Animals and experimental design
alpha-CaMKII+/- mice were obtained from Jackson Laboratories (Bar Harbor, Maine). We used heterozygous alpha-CaMKII knockout mice, because it is hard to obtain homozygotes, due to a difficulty of mating between heterozygous male and heterozygous female mice. Mice were housed one per cage in a room with a 12-hr light/dark cycle (lights on at 7:00 a.m.) with access to food and water ad libitum. Behavioral testing was performed between 9:00 a.m. and 6:00 p.m. After the tests, the apparatus were cleaned with super hypochlorous water to prevent a bias due to olfactory cues. All behavioral tests were conducted in a manner similar to those described previously [14, 56, 57]. All behavioral testing procedures were approved by the Animal Care and Use Committee of Kyoto University Graduate School of Medicine.
Neurological screen was performed with 9 to 10-wk-old male mice. 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 was performed with 9 to 10-wk-old male mice, and tested 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 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 s cut-off time.
Motor coordination and balance were tested with the rotarod test. The rotarod test, using an accelerating rotarod (UGO Basile Accelerating Rotarod), was performed by placing 11 to 12-wk-old 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.
Open field test
Locomotor activity was measured using an open field test. Open field test was performed with 10 to 11-wk-old male mice. Each mouse was placed in the center of the open field apparatus (40 × 40 × 30 cm; Accuscan Instruments, Columbus, OH). Total distance traveled (in cm), vertical activity (rearing measured by counting the number of photobeam interruptions), time spent in the center, the beam-break counts for stereotyped behaviors, and number of fecal boli were recorded. Data were collected for 120 min.
Light/dark transition test
Light/dark transition test was performed with 9 to 10-wk-old male mice, as previously described . The apparatus used for the light/dark transition test consisted of a cage (21 × 42 × 25 cm) divided into two sections of equal size by a partition containing a door (O'Hara & Co., Tokyo, Japan). 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 with the door open for 10 min. The total number of transitions between chambers, time spent in each side, first latency to enter the light side and distance traveled were recorded automatically.
Elevated plus-maze test
Elevated plus-maze test was performed with 10 to 11-wk-old male mice. The elevated plus-maze (O'Hara & Co., Tokyo, Japan) 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 were elevated to a height of 55 cm above the floor. To minimize the likelihood of animals falling from the apparatus, 3-mm high plastic ledges were provided for the open arms. Arms of the same type were arranged at opposite sides to 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, and the time spent in open and enclosed arms, were recorded. For data analysis, we used the following four measures: the percentage of entries into the open arms, the time spent in the open arms (s), the number of total entries, and total distance traveled (cm). Data acquisition and analysis were performed automatically using Image EP software.
Hot plate test
The hot plate test was used to evaluate sensitivity to a painful stimulus. 10 to 11-wk-old mice were placed on a 55.0 (± 0.3)°C hot plate (Columbus Instruments), 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.
Startle response/prepulse inhibition tests
Startle response/prepulse inhibition tests were performed with 11 to 12-wk-old male mice. A startle reflex measurement system was used (O'Hara & Co., Tokyo, Japan) to measure 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 ms) was used as the startle stimulus for all trial types. The startle response was recorded for 140 ms (measuring the response every 1 ms) starting with the onset of the prepulse stimulus. The background noise level in each chamber was 70 dB. The peak startle amplitude recorded during the 140 ms 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 ms 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 s (range: 10–20 s).
Social interaction test in a novel environment
Social interaction test in a novel environment was performed with 10 to 11-wk-old male mice. Two mice of identical genotypes that were previously housed in different cages, were placed into a box together (40 × 40 × 30 cm) and allowed to explore freely for 10 min. Social behavior was monitored by a CCD camera, which was connected to a Macintosh computer. Analysis was performed automatically using Image SI software. Total duration of contact, the number of contacts, the number of active contacts, mean duration per contact, and total distance traveled were measured. The number of active contacts was defined as follows. Images were captured at 1 frame per second, and distance traveled between two successive frames was calculated for each mouse. If the two mice contacted each other and the distance traveled by either mouse was longer than 5 cm, the behavior was considered as 'active contact'.
