Glycobiology Advance Access originally published online on September 14, 2006
Glycobiology 2007 17(1):1-9; doi:10.1093/glycob/cwl047
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Mice lacking 
1,3-fucosyltransferase IX demonstrate disappearance of Lewis x structure in brain and increased anxiety-like behaviors
2 Glycogene Function Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central-2, Open Space Laboratory, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan
3 Department of Anatomy and Embryology, Biomolecular and Integrated Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
4 Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan
5 Division of Oncological Pathology, Aichi Cancer Centre Research Institute, Chikusa-ku, Nagoya, Aichi 464-0021, Japan
6 Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan
7 Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan
1 To whom correspondence should be addressed; Tel: +81-29-861-3200; Fax: +81-29-861-3201; e-mail: h.narimatsu{at}aist.go.jp
Received on February 8, 2006; revised on August 26, 2006; accepted on September 5, 2006
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Abstract |
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The 3-fucosyl-N-acetyllactosamine [Lewis x (Lex), CD15, SSEA-1] carbohydrate structure is expressed on several glycolipids, glycoproteins, and proteoglycans of the nervous system and has been implicated in cell–cell recognition, neurite outgrowth, and neuronal migration during development. To characterize the functional role of Lex carbohydrate structure in vivo, we have generated mutant mice that lack
1,3-fucosyltransferase IX (Fut9–/–). Fut9–/– mice were unable to synthesize the Lex structure carried on glycoproteins and glycolipids in embryonic and adult brain. However, no obvious pathological differences between wild-type and Fut9–/– mice were found in brain. In behavioral tests, Fut9–/– mice exhibited increased anxiety-like responses in dark–light preference and in elevated plus maze tests. Immunohistochemical analysis showed that the number of calbindin-positive neurons was decreased in the basolateral amygdala in Fut9–/– mice. These observations indicated that the carbohydrates synthesized by Fut9 play critical roles in functional regulations of interneurons in the amygdalar subdivisions and suggested a role for the Lex structure in some aspects of emotional behavior in mice.
Key words:
1,3-fucosyltransferase
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Lewis x
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knockout mouse
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anxiety
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amygdala
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Introduction |
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Cell surface carbohydrates play important roles in cell–cell interaction and recognition. Stage-specific embryonic antigen-1 (SSEA-1), an antigenic epitope of which was defined as a Lewis x (Lex: galactose [Gal]ß1-4[Fucose {Fuc}
1-3]N-acetylglucosamine [GlcNAc]) carbohydrate structure, is widely expressed on the surface of mammalian cells and is considered to be involved in cell–cell interactions during embryogenesis, differentiation, and neurodevelopmental processes (Gotz et al. 1996
; Sajdel-Sulkowska 1998; Yoshida-Noro et al. 1999). The expression of Lex in the central nervous system (CNS) of humans, monkeys, rodents, chickens, and Xenopus is developmentally regulated and region-specifically localized (Schonlau and Mai 1995; Chou et al. 1996; Dasgupta et al. 1996; Gocht et al. 1996; Streit et al. 1996; Wiederschain et al. 1998; Allendoerfe et al. 1999; Mai et al. 1999; Shimoda et al. 2002). In rodent CNS, Lex is expressed in motor and auditory cortex, optic chiasm, hippocampus, cerebellum, brainstem, and spinal cord (Ashwell and Mai 1997a, 1997b, 1997c, 1997d; Marcus et al. 1999). This temporal and region-specific pattern of expression is consistent with the hypothesis that Lex plays an important role in neuronal development. More recently, anti-Lex monoclonal antibodies (mAbs) have been demonstrated to inhibit the adhesion of cerebellar granule cells to astrocytes in primary culture to block neurite outgrowth in explant culture (Sajdel-Sulkowska 1998; Yoshida-Noro et al. 1999), and to act as a neural stem cell marker (Capela and Temple 2002).
The expression of the Lex structure is determined by 1,3-fucosyltransfease(s) (1,3FUT or 1,3Fut). The genes encoding 1,3FUTs form a family. Human genes encoding six 1,3FUTs (FUT3, FUT4, FUT5, FUT6, FUT7, FUT9) and mouse genes encoding three 1,3Futs (Fut4, Fut7, Fut9) have been cloned and characterized. The mouse gene orthologous to the ancestral gene for human FUT3, FUT5, and FUT6 seems to be a pseudogene (Gersten et al. 1995; Costache et al. 1997). The FUT9 gene sequence is highly conserved among humans, mice, rats, and hamsters (Kudo et al. 1998; Kaneko et al. 1999; Baboval et al. 2000; Patnaik et al. 2000), indicating that it has been under strong selective pressure during evolution, and thus suggesting that it has an essential physiological role. Previously, we cloned and characterized a cDNA-encoding mouse Fut9 from a mouse brain cDNA library, using an expression cloning method (Kudo et al. 1998). The transcript for Fut9 was mainly expressed in the brain and kidney of the adult mouse (Kudo et al. 1998). In mice, in vitro fucosyltransferase assays suggested that the Fut4 and Fut9 genes control the biosynthesis of the Lex structure. The expression of Fut4 mRNA was ubiquitous, but it was not correlated with the temporal and region-specific pattern of Lex expression. We previously demonstrated that Pax6, a transcriptional factor involved in brain patterning and neurogenesis, controls Lex expression in the rat embryonic brain by regulating Fut9 (Shimoda et al. 2002). Furthermore, we performed in vitro fucosyltransferase assays to determine the acceptor substrate specificities of the fucosyltransferase family (Nishihara et al. 1999, 2003). Our immunohistochemical results using anti-Lex and anti-Fut9 mAbs strongly indicated that Fut9 is the enzyme most responsible for the synthesis of Lex in the CNS (Nishihara et al. 2003). Recently, we established and analyzed Fut9 knockout (Fut9–/–) mice (Kudo et al. 2004). Fut9–/– mice develop normally, with no gross phenotypic abnormalities, and are fertile, although expression of the SSEA-1 epitopes was completely absent in early embryos and in primordial germ cells of Fut9–/– mice despite the expression of Fut4 gene.
