Except where indicated, animals were housed at the Toronto Centre for Phenogenomics (TCP). GSK-3α null (KO) mutants and their wild-type (WT) littermates, were generated as previously described 57. GSK-3α KO mice were 6 generations backcrossed to C57BL/6 mice. GSK-3α mice were mated heterozygously and at weaning, littermates of mixed genotypes were housed by gender in groups of 3 to 5 per cage under a 12 hour light/dark cycle (lights on at 07.00) with ad libitum food (Purina mouse chow) and water. GSK-3α male and female mice were tested at 2-6 months of age. Behavioral testing was conducted between 09.00 and 18.00 hours. Experimenters were blind to genotype, which was determined after data collection. Between subjects and after all tests, all apparatus was cleaned with Clidox and 70% ethanol to prevent bias due to olfactory cues. We used 6 independent groups of KO and WT littermate control mice for behavioral tests. All behavioral testing procedures were initiated from less-stressful to more aversive, where each test was separated from each other by 2 days at least. Prior to all experiments, mice were left undisturbed in the room for 30 min to allow acclimation. The sequence of experiments for the first three sets of animals was: elevated plus maze (EPM), open field (OF), social interaction, startle response/prepulse inhibition (PPI), forced swim test (FST). The fourth set comprised: olfactory bulb test, resident intruder (RI). The fifth group: tail-flick, latent inhibition (LI). Sixth group: olfactory bulb test, tail suspension test (TST), fear conditioning (FC). In addition, six independent sets of naïve mice were used once for separate procedures: 1) MRI; 2) HPLC; 3) Basal and stress-induced corticosteroid measurements; 4) histological studies (Calbindin staining); 5) rotarod; 6) light/dark transition test, passive avoidance experiments. The latter tests were performed in Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Japan using mice shipped from Toronto. In Okazaki (NIPS), only male mice that were single housed were used. Animals were 8-9 month old when tests were conducted. Data collection for most behavioral tests (except where noted) was performed by OBSERVER 5.0 software (Noldus Information Technology, Wageningen, the Netherlands). All procedures involving animals were approved by the Animal Care and Use Committee of TCP and were conducted in accordance with the requirements of the Province of Ontario Animals for Research Act 1971 and the Canadian Council on Animal Care. The animal research protocol used in Okazaki (NIPS) was approved by the Institutional Animal Care and Use committee of National Institute for Physiological Sciences.

This test was conducted as previously described 5960. The apparatus consisted of a cage (21 cm × 42 cm × 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.

The activity cage consisted of a cubical box (Versa Max system) with a floor equipped with horizontal and vertical infrared sensors. Each mouse was placed individually into the center of the activity cage for 5 min (as described in 61) and the following behavioral measures recorded: (1) latency (seconds) to first escape from the center; (2) length of time of immobility periods (seconds); (3) time spent self grooming (seconds); (4) number of risk assessment behaviors involving the mouse stretching its body from corners/wall toward the center; (5) horizontal and (6) vertical activity. Exploratory activity and walking were recorded separately for the central and peripheral field of the open arena and the ratio between central and peripheral activity was calculated.

The activity cage was similar to that used for the new environment test with a floor equipped with horizontal and vertical infrared sensors. The chamber of the test was illuminated at 100 lux. Each mouse was placed individually into the center of the activity cage and motor activity measured by counting the number of horizontal and vertical beam breaks during the 30 min testing period.

PPI testing was conducted in four foam-lined (sound damping) isolation chambers (Med. Associates Inc., Startle Reflex System, St Albans, VT). All events were recorded and controlled by Med Associates software (Startle Reflex package). During the test, the animal was confined to the holder. Background noise was set at 65 dB. Five types of trials were used. Pulse alone trials (P) consisted of a single white noise burst (120 dB, 40 ms). The prepulse + pulse trials (PP69P, PP73P, PP81P) consisting of a prepulse of noise (20 ms at 69, 73, or 81 dB, respectively) followed 100 ms after prepulse onset by a startling pulse (120 dB, 40 ms). No-stimulus (NS) trials consisted of background noise only. Sessions were structured as follows: (1) 15-min acclimation at background noise level; (2) five P trials; (3) 10 blocks each containing all five trials (P, PP69P, PP73P, PP81P, NS) in pseudorandom order; and (4) five P trials. Intertrials intervals were distributed between 12 and 30 s. The force intensity for each trial was recorded as the startle level. The percentage PPI induced by each prepulse intensity was calculated as [1-(startle amplitude on prepulse trial)/(startle amplitude on pulse alone)] × 100%. Startle amplitude in this formula was calculated as the average response to all of the pulse alone trials, excluding the first and last blocks of five pulse alone trials.