Porsolt forced swim test
Porsolt forced swim test was performed with 11 to 12-wk-old male mice. The apparatus consisted of four plastic cylinders (20 cm height × 10 cm diameter). The cylinders were filled with water (23°C) up to a height of 7.5 cm. Mice were placed into the cylinders, and their behavior recorded over a 10-min test period. Data acquisition and analysis were performed automatically, using Image PS software (see 'Image Analysis'). Distance traveled was measured by Image OF software (see 'Image Analysis') using stored image files.
Eight-arm radial maze test
Eight-arm radial maze test was performed with 13 to 14-wk-old male mice. Fully-automated eight-arm radial maze apparatuses (O'Hara & Co., Tokyo, Japan) were used. 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 pellets sensors were able to automatically record pellet intake by the mice. The maze was elevated 75 cm above the floor and placed in a dimly-lit room with several extra-maze cues. During the experiment, the maze was maintained in a constant orientation. One week before pretraining, animals were deprived of food until their body weight was reduced to 80% to 85% of the initial level. Pretraining started on the 8th day. Each mouse was placed in the central starting platform and allowed to explore and consume food pellets scattered on the whole maze for a 30-min period (one session per mouse). After completion of the initial pretraining, mice received another pretraining to take a food pellet from each food well after being placed at the distal end of each arm. A trial was finished after the mouse consumed the pellet. This was repeated eight times, using eight different arms, for each mouse. After these pretraining trials, 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 traveling more than 5 cm from the central platform. The mice were confined at the center platform for 5 s after each arm choice. The animals went through one trial per day. 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. In the reference memory task of the eight-arm radial maze, one of the eight arms was consistently baited with one food pellet in the food well and a trial was terminated immediately after the one pellet was consumed. Data acquisition, control of guillotine doors, and data analysis were performed by Image RM software (see 'Image analysis').
Locomotor activity monitoring in home cage
Circadian rhythm monitoring in home cage was performed with 18 to 19-wk-old male mice. A system that automatically analyzes the locomotor activity of mice in their home cage was used . The system contains a home cage (29 × 18 × 12 cm) and a filtered cage top, separated by a 13-cm-high metal stand containing an infrared video camera, which is attached to the top of the stand. Each mouse was individually housed in each home cage, and their locomotor activity was monitored for at least 3 mo. Outputs from the video cameras were fed into a Macintosh computer. Images from each cage were captured at a rate of one frame per second, and distance travelled was measured automatically using Image HA software (see 'Image analysis').
The applications used for the behavioral studies (Image LD, Image EP, Image RM, Image FZ, Image SI, and Image HA) were based on the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/) and ImageJ program http://rsb.info.nih.gov/ij/, which were modified for each test by Tsuyoshi Miyakawa (available through O'Hara & Co., Tokyo, Japan).
Statistical analysis was conducted using StatView (SAS Institute, Cary, NC). Data were analyzed by two-way ANOVA, or two-way repeated measures ANOVA, unless noted otherwise. Values in tables and graphs were expressed as mean ± s.e.m.
Golgi impregnation was performed using FD Rapid GolgiStain™ kit according to the manufacturer's directions (FD NeuroTechnologies, Inc., Ellicott City, MD). Briefly, the brains of 45-wk old control mice (n = 5) and mutant mice (n = 5) were obtained after cervical dislocation, immersed in the impregnation solution for one week in the dark, and cryoprotected for 48 h. After sectioning (100 μm-thick) and mounting on adhesive-coated slides, sections were stained in a development solution. After the slides containing hippocampal sections were scanned with a light microscope (Zeiss Axioplan2) outfitted with a CCD camera, the resulting images were analyzed by the ImageJ software http://rsb.info.nih.gov/ij/. All slides were coded prior to the quantitative analysis, and the code was broken only after the analysis was completed. To be selected for analysis, Golgi-impregnated neurons had to satisfy the following criteria: (1) dark and consistent impregnation throughout the extent of the dendrites, (2) relative isolation from neighboring impregnated cells, and (3) cell bodies that were located toward the middle of the slice with dendrites lying in the same plane as the cell body. The number of dendritic intersections and dendritic length were measured in successive radial segments of 20 μm distance, using the center of the soma as a reference point (Sholl's analysis). Images of Golgi-impregnated neurons were captured using a Keyence Biozero BZ-8000.
