenter. The time the subject mouse spent investigating the OEF was recorded; investigation was defined as the subject mouse making contact with the OEF.
b. Tube dominance.
Animals were assayed in the tube dominance test as previously described 
. The Tube Dominance test assesses tendencies of social dominance and aggressive behavior in rodents without involving physical harm. Pairs of aged-matched animals from different genotypes (WT (n
5) and KO (n
8)) are formed. Subjects of each pair were simultaneously released facing each other into opposite ends of a clear, narrow tube. The more dominant animal forced its opponent out of the tube. When one animal had all four paws out of the tube, it was declared the loser. Each mouse was tested in 4 trials with a 10-min ITI. No subject faced the same opponent twice. The number of wins was reported as a percentage of total number of matches.
c. Two-day social test.
This test was used to study detailed social behavior in a neutral environment. Two days prior to testing, each mouse was habituated to the testing arena (a large clean cage with bedding) 10 minutes daily. On the first day of testing, WT (n
5), HET (n
10), and KO (n
8) mice were placed in the testing arena with an unfamiliar C57Bl/6 male (age-matched) for 10 minutes. No obvious body weight difference was observed; historical data from our laboratory indicate that the average body weight of our Npas4 HET and WT mice at 12 weeks old were 31.00±3.46 g and 30.60±1.94 g, respectively, while the average body weight of a C57BL/6J from Jackson Laboratory at 12 weeks is 27.82±1.58 g (http://jaxmice.jax.org/support/weight/000664.html
). Twenty-four hours after the 1st
trial, subject animals were placed back in the testing arena with the C57Bl/6 male encountered during the 1st
trial. At the end of each trial the testing arena was changed for a new clean one. For each trial, the activity of the mice was recorded using an overhead camera. The behavior of the subject animals was analyzed offline using the Annotation software (Saysosoft; http://www.saysosoft.com
). Detailed social behaviors were recorded: (1) active social behavior: the subject animal actively sniffs, approaches/follows, or touches the C57Bl/6 mouse; (2) avoidance of social contact: the subject animal actively avoids the C57Bl/6 mouse; (3) proximity: mice are in body contact without sniffing each other; (4) non-social behavior.
- Tests of cognitive abilities
a. Spontaneous alternation.
Spontaneous alternation, as an indicator of working memory, was assessed using a modified version of the procedure developed by Lalonde (2002) in WT (n
21), HET (n
22), and KO (n
10). The apparatus consists of a gray plastic maze formed by three arms (A, B, and C) so as to form a Y shape (arm length: 40 cm; width: 8 cm; height: 15 cm). Each animal was placed in the apparatus for 8 minutes during which it was allowed to explore the entire maze. This test is based on a strong tendency in rodents to alternate arm entries, explained by their natural propensity to explore a novel environment over a recently explored one. The series of arm entries (e.g. ACBCABCBCA) was recorded using an overhead camera and videos were scored by an observer unaware of the animal's genotype. Alternation was defined as successive entry into the three arms on overlapping triplet sets (e.g. in the sequence ACBCABCBCA, five alternations were recorded). Percent alternation was calculated as the ratio of actual alternations to possible alternations (defined as the total number of arm entries minus two), multiplied by 100.
b. Place and reversal learning.
A place and reversal learning test was used to assess for spatial memory and cognitive flexibility. Only WT (n
15) and HET (n
17) animals were tested; because of the inability of KO animals to swim and because of their early age of death, Npas4-KO mice were not tested in this paradigm. The test took place in a Y-maze filled with tap water. The water was made opaque using non-toxic white paint. A clear escape platform was submerged 8 mm from the water's surface and external-maze visual cues were placed around the maze.
Before the first day of testing, animals were exposed to the water Y-maze without the platform to detect a possible turning bias. Mice were released from the start arm and explored the maze for 6 30-second trials with an ITI of 15 minutes. The first arm entered (left or right) was recorded. Six entries into the same arm constituted a turning bias.