The LI procedure was used as previously described 62. All events were programmed by the MED-PC software. Before the beginning of each LI experiment, mice were weighed and water was removed from the cages for 24 h. They were then trained to drink in the experimental chamber for 5 days, 15 min per day (training period). Body weights were monitored daily throughout all behavioral testing and maintained at no lower than 80% of the initial body weight. For each daily training session, mice were acclimated to the chamber without access to the sipper tube for 5 min then the guillotine door was opened. Latency to the first lick and number of licks were recorded for 15 min. The LI procedure was conducted on days 6-9 and consisted of the following stages:

Fear-conditioning evaluation was performed in a sound-attenuated chamber (Med Associates, St. Albans, VT) equipped with a computer-controlled fear conditioning system (Actimetrics, Wilmette, IL). Freezing behavior, defined as the complete absence of any movement except for respiration and heartbeat, was measured during the context and cued conditioning tests at 0.25 second intervals by using FreezeFrame automated fear conditioning software (ACTIMETRICS software, FREEZEFRAME v. 1.6e). Tests subjects were removed from their home cage and allowed to explore for 2 min. Conditioning consisted of a single pairing of an auditory cue (3600 Hz, 80 dB) with a foot shock (1 mV scrambled). The auditory cue was present 2 min after the training session started and was 30 seconds in duration. The foot shock was delivered continuously during the last 2 seconds of the auditory cue. The subject was removed from the chamber 30 seconds later and returned to its home cage. Approximately 24 h later, each subject was returned to the test chamber and monitored for 5 min. Two hours later, the context was altered and each subject was placed into the altered chamber and allowed 3 min for exploration, after which the auditory tone cue of 3 min was delivered.

Passive avoidance test was assessed in a two-compartment box with a shock grid (O'Hara & Co., Tokyo, Japan) 63. This task allows measurement of avoidance behavior and learning abilities in mice. The box consisted of a bright compartment and a dark compartment, separated by a guillotine door. After 30 min adaptation to an experimental room, the subject was placed in the light part, and latency to enter the dark compartment was measured. On entry into the dark compartment, the door was closed and a 3 s footshock (0.3 mA) was applied. The mouse was then removed from the box and placed in its home cage. Twenty-four hours later, the mouse was again placed in the light box and latency to enter the dark compartment was measured. The retention test was performed 35 days after second test.

The protocol was performed as described by Cryan et al. 64. The mice were released individually into a transparent plastic cylinder (25 cm height, 18 cm diameter), which contained water at 25°C to the depth of 18 cm. The experiment lasted 6 min, and an observer scored the following parameters in the last 4 min of the trial: (1) active swimming (including crossing the quadrants of the container) and (2) floating (no limb movement and making only minimal movements to keep the head above the water). Each mouse was allowed to dry after the test, and the water was changed between subjects.

This procedure was followed as previously described by Steru et al. 65. Scotch tape pasted to the tip of the tail (~1 cm) was used to securely fasten the mice to a flat wooden surface located 50 cm above the ground. Active hanging and immobility, defined by presence or absence of limb movement, were recorded over a 6 min period.

For this experiment, an Economex Rotarod apparatus was used (Columbus Instruments, Columbus, OH, USA). The original 3 cm ribbed plastic rotating axle was divided into four adjustable flanges, which enabled testing a maximum of four mice simultaneously. The rod is suspended at a height of 30 cm above the plastic surface. Mice are placed on top of the rod, facing away from the experimenter. In this orientation, forward locomotion opposite to rotation of the rod is necessary to avoid falling. During the stationary mode, each mouse was first observed for 10 seconds without any rotation. The axle was then adjusted for a constant motor speed of 5 r.p.m., and each mouse observed for a total of 10 seconds (fixed speed mode). Next, beginning at 5 r.p.m., the rotation gradually increased in increments of 0.1 r.p.m. per second and the latency to fall off the axle was recorded in seconds for each mouse for the maximum period of 300 seconds (accelerating speed mode). The mean latency was then calculated by averaging the latency for three consecutive trials 66. The stationary and fixed speed mode sessions were training periods and allowed the animals to become accustomed to the apparatus. Impaired performance in these sessions served as early indicators of abnormality. The accelerated Rotarod procedure was repeated for 3 constitutive days by 3 trials per day to measure motor learning.