Biochemical assay of monoamine levels
Levels of dopamine, noradrenaline, serotonin, and their metabolites in the brain samples were determined by high-performance liquid chromatography (HPLC) with electrochemical detection . Control mice (n = 9) and mutant mice (n = 9) (10 to 12-wk-old) were killed by decapitation after which their hippocampus, striatum, nucleus accumbens, and medial prefrontal cortex were immediately dissected and frozen in liquid nitrogen. Wet tissue samples were weighed and stored at -80°C until subsequent analysis. Tissues were homogenized in 9 volumes of 0.2 M perchloric acid prior to centrifugation at 20,000 g for 10 min. After the supernatant was neutralized with sodium acetate, the samples were analyzed by HPLC.
Adult male alpha-CaMKII+/- mice and their wild-type littermates (13 to 18 wk old) were used for electrophysiological experiments. Mice were decapitated under halothane anesthesia and both hippocampi were isolated. Transverse hippocampal slices (370 mm) were cut using a tissue slicer (Leica VT1000S, Nussloch, Germany) in sucrose-containing saline composed of (in mM): sucrose 72, NaCl 80; KCl, 2.5; NaH2PO4, 1.0; NaHCO3, 26.2; glucose, 20;CaCl2, 0.5; MgCl2, 7; kynurenic acid, 0.1 – 0.2 (equilibrated with 95% O2/5% CO2). Slices were then incubated for 30 min at 30°C in standard saline (see below) and maintained in a humidified interface holding chamber at room temperature (24 – 27°C) before use. Electrophysiological recordings were made from slices placed in a submersion-type chamber superfused at 2 ml/min with the standard saline composed of (in mM): NaCl 125; KCl, 2.5; NaH2PO4, 1.0; NaHCO3, 26.2; glucose, 11;CaCl2, 2.5; MgCl2, 1.3 (equilibrated with 95% O2/5% CO2). Bath temperature was maintained at 26.5 – 27.5°C using an automated temperature controller. All procedures were approved by the institutional Animal Care and Use Committee.
Whole-cell recordings were made from granule cells in the dentate gyrus by using the blind whole-cell patch-clamp technique. Current-clamp recordings were made with a pipette filled with a solution composed of (in mM) potassium gluconate 140, HEPES 20, NaCl 8, MgATP 2, Na2GTP 0.3, EGTA 0.05 (pH adjusted to 7.2 with KOH). The recording pipette was placed in the middle third of the granule cell layer. Resting membrane potentials were measured immediately after rupture of the patch membrane. Liquid junction potentials were not corrected. Hyperpolarizing currents (4 – 16 pA, 400 ms) were injected through the recording pipette to measure the input resistance. Depolarizing currents (400 ms) were injected to evaluate action potential firing properties. The current intensity was increased by 20 to 40 pA steps, and the number of action potentials and the latency of the first action potential were measured.
Field excitatory postsynaptic potentials (EPSPs) arising from mossy fibers (MFs) were recorded from the CA3 region of the hippocampus. Bipolar tungsten stimulating electrodes were placed in the dentate gyrus granule cell layer to activate MFs and a recording glass electrode filled with 2 M NaCl was placed in the stratum lucidum of the CA3 region. Single electrical stimulations were delivered at a frequency of 0.05 Hz unless otherwise specified. An agonist of group II metabotropic glutamate receptors, (2S,2'R,3'R)-2-(2',3'-Dicarboxycyclopropyl)glycine (DCG-IV), which selectively suppresses the MF input, was applied at the end of each experiment. DCG-IV was less effective in mutant mice (92.8 ± 1.0% block in wild-type mice, n = 20; 87.6 ± 0.5% block in mutant mice, n = 23; p < 0.0001). Given changes in the expression level of various genes in these mutant mice, this reduction of the DCG-IV effect might be due to altered expression of metabotropic glutamate receptors and/or molecules involved in downstream signaling. The EPSP amplitude was measured throughout the study following a previously described method unless otherwise specified . In most experiments, the experimenter was blind to the genotype of the mice. All recordings were made using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA), filtered at 2 to 10 kHz and stored in a personal computer via a digital interface (digitized at 5 – 20 kHz). DCG-IV was purchased from Tocris (Bristol, UK). All values are expressed as means ± s.e.m. Statistical significance was evaluated by two-tailed Student's t-test with a significance level of p less than 0.05.
