Conditional calcineurin knockout mice exhibit multiple abnormal behaviors related to schizophrenia -- Data Supplement - HTML Page - 2926SuppText.htslp -- Proceedings of the National Academy of Sciences

Supporting Text
Supporting Materials and Methods
Rotarod Test and Hot Plate Test.
The rotarod test was performed using an accelerating rotarod (UGO Basile Accelerating Rotarod) and consisted of placing a mouse on a rotating drum (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 hot plate test was used to evaluate sensitivity to a painful stimulus. Mice were placed on a 55.0ºC (±0.3ºC) hot plate (Columbus Instruments, Columbus, OH), and latency to the first hind-paw response was recorded. The hind-paw response was either a foot shake or a paw lick. Light/Dark Transition Test.
The apparatus used for the light/dark transition test (1) consisted of a cage (21 × 42 × 25 cm) divided into two sections of equal size by a black partition containing a small opening (O’Hara & Co., Tokyo). One chamber was brightly illuminated, whereas the other chamber was dark. Mice were placed into the illuminated side and allowed to move freely between the two chambers for 10 min. The total number of transitions, time spent in the dark side, and distance traveled were recorded by IMAGE LD4 software. Elevated Plus Maze Test.
The apparatus used for the elevated plus-maze test (2) consisted of two open arms (30 × 5 cm) with 3-mm high ledges and two enclosed arms of the same size, with 15 cm high transparent walls (O’Hara & Co, Tokyo). The arms and central square were made of white plastic plates and were elevated to a height of 100 cm above the floor. Each mouse was placed in the central square of the maze (5 × 5 cm), facing one of the open arms. Mouse behavior was recorded during a 10-min test period. Data acquisition and analysis were performed automatically, by using IMAGE EP software. Object Exploration Test.
The object exploration test was performed in a manner similar to published methods (3, 4). The test consisted of 5 trials (10 min per trial). Mice were introduced into a box (40 × 40 × 30 cm) made of white Plexiglas and allowed to explore freely on the first day (trial 1) and the second day (trial 2) without objects. On the third day, they were placed in the box in the presence of two identical objects (object A; trial 3). Ten min after trial 3, one of the objects was replaced by a novel object (object B), and mice were allowed to explore the box with the two different objects (object A and object B; trial 4). On the following day, object B was replaced by another novel object (object A and object C; trial 5). The behavior was monitored by a color charge-coupled device (CCD) camera (Sony DXC-151A) which was connected to a Macintosh computer. Locomotor activity and the time each animal spent around the objects, as well as the time spent in the center part of the field were recorded. The regions of interest (ROI) around the objects were defined as circles with an 8-cm radius from the center of the object position. When the center of the mouse image was within the defined ROI for each object, the mouse was considered to be "around the object." Analysis was performed automatically by using IMAGE OE software (see Image Analysis). The recognition index (RI) was defined as (tB/(tA + tB))/100 as an index for memory on the objects. Social Interaction Test in a Novel Environment.
The social interaction test in a novel environment was done in a manner similar to published methods (5–7). Two mice of identical genotypes, which 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 (Sony DXC-151A), which was connected to a Macintosh computer. Analysis was performed automatically by using IMAGE SI software (see Image Analysis). The number of contacts, mean duration per contact, and total distance traveled were measured. Image Analysis in Social Interaction Test in Home Cage.