Two identical transparent Perspex cylinders (each 8 cm diameter, 13 cm tall) with removable, black Perspex lids stood vertically inside the apparatus, one in each side chamber. Each cylinder was perforated with multiple holes (1 cm diameter) to allow for air exchange between the interior and exterior of the cylinder. Briefly, the subject mouse was first placed at the center of the middle chamber. After a 5 min adaptation period in which the subject was free to explore each chamber, an unfamiliar mouse (male C57BL/6J, "stranger 1") was placed inside a cylinder in one of the side chambers. Containing the stranger mouse in the cylinder ensures that social approaches were initiated by the subject mouse and was investigatory only, without direct physical contact. Time spent by the test mouse in each side chamber was recorded over a 10-min period, to estimate social novelty and motivation (session I). Another unfamiliar mouse ("stranger 2") was then placed inside an identical cylinder in the opposite side chamber, and the activity of the test mouse was likewise recorded for a further 10 min, to evaluate social memory (session II). The test mouse was considered to be in the chamber when its head and two front paws entered the chamber.

Aggression was assessed using the resident-intruder test in isolated male mice, essentially as previously described 67. Males were housed individually for 4 weeks before assessment, which was performed over three sessions in the same day. Intruders (unfamiliar socially housed C57BL/6J male mice, age and weight matched with each resident) were individually placed in the resident home cage for a 10-min test session and observation was started. The latency, duration and number of events were recorded as: aggressive behavior (contact between the resident and the intruder such as biting or wrestling and aggressive grooming of partner) or social interest behavior (following and sniffing of partner). A different intruder animal was used for each resident. Animals that did not attack the intruder were given an attack latency of 10 min.

Levels of catecholamine neurotransmitters and their metabolites, including dopamine (DA), 4-hydroxy-3- methoxyphenylacetic acid (HVA), norepinephrine (NE), normetanephrine (NM), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), were quantified by HPLC with electrochemical detection. Analyses were performed on a system consisting of a Thermo Separation Products (TSP) P4000 pump, a TSP AS3000 autosampler with cooling unit, an ESA Coulochem II electrochemical detector (ESA 5011A Analytical Cell and 5020 Guard Cell) and a Spectra Physics 4290 Integrator connected to a PC running TSP WOW chromatography software. The mobile phase, an aqueous mixture of 0.098 M glacial acetic acid, 0.09 M sodium acetate (pH 3.7), 0.118 mM EDTA, 8% methanol and 0.8 mM octane sulphonate was delivered at a flow rate of 0.8 ml/min. Separation of the 100 μl samples was performed on an ACE 250 × 4.6 mm column with Ace C18, 5 μm stationary phase. Peak heights recorded at E2 were detected using electrode potentials as follows (Guard cell +450 mV: Analytical cell E1+100 mV; E2 -400 mV). Quantification of monoamines was performed on 0.1 N perchloric acid extracts in a procedure involving two 30 min. runs per sample. An appropriately diluted sample was run followed by a second run consisting of 1/2 sample and 1/2 standard cocktail (pure monoamines and metabolites in concentrations of 10 pg/μl). Monoamine and metabolite levels in ng/mg tissue wet weight were then calculated.

To detect Purkinje cells, Calbindin immunohistochemistry was performed on sections of the cerebellum. Eight-week old KO and their WT littermate controls (n = 3 of each) were sacrificed and brains were removed and putt into 4% PFA. Paraffin-embedded coronal sections of cerebellum were deparaffinized with xylene, rehydrated in graduated ethanols, and then washed in phosphate-buffered saline. After citrate buffer antigen retrieval, the endogenous peroxidase activity was quenched by incubating the sections in 0.3% hydrogen peroxide for 30 min. Sections were blocked with Universal Blocking Buffer (Daco) for 10 min, following overnight incubation with specific antibodies against Calbindin antibody (1:200; Cell Signaling Technologies). Then tissues were incubated with biotinylated anti-rabbit IgG (Vector) for 1 h at room temperature, following incubation with Vestatin ABC solution. POD reaction was done by using DAB substrate (Vector). Counterstaining was performed with hematoxylin.