Microarray experiments were performed with 12-wk-old male mice (3 control mice and 3 mutant mice) and 26 to 40-wk old mice (6 control mice and 15 mutant mice). The older mice had been tested in the behavioral test battery, including general health and neurological screen, light/dark transition test, open field test, elevated plus maze test, social interaction test, rotarod, prepulse inhibition, and Porsolt forced swim test. RNA was isolated by using the TRIzol method (Invitrogen, Carlsbad, CA) from the hippocampus of control mice (n = 9) and alpha-CaMKII+/- mice (n = 18), followed by purification, using RNeasy columns (Qiagen, Valencia, CA). Double-stranded cDNA was synthesized from the total RNA, and in vitro transcription reaction was then performed on biotin-labeled RNA that was made from the cDNA. Labeled RNA was hybridized with Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA) containing 45101 probe sets, and washed according to the manufacturer's recommendations. The hybridized probe array was then stained with streptavidin-conjugated phycoerythrin, and each GeneChip was scanned by an Affymetrix GeneChip Scanner 3000 (GCS3000). GeneChip analysis was performed with Microarray Analysis Suite version 5.0. All of the genes represented on the GeneChip were globally normalized. Two-way analysis of variance was performed for each probe, and the probes, for which the expression levels were significantly changed (genotype effect, p < 0.05) and not affected by the interaction between genotype and age (p > 0.05), were identified. Expression data and other information used in this paper will be available at the ArrayExpress database (http://www.ebi.ac.uk/microarray-as/aer/, accession number: E-MEXP-962).
RT-PCR was performed to validate the microarray results. Total RNA was isolated from 27 to 29-wk-old control mice and alpha-CaMKII+/- mice. First-strand cDNA was prepared from 2 ug of DNase I-treated total RNA using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA). The expression of related genes was quantified using the SYBR green reagent (2× SYBR Green PCR Master Mix; Qiagen, Valencia, CA) following the instructions of the manufacturer. Quantitative PCR was performed using DNA Engine Opticon 2 Real-Time PCR Detection System(Bio-Rad, Hercules, CA) with conditions as follows: 15 min at 95°C, then 45 cycles of 15 sec at 94°C, 30 sec at 60°C, 30 sec at 72°C and 1 min at 65°C. β-actin was amplified from all samples to normalize expression. The following primers were used: tryptophan 2,3-dioxygenase(1–105), 5'-ATGAGTGGGTGCCCGTTTG and 5'-GGCTCTGTTTACACCAGTTTGAG; desmoplakin(7–113), 5'-GCTGAAGAACACTCTAGCCCA and 5'-ACTGCTGTTTCCTCTGAGACA; interleukin 1 receptor, type I(16–149), 5'-GTGCTACTGGGGCTCATTTGT and 5'-GGAGTAAGAGGACACTTGCGAAT; nephronectin(88–206), 5'-GTTTCTTCAATCGGCCTATGTCG and 5'-ATCCTTGCTAGTCTCTGCCTT; pregnancy upregulated non-ubiquitously expressed CaM kinase(142–293), 5'-AAGAAAGCACTTCGGGGCAA and 5'-AGTTCACCACCTGTTACCAGC; solute carrier family 39 (metal ion transporter), member 6(419–524), 5'-GTCACACGGTTGCTGGTAAAA and 5'-GGGCGAGATCCTTTCCCTAGA; β-actin(851–962), 5'-AGTGTGACGTTGACATCCGTA and 5'-GCCAGAGCAGTAATCTCCTTCT. Ct values used were the means of duplicate replicates.