Captured images were processed before counting the number of particles as follows. First, images were captured at 256 level grayscale (Fig. 7B). Second, the images were converted to binary with a threshold level. Two different threshold levels were used during the light cycle and during the dark cycle. Whether it was the light cycle or dark cycle was determined every second by assessing the average brightness of a certain field of a captured image, in which the brightness was unaffected by the position of the mice (Fig. 7 B and C). Such a binary image often contained noise (Fig. 7D). Also, an image of a mouse was sometimes divided into a few particles due to unequal brightness of their coat or tails (upper left cage in Fig. 7D). To reduce such noise and unite the images of a mouse, images were processed by a rank filter, "Erode," four times and were subsequently processed by another rank filter, "Dilate," six times (Fig. 7E). For the details of the commands, see the manual of NIH IMAGE, which can be downloaded on the web site of NIH IMAGE at http://rsb.info.nih.gov/nih-image/index.html. After the image processing, the number of particles in a cage was counted. Porsolt Forced Swim Test.
The Porsolt forced swim test was done in a manner similar to published methods (7, 8). The apparatus consisted of four glass beakers (15 cm height × 10 cm diameter). The cylinders were separated from each other by a nontransparent panel to prevent mice from seeing each other. The cylinders were filled with water (23ºC), up to a height of 7.5 cm. Mice were placed into the cylinders, and their behavior was 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. Prepulse Inhibition Test.
A startle reflex measurement system was used (MED Associates, St. Albans, VT). A test session began by placing a mouse in a Plexiglas cylinder, where it was left undisturbed for 5 min. The duration of white noise that was used as the startle stimulus was 40 msec for all trial types. The startle response was recorded for 160 msec (measuring the response every 1 msec) 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 160-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 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 employed (74–110, 78–110, 74–120, and 78–120). Six blocks of the six trial types (four trial types with the combinations of prepulse and startle stimulus and two startle stimulus only trials) were presented in pseudorandom order such that each trial type was presented once within a block. The average intertrial interval was 15 sec (range: 10–20 sec). HPLC Assessment of Brain Content of Monoamines and Metabolites.
The striatum or frontal cortex of mice was homogenized in 0.1 M HClO₄ containing 100 ng/ml 3,4-dihydroxybenzylamine as an internal standard. Homogenates were centrifuged for 10 min at 10,000 × g. Supernatants were filtered through a 0.22-μm filter and analyzed for levels of dopamine, serotonin, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid with the use of HPLC with electrochemical detection. Monoamines and metabolites were separated on a microbore reverse-phase column (C-18, 5 μm, 1 × 150 mm, Unijet; BAS, West Lafayette, IN) with a mobile phase consisting of a 0.03 M citrate-phosphate buffer with 2.1 mM octyl sodium sulfate, 0.1 mM EDTA, 10 mM NaCl, and 17% methanol (pH 3.6) at a flow rate of 90 μl/min and detected by a 3-mm glass carbon electrode (Unijet; BAS) set at +0.8 V. The volume of injection was 5 μl. In Vivo
Microdialysis. Mice were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic frame. Dialysis probes (2-mm membrane length, 0.24-mm o.d., Cuprophane, 6-kDa cutoff, CMA-11; CMA/Microdialysis, Solna, Sweden) with CMA-11 guide cannulae were implanted in the right striatum. The stereotaxic coordinates for implantation of microdialysis probes were 0.0 mm AP, 4.4 DV mm, L2.5 relative to bregma. Placement of the probe was verified by histological examination subsequent to the experiments.

