Neuroligin-2 knockout (KO) mice were generated using SM1 embryonic stem cell clone derived from 129S6/SvEvTac mouse, and the resulting chimeric mice were bred with C57BL/6J mice to obtain F1 heterozygous KO mice. Thus, the F1 mice were on a 129S6/SvEvTac/C57BL/6J hybrid background. KO mice were maintained by interbreeding mice heterozygous for the NL2 allele for approximately 30 generations. Littermates were used for the breeding in some generations, though this was avoided as much as possible. Prior to the behavioral study, KO mice were backcrossed to C57BL/6NCrl mice for two generations and subsequently to 129S2/SvPasCrlf mice for another two generations. Resulting NL2 heterozygote mice were interbred and resulting age and sex-matched littermate pair offspring were used for behavioral studies. The use of such a hybrid background minimizes the possibility of deleterious recessive mutations that occur in inbred strains being homozygous in the experimental mice.
Male NL2 knockout and littermate control mice were anesthetized and perfusion-fixed with 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4). Brains were removed and immersion-fixed for 4 hours in 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4) and cryoprotected with 30% sucrose in PBS for 2 days at 4 °C. Labels on glass vials storing brain samples were removed and coded for blind experiment. 30 μm serial parasagittal sections were cut on a cryomicrotome and blocked with 3% goat serum/0.3% Triton X-100 in PBS and incubated with anti-synaptophysin monoclonal antibody (Millipore, Billerica, MA), anti-VGlut1 monoclonal antibody (Synaptic System, Göttingen, Germany), and/or anti-VGAT polyclonal antibody (Millipore, Billerica, MA) overnight at 4 °C, followed by incubation with Alexa Fluor 488 or 633 conjugated goat anti-mouse IgG (Invitrogen, Eugene, OR). Sections were transferred onto SuperFrost slides and mounted under glass coverslips with Vectashield with DAPI (Vector Laboratories, Burlingame, CA). For each brain section, areas including the center portion of the CA1 and CA3 subfields of the hippocampus were imaged with a Leica TCS2 laser-scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) at 63×, and the stratum radiatum layer of the CA1 and CA3 regions (where the dendrites of pyramidal neurons receive synaptic inputs) was magnified fivefold. For each protein of interest, images were acquired with identical settings for laser power, photomultiplier gain and offset with a pinhole diameter. Images of identical regions (specified above) were acquired from 15 sections from each of 3 animals/genotype. Images were imported into ImageJ software for morphometric analysis. In the software, images were converted into binary data and thresholded to outline immunopositive particles.
Thresholds were determined to outline as many immunopositive puncta as possible throughout all images. Identical thresholds were used for the same sets of experiments (threshold = 60 for synapsin and VGlut1 staining, threshold = 20 for VGAT staining). The number and size of puncta were detected using the “analyze particle” module of the program. The average number and size of immunopositive puncta were normalized with data from wild-type to determine synaptic density and size, respectively. Statistical significance was determined by Student’s t test. All of the data shown are means ± SEM.
Male NL2 KO littermates (4 WT and 4 KO, 8 weeks of age) were anesthetized and vascularly perfused through the heart with 2% paraformaldehyde and 1% glutaraldehyde in 100 mM phosphate buffer (pH 7.4) for the first 15 min. Brains were removed and immersion fixed with 2% paraformaldehyde and 2% glutaraldehyde in the 100 mM cacodylate buffer overnight at 4 °C. The tissue was sectioned using a vibratome at 200 μm thickness. The hippocampus of each section was dissected out before the post-fixation (1 hr) with 1% OsO4, 0.8% potassium ferricyanide and en bloc stained with 2% uranyl acetate for 15 min. After dehydration in a series of ethanol up to 100%, slices were embedded in Poly/bed 812 (Polysciences Inc., Warrington, PA) for 24 hr. Thin sections (65 nm) were made and post-stained with uranyl acetate and lead citrate, and viewed under a FEI Tecnai transmission electron microscope at 120 kV accelerating voltage. All EM images were captured by a 4k × 4k CCD camera at magnifications of 30,000, and quantitative analyses were conducted on the digital EM micrographs of the same magnification. Images were taken in the stratum radiatum layer of the CA1 hippocampal region, and all images were within 20–30 μm of the inner layer of pyramidal neuron cell bodies. A total of 258 EM micrographs were analyzed. From the 4 KO mice, 30, 45, 25, and 25 images were randomly selected for analysis, and from the 4 WT mice, 42, 42, 26, and 36 images were randomly selected for analysis. The measurement was performed without knowledge of the genotyping and was assisted by MetaMorph software (Molecular Devices, Union City, CA). The final data were derived from the number of synapses in the following sequence: Asymmetric/symmetric/unidentifiable. The statistical significance was calculated with SigmaPlot and Microsoft Excel.
Protein compositions were determined by immunoblotting on whole brain tissues homogenized in PBS, 10 mM EDTA, and proteinase inhibitors from four pairs of P40 littermate mice per genotype. 40 μg of proteins were loaded per lane and blotted with antibodies for synaptic proteins and internal controls (β-actin or GDI). Blots were reacted with 125I-labeled secondary antibodies followed by PhosphoImager (STORM 860 Amersham Pharmacia Biotech) detection.