Human genetic study
In the association analyses, two independent samples were examined: for the initial screening, 376 patients with schizophrenia (195 males and 181 females; mean age ± standard deviation (SD) 42.5 ± 14.8 y), 115 patients with bipolar disorder (56 males and 59 females; 46.2 ± 13.7 y), and 347 controls (194 males and 153 females; 34.6 ± 13.6 y), and for the following confirmation analysis, 368 patients with schizophrenia (204 males and 164 females; 52.8 ± 13.7 y), 149 patients with bipolar disorder (76 males and 73 females; 47.1 ± 15.2 y), and 382 controls (202 males and 180 females; 40.3 ± 13.6 y).
Sixty-four controls were used as subjects for linkage disequilibrium (LD) evaluation. These subjects were also included in the initial screening scan. Characterization details and psychiatric assessment of these subjects were identical to those published elsewhere [61, 62]. All subjects were unrelated to each other and ethnically Japanese.
After the study was described to the subjects, written informed consent was obtained from each subject. This study was approved by the Ethics Committee at Fujita Health University and Nagoya University School of Medicine.
Primer pairs were designed using information from the GenBank sequence (accession number: NT-029289.10) and 23 amplified regions, which covered the coding exon and 5'-flanking region 968 bp upstream. The details are as previously described . Sequences of primer pairs are available on request.
Single nucleotide polymorphism (SNP) selection and Linkage Disequilibrium (LD) evaluation
For the evaluation of LD, we included SNPs from databases (dbSNP, NCBI; and SNPbrowser2.0, Applied Biosystems, Foster City, CA). First we determined 'LD blocks' with the criteria D' > 0.8, using HAPLOVIEW ver 3.0 software . We excluded the minor allele frequencies of SNPs less than 0.05. Next, haplotype-tagging SNPs were selected within each LD block for 90% haplotype coverage using SNPtagger software .
We used TaqMan assays, primer extension using denaturing high performance liquid chromatography, PCR-RFLP assays, and direct sequencing. Primer pair sequences are available upon request.
Genotype deviation from the Hardy-Weinberg equilibrium (HWE) was evaluated by c2 test (SAS/Genetics, release 8.2, SAS Institute Japan Inc., Tokyo, Japan). Marker-trait association was evaluated allele/genotype-wise with c2 test (SPSS 10.0 J, SPSS Japan Inc.), and haplotype-wise with the log likelihood ratio test (COCAPHASE 2.403 program ). Rare haplotypes found in less than 5% of cases and control subjects were excluded from the association analysis to provide greater sensitivity and accuracy. The significance level for all statistical tests was 0.05.
Frozen brains were obtained from 25-wk-old alpha-CaMKII+/- mice and control mice, and were cut into 10-mm-thick coronal sections with a HM560 cryotome (Carl Zeiss, Oberkochen, Germany). The sections were mounted on slide glass (Matsunami Glass, Osaka, Japan) and stored at -80°C until analyzed. Dopamine D1 and NMDA receptor levels were determined analyzing specific binding of [3H]SCH233090 (1 nM) and [3H]MK801 (5 nM), respectively, to the brain slices using autoradiography. The buffers and incubation times were as described in previous reports [67, 68]. Non-specific binding of [3H]SCH23390 and [3H]MK801 was determined by adding 1 mM of flupenxiol and 200 mM of ketamine, respectively, to the reaction. Dopamine D2, dopamine transporter, serotonin transporter, and central benzodiazepine binding site levels were determined by [3H]raclopride (2 nM), [3H]GBR12935 (1 nM), [3H]citalopram (1 nM), and [3H]flumazenil (1 nM), respectively. The buffers and incubation times were as described in previous reports [67, 69–71]. Non-specific binding of [3H]raclopride, [3H]GBR12935, [3H]citalopram and [3H]flumazenil were determined by adding 1 mM of butaclamol, 10 mM of nomifensine, 1 mM of fluoxetine, and 10 mM of flunitrazepam, respectively. Following the incubation, the samples were rinsed with ice-cold buffer, and desalted with ice-cold distilled water. The slices were subsequently dried under warm blowing air and exposed to an imaging plate (Fuji Film, Tokyo, Japan) for 10 to 14 days. The imaging plate was subsequently scanned with a BAS5000 system (Fuji Film). Regions of interest (ROIs) were defined on the images using a Multi Gauge® software (Fuji Film), and densitometric assay for each ROI was performed using autoradiographic [3H]micro-scales (GE Healthcare Bio-Sciences Corp., Piscataway, NJ).