Images were digitally captured by a 12-megapixel Olympus DP71 camera for analysis. The cell density was analyzed by measuring the number of Purkinje cells per cell layer (linear density) using Metamorph software (Universal Imaging Corporation). For morphological analysis of Purkinje cells, the long axis of cell body was measured and taken as an indicator of cell size. All analysis were performed blind to the animals used. Results are reported as mean and S.E.M. Statistical significance was determined by Kolmogorov-Smivnov test and defined at P < 0.05.

Corticosteroid levels were measured in 10-week old KO (n = 19) and littermate WT (n = 12) control animals (both genders). Blood samples were taken in the morning (9.00 am) and evening (6.00 pm). Stress-induced corticosteroid levels were evaluated in blood samples from mice before and after physical restraint. Briefly, each mouse was individually placed in a plastic film decapicone (Braintree scientific inc., Braintree, MA, USA) for 30 min in a horizontal position. The animals were capable of breathing through the breathing hole at the smaller end and were immobilized without being squeezed. The larger end was tightly closed with a paper clip. All the subjects were checked to prevent suffocation. Serum concentrations of corticosteroids were measured using a commercially available Corticosterone 125I RIA kit (MP Biomedicals, USA), kindly provided by Dr. Daniel Drucker (SLRI).

Antinociceptive effects of the test substances were determined by the hot tail-flick method described by Sewell and Spencer 77. One to two cm of the tail of a mouse was immersed in warm water kept at 50°C. The reaction time was the time taken by the mice to deflect their tails. The first reading is discarded and the reaction time was taken as a mean of the next two readings. The latent period of the tail-flick response was taken as the index of antinociception.

Mice were first habituated to the food (Bud's Best Cookies, Hoover, AL, USA) by the experimenter leaving food pellets (1 × 1 × 0.5 cm) in the home cage overnight. The next day, rodent food pellets were removed from the home cage, and the mice were food deprived for 24 hr. The test was conducted in a standard polycarbonate cage (30 × 17 × 12 cm). A piece of chow was placed in a randomly chosen area on the cage floor and then the entire cage floor was covered with corncob bedding to a depth of 2.5 cm. The subject was then placed into the cage and latency to find the food was recorded up to a maximum time limit of 15 min.

For wet brain weight determination of KO and WT mice (n = 25 in each group) at 24 weeks old were sacrificed. The entire brain with olfactory bulbs and cerebellum were weighted.

Data were analyzed by two-tailed t tests for independent samples; two- and three-way ANOVAs followed by Fisher least significance difference (LSD) post-hoc test. PPI was analyzed by two-way ANOVA with gene and gender as a between-subjects factors, and prepulse intensity as a repeated measurement factor. Latent Inhibition was by two-way ANOVAs with main factors of pre-exposure (0,40) and genotype (WT; GSK-3α KO). Values in figures are expressed as Mean ± S.E.M.

The elevated plus maze (EPM) is a widely used test to measure anxiety-like behavior. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced stress in mice 58. GSK-3α KO females spent significantly more time in the closed arms and less in the open arms of EPM than WT littermates, suggesting increased stress behaviors in the KO females (Table 1). However, KO males in EPM behaved similarly to WT animals. Innate avoidance of the bright environment was measured by using an automated light/dark transition test. The number of transitions, distance traveled and latency to light in the light/dark transition test of GSK-3α KO males were all similar to WT littermate controls (Figure 1), confirming normal behavior of GSK-3α KO males in tests associated with increased stress.

To estimate emotional responsiveness in both groups, mice were subjected to a bright open-field arena (~200 lux) in a dim testing room for 5 minutes. GSK-3α KO animals of both genders showed a significant increase in freezing and grooming behavior (Table 2), indicating elevated instinctive behaviours in these animals compared with their littermate controls.

In the forced swim test (FST), rodents are placed in an inescapable cylinder partially filled with water, and the time spent immobile is measured over a short time window 78; this test is a well-established paradigm for assessing motivational behaviors in rodents 79 and is used to predict antidepressant drug efficacy in both mice and rats 78. Mice will generally swim in the water but after a while will stop swimming and float on the surface of the water, appearing to have given up trying to find solid ground. The duration of immobile floating on the surface of the water was decreased in GSK-3 mutants compared with WT littermates (Figure 2A). This finding was confirmed using a tail suspension test (TST) - a second paradigm commonly used to evaluate effects of antidepressants in rodents 6579. Mice are suspended by their tails for six minutes, during which time both activity and immobility are measured. Immobility time was significantly reduced for GSK-3α KO mice (Figure 2B).