The following day, spatial place learning took place. The platform was placed at the end of the left or right arm in a counterbalanced fashion unless the mouse had a turning bias, in which case the platform was placed on the opposite side of the bias. A trial began by placing a mouse at the end of the start arm and ended when the mouse climbed onto the platform and remained there for 2 s, or after 60 s had elapsed. Mice that did not escape to the platform after 60 s were gently guided to it. Mice were then left on the platform for an extra 15 s and then returned to their home cage. Each day, animals were tested 8 times (8 trials) with an ITI of 15 minutes. A correct response was defined as reaching the platform without entry into the opposite arm. The criterion for acquiring spatial learning was seven or eight correct responses per day for three consecutive days. Once this criterion was reached, the reversal learning began after a 24-hour ITI. The same procedure as used during spatial place learning was employed during reversal learning, except the platform was placed at the end of the opposite arm. The same criterion for acquisition and the same dependent measures were used (seven or eight correct responses per day for three consecutive days).
c. Novel object recognition.
The novel object recognition test was used to assess long-term memory. WT (n
13), HET (n
15), and KO (n
3) mice were tested in a 20×40 cm arena to which they were habituated for 15-min the day prior to testing. The arena contained visual cues on two of its walls. On the first day of testing, animals were placed in the arena with 2 identical unfamiliar objects positioned 5 cm away from the walls. Animals were allowed to explore the arena and the objects during a 10-min trial (training session). Twenty-four hours later, one of the objects was changed for a new unfamiliar one while the other object was identical to the ones used during the training session. Again, animals were allowed to explore the arena and the objects during a 10-min trial (testing session). Each trial was recorded using an overhead camera. The amount of time spent sniffing and with the head within 1 cm of the objects was scored as exploration of the objects. At the end of each trial, the arena and the objects were cleaned using 70% ethanol.
- Test of sensorimotor gating – Pre-pulse inhibition (PPI)
Sensorimotor gating was tested in the PPI test in WT (n
5), HET (n
10), and KO (n
8) mice using a startle apparatus (Med Associates, Georgia, VT, USA). During this test, two KO mice developed severe seizures and died. Thus, only 6 KO mice were tested. Seizures in KO mice have been observed by other experimenters 
. Each mouse was habituated to the equipment by being placed in the restraint device for 15 minutes per day for 3 days prior to the experiment. On testing day, subjects were exposed to a 70 dB white background noise for 5 minutes, and then exposed to three blocks of different startle pulses. In the first block, each animal was given five 120 dB startle pulses lasting 40 ms (no prepulses) with randomly variable inter-trial intervals of 10-20 s. In the second block, the animals encountered the same startle pulses with the addition of prepulse tones just prior to them. Each subject was given 12 exposures to each of the 6 different prepulses (0, 74, 78, 82, 86, and 90 dB), presented in random order for a total of 72 trials. The duration of the prepulse tone was 20 ms for all pre-pulses followed by a fixed interval of 100 ms (startle delay). After each prepulse tone, the animals received 40 ms of 120 dB startle pulse alone and the startle response of the animals was recorded. Inter-trial intervals for all 72 trials of Block 2 were randomly variable from 10–20 s. The maximum amplitude of the startle response in each trial was recorded for analysis. Block 1 and 2 were used for PPI measurements. For each PPI dB level (block 2), the PPI% was calculated using the following equation: [(startle response with startle pulse alone – startle response with prepulse and subsequent startle pulse)/ startle response with startle pulse alone] X 100. In the third block, each animal received 25 trials with 10–20 s randomly variable inter-trial intervals. Five different intensities of startle pulses (0, 90, 100, 110, 120 dB) lasting 40 ms were used for this block. Each animal was exposed 5 times to each intensity of startle pulses, which were given in random order. One ms after the onset of the startle stimulus, the startle response of the subject was recorded for 65
ms. This block 3 was used to assess acoustic startle. The animal holder of the apparatus was cleaned with 20% alcohol between each animal.
a. Npas4 immunohistochemistry.