After the brains of 11 to 12-wk-old control mice (n = 6) and mutant mice (n = 6) were dissected, hippocampus (HF), striatum (Str), cingulate cortex (CC), and amygdala (AM) tissues were homogenized in 300 μl of homogenizing buffer containing 50 mM Tris-HCl (pH 7.4), 0.5% Triton X-100, 4 mM EGTA, 10 mM EDTA, 1 mM Na3VO4, 40 mM sodium pyrophosphate, 50 mM NaF, 100 nM calyculin A, 50 μg/ml leupeptin, 25 μg/ml pepstatin A, 50 μg/ml trypsin inhibitor, and 1 mM dithiothreitol. Insoluble material was removed by a 10-min centrifugation at 15,000 rpm. After determining protein concentration in supernatants using Bradford's solution, samples were boiled for 3 min in Laemmli's sample buffer . Samples containing equivalent amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to an Immobilon PVDF membrane for 2 h at 70 V. After blocking with TTBS solution (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 2.5% bovine serum albumin for 1 h at room temperature, membranes were incubated overnight at 4°C with anti-phospho CaMKII (1:5000) , anti-CaMKII (1:5000) , calcineurin A (1:2000) , anti-phospho-ERK (1:2000, Cell Signaling, Beverly, MA). Bound antibodies were visualized using the enhanced chemiluminescence detection system (Amersham Life Science, Buckinghamshire, UK) and analyzed semiquantitatively using the National Institutes of Health Image program.
For the analysis of the expression of markers related to neurogenesis and differentiation, at least five (7-wk-old) wild-type and heterozygote mice were examined. There was a consistent difference between the genotypes. Mice were deeply anesthetized with sodium pentobarbital and transcardially perfused with 4% paraformaldehyde (PFA) and 0.5% picric acid in 0.1 M phosphate buffered saline (PBS). The brains were removed and further immersion-fixed in the same fixative at 4°C for 2 h and 14-mm-thick coronal sections were prepared on a cryostat (Leica). The sections were washed with Tris-buffered saline containing Tween 20 (pH 7.4). For immunostaining, the cryostat sections were incubated at 4°C for 18 h with the following primary antibodies: mouse anti-polysialic acid-NCAM (PSA-NCAM) monoclonal antibody (IgM, 1:500 dilution; CHEMICON, Temecula, CA), rabbit anti-calretinin 7699/4 polyclonal antibody (1:3000 dilution; SWANT, Bellinzona, Switzerland), rabbit anti-calbindin D-28k polyclonal antibody (1:3000 dilution; SWANT), and mouse anti-calmodulin-dependent protein kinase II (CAMKII) monoclonal antibody (IgG, 1:500 dilution; CHEMICON). For detection of the antigen localization, the sections were incubated at 4°C for 2 h with Alexa Fluor (488 or 594)-conjugated goat anti-mouse IgM or IgG (1:400 dilution; Invitrogen) and/or Alexa Fluor (488 or 594)-conjugated goat anti-rabbit IgG (1:400 dilution; Invitrogen). Fluorescent signals were detected using a confocal laser-scanning microscope (LSM5 Pascal, Zeiss) or a fluorescence microscope (Axioplan-2, Zeiss).