Changes in general activity can affect animal behavior in FST and TST 7980. Locomotor activity in response to a novel environment was monitored in the open field. Compared to WT littermates, GSK-3α KO mice displayed reduced horizontal and vertical activity throughout the 30 minutes of the test period, indicating decreased locomotion and exploratory behavior in KO vs. WT animals (Figure 2C-D). Since the FST involves exposure to water, it increases stress in the animals as the animals prefer to avoid immersion. Stress stimulates the hypothalamus-pituitary-adrenal (HPA) axis, resulting in elevation of corticosteroid hormones. To determine whether the HPA response to stress was altered in GSK-3α KO mice, basal and stress-induced levels of corticosteroid (CORT) hormones were measured (Figure 2E). To check the status of basal levels of CORT in GSK-3α KO animals and their littermate controls, morning and evening measurements were taken. Basal CORT levels were significantly decreased in KO animals compared with WT in the morning as well as in the evening. After being subjected to restraint-induced stress, blood CORT concentrations increased and reached similar elevated levels in both groups (Figure 2E). These data indicate that GSK-3α KO mice have enhanced basal reactivity to stressful situations.

Sociability and social novelty were estimated using two phases of social interaction tests (session I and session II). Unlike wild-type mice, GSK-3α mutants did not prefer the chamber containing an unfamiliar mouse (stranger 1) over an empty chamber in session I (Figure 3A). In addition, KO males did not favor the chamber containing a newly introduced mouse (stranger 2) over a chamber containing a now familiar mouse (stranger 1) in session II. Indifferent behavior of KO mice in this test is indicative of decreased social motivation and novelty (Figure 3A).

GSK-3α KO mice showed a significant decrease in duration of fighting and aggressive grooming and sniffing of the unknown intruder mouse compared with WT mice (Figure 3B). The time spent by GSK-3α KO males either freezing or running away from the opponent was increased vs. WT (Figure 3B). These findings indicate reduced aggression-like behavior in GSK-3α KO animals.

The lower social interaction and aggressive behavior of GSK-3α KO mice was not a result of diminished olfaction, as we found that the time required to find buried food in an olfactory test was not significantly different between the genotypes (Figure 3C).

Prepulse inhibition (PPI) and latent inhibition (LI) are the most common methods to quantify information processing in both humans and mice 81. Sensorimotor gating is the process by which inhibitory neural pathways filter multiple stimuli and allow attention to be focused on one stimulus 82. Sensorimotor gating can be measured by prepulse inhibition of startle, which is the modulation of the startle response by a weak prepulse 83. The acoustic startle reflex is a simple neural circuit, involving approximately five synapses 84, and prepulse modification of this circuit has been postulated to arise from diverse cortical, midbrain, and hindbrain centers 85. PPI is defined as the degree (%) to which the acoustic startle response is reduced when the startle-eliciting stimulus is preceded by a brief, low intensity, non-eliciting stimulus 86.

GSK-3α KO mice were tested for sensorimotor gating deficits in the prepulse inhibition test (Figure 4A-B). We found that GSK-3α KO mice have facilitated PPI at the lowest prepulse (69 dB) in KO females vs. WT and at 69 and 81 dB in KO males vs. WT (Figure 4A). The amplitude of the acoustic startle response was similar between KO and WT, however, a gender effect on ASR was observed (Figure 4B).

Latent inhibition refers to the phenomenon by which pre-exposure to stimuli (such as tones) retards subsequent conditioning to the same stimulus. The reduced conditioning is believed to result from attention filtering so that decreased attention is given to a familiar stimulus that the animal has learned to ignore during pre-exposure 87. WT mice that were pre-exposed (PE) to the tone showed less freezing to the tone on the test day compared to non pre-exposed (NPE) animals (Figure 4C), indicating significant latent inhibition in control mice. The suppression ratio of pre-exposed GSK-3α KO mice was similar to that of non-exposed GSK-3α KO mice (Figure 4C), suggesting a deficit of the GSK-3α null mutant in latent inhibition.

Recently, GSK-3 has been reported to be involved in synaptic plasticity 24252627 and memory reconsolidation 53. To examine whether the loss of GSK-3α was associated with cognitive deficits, we assessed memory and learning in GSK-3α KO mice in a Pavlovian fear conditioning paradigm as well as in a passive avoidance test (Figure 5).