Eight male C57Bl/6J mice were used in this experiment. One week prior to the experiment, mice were singly-housed. One group of mice (n
4) was placed in a social situation while the other group of mice (n
4) was used as controls. For the social situation, each mouse was placed in a new clean cage with a never-before met male adult mouse. Each pair was left undisturbed for 10 minutes. At the end of the 10 minutes, mice were placed back in their home-cage and left undisturbed for 45 minutes. The control mice were placed individually in a new clean cage for 10 minutes. After 10 minutes, they were placed back in their home-cage and left undisturbed for 45 minutes. Animals were then anaesthetized with isoflurane and perfused transcardially with 50 ml of 0.1 M phosphate-buffered saline (pH 7.4) followed by 50 ml of fresh 4% paraformaldehyde (PFA). Brains were removed and post-fixed overnight in PFA at 4°C, placed in 30% sucrose until they sank, then frozen on dry ice and sectioned at 50 µm using a cryostat. Tissues were stored in cryoprotectant solution at −20°C until Npas4 staining was performed. For immunohistochemistry, free-floating sections were labeled with a rabbit anti-Npas4 primary antibody (dilution 1
15,000), detected using Vectastain Elite ABC reagents (Vector Laboratories, Burlingame, CA, USA) with nickel intensified diaminobenzidine.
b. c-Fos immunohistochemistry.
Three WT, 3 HET, and 3 KO mice were stained for c-Fos after novel object recognition, a hippocampal-dependent memory task. Animals were tested in this task as described earlier. Ninety minutes after the testing session, animals were anaesthetized with isoflurane and perfused transcardially as described earlier. Brains were treated as previously described. For immunohistochemistry, free-floating sections were labeled with a rabbit anti-c-Fos primary antibody (dilution 1
10,000; Ab-5; Calbiochem/EMD, San Diegeo, CA, USA), detected using Vectastain Elite ABC reagents (Vector Laboratories, Burlingame, CA, USA) with nickel intensified diaminobenzidine.
c. Image analysis.
Images were captured with a Zeiss Imager M2 microscope (Carl Zeiss Vision, Munich, Germany). The quantitative analysis of Npas4 expression in the hippocampus of C57Bl/6j mice was achieved using an optical density analysis as previously described 
. Briefly, images were taken with a 5x objective at four different Bregma levels of the dorsal hippocampus (Bregma −1.22 mm; Bregma −1.32 mm; Bregma −1.94 mm; Bregma −2.30 mm), two different levels of the ventral hippocampus (Bregma −2.54 mm; Bregma −2.70 mm) and two different levels of the amygdala (Bregma −1.06 mm; Bregma −1.82 mm) according to the Mouse Brain Atlas of Franklin and Paxinos 
. Images were analyzed using Image J software (http://rsbweb.nih.gov/ij/
). In this program, a grey level of 0 represents black (no transmission) and 255 is white (full transmission). The measured parameters included the mean grayscale value of each drawn region of interest (CA1, CA2, CA3, and dentate gyrus [DG] in the hippocampus and medial amygdala [MeA], central amygdala [CeA], and basolateral amygdala [BLA] in the amygdala). Three measures were taken from each image capture: (1) a non tissue area that contained mounting media and coverslip but no tissue; this provided the measurement of the incident light, (2) connective tissue containing no cells to provide the measurement of background; (3) the region of interest. The optical density (OD) was derived using the formula: OD
(incident light/transmitted light). This formula inverts the grey level scaling so that intense (dark) signal results in a high OD value. The background OD value was subtracted from each OD value measured at the region of interest to normalize the data.
The quantitative analysis of c-Fos expression in the hippocampus of Npas4 mice was achieved using the stereology method. The total number of c-Fos positive cells in the CA1, CA2, CA3, and DG of the dorsal hippocampus was quantified using the optical fractionator method 
, with assistance from the StereoInvestigator software from MBF Bioscience (Williston, VT, USA). Cells were counted in every 4 sections. Regions of interest were outlined under low magnification (5x), and c-Fos positive cells counted at high magnification (63x oil immersion). The counting criteria were determined so as to obtain a mean coefficient of error (CE, 
) below 0.11.
Data were analyzed using the software Prism 5.01 (GraphPad Software Inc., CA, USA). All data were tested for normality using the Kolmogorov-Smirnof test and then analyzed using appropriate tests as described in the results section. Statistical significance was set at p≤0.05. All data are presented as mean ± standard error of the mean (SEM).