Mice were age/sex-matched littermate progeny of heterozygous/heterozygous matings tested behaviorally in two groups. Experimenters were blind to genotype. For all behavioral tests, the number of NL2 KO littermate pairs was 22 (total of 44 mice). No significant sex × genotype interactions were found during the statistical analysis of any test (, NL2, n = 10 male pairs, 12 female pairs). For shock threshold, pain sensitivity, and the test of olfaction, however, only 15 littermate pairs were tested (30 mice total) as some of the mice were removed for histological studies. All mice ranged from 2–4 months of age during the behavioral testing, and within each group mice were born within 4 weeks of each other. Less stressful behaviors were tested first, with more stressful procedures at the end. The order of tests was as follows: locomotor, dark/light box, open field, accelerating rotarod, social interaction with a juvenile, social learning, social vs. inanimate preference test, preference for social novelty test, social interaction with an adult caged conspecific, hot plate sensitivity, and shock threshold. Mice were moved within the animal facility to the testing room and allowed to habituate to the new location for at least one hour prior to behavioral testing. Significance was taken as p < 0.05 for all experiments and a complete description of statistical results can be found in .
Detailed Statistical Analysis
Anxiety-like Behavioral Tests
The dark/light and open field tests were performed essentially as described (Powell et al., 2004
). In the dark/light test, one side of the apparatus was kept dark (room light entry limited) while a light built into the top lit the other side (1700 lux, each chamber 25 cm × 26 cm). Mice were placed in the dark side and allowed to freely explore the light and dark sides for 10 minutes. Anxiety-like behavior was measured using latency to enter the light side, time in the dark side, and number of crosses into the light side. Locomotor activity was also examined in both the light and dark sides of the apparatus. The open field test was performed for 10 min in a brightly lit (~800 lux), 48 × 48 × 48 cm white plastic arena using video tracking software from Noldus (Ethovision 2.3.19). Time spent in the center zone (15 × 15 cm) and frequency to enter the center was recorded. Locomotor activity was also measured during the open field test. Data were analyzed with a 2-way ANOVA for genotype and sex.
An accelerating rotarod designed for mice (IITC Life Sciences) was used essentially as described (Powell et al., 2004
) except 3 sets of 3 trials were performed per day over 3 days. Briefly, the rotarod was activated after placing a mouse on the motionless rod. The rod accelerated from 0 to 45 revolutions per min in 60 s. Time to fall off the rod or to turn one full revolution was measured. Data were analyzed with a mixed ANOVA for genotype, sex, and the repeated measures of trial.
Hot Plate Sensitivity
Mice were placed on a black, anodized, constant-temperature plate of 52 °C (IITC Model 39 Hot Plate) covered with a Plexiglas enclosure. Latency to lick any paw was measured. Mice were removed upon first paw lick or after 30 s if no response was elicited, and the plate was cleaned with water between mice and allowed to return to room temperature. Data were analyzed with a 2-way ANOVA for genotype and sex.
Footshock threshold analysis was performed by placing mice in the fear conditioning apparatus (described in (Powell et al., 2004
) for a two min habituation followed by a two s footshock with an interstimulus interval of 20 s of gradually increasing intensity from 0.05 mA at 0.05 mA intervals. The intensity required to elicit flinching, jumping and vocalizing was recorded by an observer blind to genotype. Data were analyzed with a 2-way ANOVA for genotype and sex.
Social Interaction and Social Learning
Direct social interaction with a juvenile took place in a novel, empty, clear, plastic mouse cage under red light (Kwon et al., 2006
). Following a 15 min habituation in the dark, the experimental and target mice were placed in the neutral cage for two min and allowed to directly interact. Time spent interacting with the juvenile was scored by an observer blind to genotype. Social learning was assessed three days later by allowing mice to interact with the same juvenile for an additional two min. Again, time spent interacting with the juvenile was scored. Data were analyzed with a 3-way mixed ANOVA with genotype and sex as between subjects factors and test session as a within subjects factor.
Social versus inanimate preference and preference for social novelty analyses were performed as described (S. S. Moy et al., 2004; J. J. Nadler et al., 2004) except room and door dimensions were different (15 × 90 × 18.5 cm divided into three compartments of 15 × 29 cm separated by dividers with a central 3.8 × 3.8 cm door), and videotracking software from Noldus (Ethovision 2.3.19) was used to record mouse behavior (Kwon et al., 2006
). In the test, mice were initially allowed to explore the apparatus for 10 min. Then, mice were allowed to interact with an empty cage in one compartment versus a caged social target in the far compartment for another 10 min. The test for social novelty involved a subsequent 10 min test in which mice were allowed to interact with the familiar caged adult, or a novel caged adult. Location of empty cages and target mouse as well as novel versus familiar mouse was counterbalanced. The test was done under red light and the box was wiped with 70% ethanol and air-dried between mice. Data were analyzed with a 3-way mixed ANOVA with genotype and sex as between subjects factors and interaction target as a within subjects factor.