Analysis of c-Fos expression following footshock
For the analysis of c-Fos expression, electric shocks (0.6 mA, 125 V) were delivered to control mice (8 wk-old, n = 4; 13 wk-old, n = 8) and alpha-CaMKII+/- mice (8 wk-old, n = 4; 13 wk-old, n = 6) 10 times through the metal grids in the bottom of a chamber. The duration of each foot shock was 1 s, and the interval between pairs of shocks was 30 s. Two hours after exposure to the last foot shock, mice were perfusion-fixed with 4% PFA and 0.5% picric acid in 0.1 M PBS. The brains were removed and further immersion-fixed in the same fixative at 4°C overnight and 70-μm-thick coronal sections were prepared on a Vibratome. The immunostaining was performed by incubating free-floating sections with rabbit anti-c-Fos polyclonal antibody (Santa Cruz Biotechnology, 1:5000 dilution) at 4°C overnight. After rinse with PBS, the sections were incubated at 4°C overnight with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:300 dilution; Invitrogen), and the fluorescent signals were detected as described above. The number of c-Fos-immunoreactive cells in the specified regions or subdivisions were counted on the confocal images in a blinded manner.
BrdU labeling analyses
Mice (7-wk-old) received four injections of 5-bromo-2-deoxyuridine (BrdU) (Sigma, St. Louis, MO), one every 2 h, at 50 mg/kg body weight (10 mg/ml in PBS). Mice were perfusion-fixed with 4% PFA and 0.5% picric acid in 0.1 M PBS 1 d after the BrdU injections. The brains were removed and further immersion-fixed in the same fixative at 4°C for 2 h and 14-μm-thick coronal sections were prepared on a cryostat (Leica). The sections were boiled in 0.01 M citric acid for 10 min and incubated in 2N HCl for 10 min at 37°C, and washed with PBS. The sections were incubated at 4°C for 18 h with mouse BrdU monoclonal antibody (IgG, 1:100 dilution; Becton Dickinson, San Jose, CA). After washing with PBS, the sections were incubated at 4°C for 2 h with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:400 dilution; Invitrogen), and the fluorescent signals were detected as described above. For quantitative analysis, every fourth section (14 mm; throughout the DG in its rostrocaudal extension) was used for counting, and the total cell number was obtained by multiplying the number of counted cells by 4 . The cells were counted in a blind manner.
The mice (8 to 13-wk-old) were perfused transcardially with a solution of 2% PFA and 2.5% glutaraldehyde in PBS. The brains were removed and coronal sections (100 mm thick) were prepared on a Vibratome. Sections containing the dorsal hippocampus were osmicated, dehydrated, and embedded in epoxy resin. Ultrathin sections of the hippocampal CA3 region were prepared and stained with lead citrate and uranyl acetate, and observed under a Hitachi H-7000 electron microscope.
Gene expression studies using postmortem brain tissue
The expression data for 166 hippocampal genes from the BioExpress database [76, 77] (GeneLogic) were used in this study (see Additional file 2, Table S3). Differential analysis of mouse GeneChip data indicates that 130 mouse probes (n = 3, p < 0.05 [t-test], fold change > 1.5) were affected in mutants, independent of age or experience. Fifty-seven human orthologues corresponding to the 130 mouse probes were present in at least 10% of the 166 hippocampi and were identified as biomarkers. Of the 166 hippocampi, there were 18 from patients with schizophrenia, 2 with bipolar disorder, and 1 with schizoaffective disorder (see Additional file 2, Table S3). Variance analysis of hippocampal expression levels was performed to identify the 10 best genes of the 57 biomarkers so that the 21 hippocampi of patients with psychiatric disorders would be distinguished from the remaining 147 hippocampi (see Additional file 2, Table S4). Cluster analysis of the 166 hippocampi based on the expression levels of the 10 best biomarkers was performed using Spotfire DecisionSite™ For Functional Genomics http://spotfire.tibco.com/. Of the 166 hippocampi, 44 were obtained from donors with no known history of CNS-related illness. Of the 44 donors with no CNS-related illness, 28 were over 50 years old and 26 were male. We defined age- and gender-affected probes by differential expression analysis of older (over 50 years old) vs. younger donors with no CNS-related illness and of male vs. female donors with no CNS-related illness, respectively (p < 0.05, fold change > 1.8). For cluster-based differential gene expression analysis, age- and gender-affected probes were eliminated and expression levels of other probes in clustered psychiatric hippocampi were compared with those in the other cluster obtained from donors with no CNS-related illness as specific controls (p < 0.01, fold change > 2.0). In addition, only probes whose present flags were marked in over 75% of hippocampi in at least one of the two groups (case and specific controls) were considered significant.