After surgery, animals were returned to their home cages with free access to food and water. Twenty-four hours after surgery, the dialysis probe was connected to a syringe pump and perfused at 1 μl/min with artificial cerebrospinal fluid (147 mM NaCl/2.7 mM KCl/1.2 mM CaCl₂/0.85 mM MgCl₂) (CMA/Microdialysis). After an equilibration period of 1 h, the flow rates were reduced to 70 nl/min and the perfusates were collected every 90 min in vials containing 2 μl 0.5M HClO₄.

Supporting Results
Sequence of Behavioral Experiments.
Sequences of behavioral experiments are listed in Table 1. Seven groups of animals were used. Animals belonging to a group experienced the same set of experiments in an identical sequence. Due to sudden deaths of animals [mostly calcineurin (CN) mutants] and some accidental technical errors, the number of animals used for each experiment may differ from those for other experiments in a given group. Abnormal Anxiety-Like Behaviors.
In the light/dark transition test, the distance traveled in the light chamber, the number of transitions between the two boxes, and time spent in the light chamber were significantly decreased in CN mutants relative to control mice (Fig. 9 A–C, P = 0.0004, P < 0.0001, and P = 0.0003, respectively). Also, it took the mice a significantly longer period of time to enter the light chamber after being placed into the dark chamber at the start of the experiment (Fig. 9D, P = 0.0014). Because CN mice were found to be hyperactive by numerous indices of locomotor activity in several tests, the decreased number of light/dark transitions of CN mutant mice is particularly suggestive of increased fear or anxiety in these mice. CN mutants spent a significantly shorter period of time in the central region of the open field apparatus (Fig. 1D; first 30 min, P = 0.0011), another index of anxiety (9). In addition, despite their pronounced hyperactivity, CN mutants displayed a consistent, characteristic time course of locomotor activity. They were less active than control mice during the initial one minute of the first object exploration trial and made fewer contacts in the social interaction test (Figs. 10A and 11B, respectively, suggesting their increased neophobia.

In contrast, in the elevated plus maze test for anxiety, the number of total entries into arms, percentage of entries into open arms, and percentage of time spent in open arms were all significantly greater in CN mutants compared to controls (Fig. 9 E, F, and G, respectively; P < 0.0001 for every index). In this test, a greater value of these indices is usually interpreted as an indication of reduced anxiety.

Interpretation of the Performances in the Two Anxiety Tests.
The results obtained in the elevated plus maze test seemingly contradict the increased anxiety-like behaviors observed in the other tests. One possible explanation for the apparent discrepancy is increased flight behavior of CN mutants. The mutant mice had higher tendency to escape from the cage or experimental chambers. Also, experimenters experienced great difficulties in catching them during cage change or from experimental chambers because of their tendency to avoid being caught, suggesting an increased flight behavior. Moreover, after being caught, CN mutant mice often vocalized and bit experimenters. We seldom observed this kind of behavior in normal mice. Recently, Holmes et al. (10) tested a wild-derived population of house mice (wild mice) in an elevated plus maze test. These mice were previously reported by others to have markedly increased levels of flight behavior and escape attempts in a mouse defense test battery. The mice showed a larger percentage of time spent in open arms, a larger percentage of entries into open arms, and a greater number of total entries into arms. At the same time, these mice showed either actual or attempted jumps from the maze, had a high incidence of spontaneous freezing, and exploration of the upper ledges of closed arms. Based on these findings, the authors raised the possibility that the behaviors exhibited by the wild mice may rather reflect a higher level of anxiety. Therefore, with the data we have available, it is reasonable to assume that the increased flight behavior of CN mutant mice contributed to an increased exploration of open arms, arms that have no walls and therefore provide a potential escape route.

Although increased anxiety is not a core symptom of schizophrenia, it has been reported that panic disorder (11) and chronic anxiety (12) are associated with schizophrenia. Thus, the abnormal anxiety behavior observed in the CN-mutant mice is consistent with some aspects of schizophrenia symptomatology.

CN-Related Genes Found to be Differentially Expressed in Postmortem Brains of Schizophreniac Patients.
Recently, DNA microarray analysis was used to assay gene expression levels in postmortem brain of schizophrenic patients (13). The list of genes that showed an altered expression level in that study contained at least 10 genes whose functions are known to be related to CN, including CN A and calmodulin. Expression of IP3 receptor, which is physically coupled with CN, was also found to be altered. IP3 receptors and CN interact functionally (14), and CN controls the expression of the receptor (15). ZAKI-4 is an endogenous CN inhibitor (16). Amphiphysin and clathrin are dephosphorylated by CN and are involved in synaptic vesicle endocytosis and long-term depression (17–20). HSP70 was reported to activate CN (21). MARCKS (22), GAP-43 (23), and GAD (GAD65 and/or GAD67) (24) have been reported to be substrates of CN.

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