GSK-3α KO mice showed similar freezing times in response to the unconditioning stimulus (US) compared to their littermate controls (baseline in Figure 5A and 5B). Memory performance in KO and WT groups was examined 24 hours after exposure to the US, by measuring freezing time (Figure 5). In the following trial (using the same chamber as used in the previous conditioning, without the sound), the learning freezing response of GSK-3α KO-context group was decreased (Figure 5A). In the cued trial (placing the mice into a different shape of chamber from that used for the conditioning, but with the presence of sound as was used for the conditioning), the GSK-3α KO-cue group showed a decreased level of freezing compare to WT, suggesting that GSK-3α mutant animals have an impaired ability to form and consolidate memory (Figure 5B). The decreased percent of freezing of GSK-3α KO mice in the FC test was not a result of diminished sensitivity, as we found that the latency in the tail flick test was not significantly different between the genotypes (Figure 5C).

However, no difference was found in the passive avoidance test (Figure 5D) between KO and WT groups; suggesting that the learning ability of KO was similar to WT and the behavior of the mutant group in the FC test might be interpreted as active avoidance of a fear-related situation leading to them freezing less than WT animals.

To investigate whether altered levels of monoamines could contribute to some of the social, cognitive and memory deficits observed in GSK-3α KO mice, levels of catecholamines and their metabolites were measured using HPLC-electrochemical detection (Table 3). Slight but significant decreases were observed in the levels of HVA (in PFC) and NE (in hippocampus). However, the level of NM was increased in the amygdala (Table 3). Analysis of striatum revealed no significant alterations between genotypes in the profile of monoamines (Table 3).

We examined 9-10 week old GSK-3α mice for structural brain abnormalities using very high resolution MRI combined with computer modeling (Figure 6). Analysis by MRI revealed no overall brain size difference. We used a statistical map of the Jacobian determinant that illustrates the expansion and contraction of tissue based on genotype, to find regions of significant change. In order to account for an inflated amount of false positive findings due to the number of statistical tests employed, the FDR technique was applied with a 10% FDR threshold. The arbor vita of the cerebellum of the GSK-3α knockout mice was observed to be significantly enlarged by 9% compared to wild type mice (Figure 6). The mean volume was 12.8 ± 0.55 mm³ in the knock out animals and 11.7 ± 0.4 mm³ in wild types. The pons was found to be significantly larger by 5% in the knock out animals. Mean volume was 18.1 ± 0.67 mm³ in the knock outs and 17.2 ± 0.4 mm³ in wild type samples (Figure 6). Slightly increased volume of medulla oblongata was observed in KO (24.48 mm³) vs WT (23.65 mm³) mice (Figure 6). Small regions of significantly expanded voxels were also found in the cerebellar cortex, the cerebral aquaduct, the cerebral cortex, the cerebellar peduncle, the left striatum and the right olfactory bulb, as well as a small region of significantly contracted voxels in the parieto-temporal lobe of the cerebral cortex. No gender related differences were found.

Brain weight analysis of 6 months old animals revealed slightly but significantly increased brain weight of KO males and females compared with their littermate controls (Figure 7A). Thus, structural changes (especially in the cerebellum) found by MRI may correlate with increased brain weight of KO animals.

The cerebellum is an important component of the brain motor system, involved in the control and adaptability of movement. To elucidate the involvement of the cerebellum in morphological changes in GSK-3α null mice, we performed a cerebellum-dependent behavior test (Figure 7B). Associative motor learning, coordination and balance were examined by a rotarod test in KO and WT animals. A significant coordination deficit was observed in the KO group, as assessed by decreased latency to fall, compare to WT littermates. During three day trials, relative improvements in rotarod performance were found to be comparable between GSK-3α KO and WT mice (Figure 7B).

The cerebellar cortex contains eight types of neurons: Purkinje, granule, stellate, basket, Golgi, Lugaro, unipolar brush cells and candelabrum cells 888990. Purkinje cells provide the sole output of the cerebellar cortex and are the pivotal element around which the cerebellar circuit is organized 88. Most Purkinje cells express calbindin, which is expressed when neurons start to migrate and differentiate 91. By using anti-calbindin immunohistochemistry, we examined the Purkinje cell composition of the cerebellum in 9-10 week old KO and WT animals (Figure 7C). We quantified cell density and shape, analyzing more than 1500 calbindin-positive cells per sample. Significant decreases in Purkinje cell density and size were observed in GSK-3α KO mice (Figure 7D-E).