To directly determine whether the Lex structure contributes to differentiation and development of the CNS, we carried out a characterization of Fut9–/– mice. No obvious differences in the architecture of the cerebrum and cerebellum was observed in Fut9–/– mice, but an increase in anxiety-like behavior and a decrease in the number of calbindin-immunoreactive cells in the amygdalar subdivision were observed. These observations imply that Fut9 synthesizes the Lex structure in the CNS and that the carbohydrates synthesized by Fut9 may be involved in emotional behavior and neural development.
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Results and discussion |
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Fut9–/– mice lack the Lewis x carbohydrate structure in the CNS
In the developing brain, the Lex structure is carried primarily by glycolipids during embryonic development and on glycoproteins and proteoglycans in postnatal and adult tissue (Allendoerfer et al. 1995
). Two Lex synthase candidate enzymes, Fut4 and Fut9, are expressed in rodent CNS (Nishihara et al. 2003) and developmentally regulated in rat postnatal cerebellum (Baboval et al. 2000). Our previous report predicted that Fut9 was the prime candidate enzyme for the biosynthesis of the Lex structure in the mouse CNS (Nishihara et al. 2003). We established Fut9 knockout (Fut9–/–) mice to elucidate the physiological function of the Lex structure (Kudo et al. 2004). Fut9–/– mice exhibited normal gross development and brain size (data not shown). Immunohistochemical staining of adult brain paraffin sections with anti-SSEA-1 mAb revealed the Lex structure expression widely in wild-type mice (Figure 1A and C). In contrast, the immunoreactivity of the Lex structure in Fut9–/– mice was completely lost in all regions of the CNS (Figure 1B and D). The Lex expression in Fut9–/– mouse brain was also absent in frozen sections (data not shown). The same results were obtained using other anti-Lex antibodies (data not shown). These findings indicate that Fut9 is the principal enzyme responsible for the biosynthesis of the Lex structure in the mature brain in vivo.
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Expression of glycoproteins and glycolipids carrying the Lex structure in the brains of wild-type and Fut9–/– mice
The Lex structure is present on cell surface proteoglycans and glycolipids and varies with cell type and stage of differentiation (Allendoerfer et al. 1995
). The SSEA-1 was visualized as a single high-molecular-mass (>250 kDa) band in immunoblots of wild-type adult mouse brain extract by SDS–PAGE (Figure 2A). No positive bands could be detected in the Fut9–/– mouse brain. Allendoerfer et al. (1995) reported that the high-molecular-weight Lex structures recognized by FORSE-1 mAb were some of mannose-linked oligosaccharides on phosphacan in the embryonic and postnatal rat brain (Krusius et al. 1986). Phosphacan, a soluble nervous tissue-specific chondroitin sulfate proteoglycan, is an alternative splicing product representing the entire extracellular domain of a transmembrane receptor-type protein–tyrosine phosphatase (RPTPß) and binds neural cell adhesion molecules. However, the RPTPß-deficient mice are normal in their gross general behavior and with respect to fertility, body weight, and life span (Harroch et al. 2000). Recently, Comelli et al. (2006) identified the Lex structure on N-glycans in the mouse brain and kidney using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Moreover, the expression of Fut9 transcript and Lex structure on N-glycans are not detected in liver, lung, spleen, and thymus. The Lex expression on glycolipids in the embryonic brain is determined by lactosylceramide N-acetylglucosaminyltransferase (ß3Gn-T5), which synthesizes the root structure of the glycolipids carrying Lex (Togayachi et al. 2001). Anti-SSEA-1 mAb detected three positive bands of neutral glycolipids extracted from the embryonic brain of wild-type mice (E16.5 and E18.5) (Figure 2B). These bands, from the top to the bottom of the immuno-thin layer chromatography (TLC) gel, correspond to Galß1-4(Fuc1-3)GlcNAcß1-3Ga1ßl-4Glc1-1'Cer (III3FucnLc4), Galß1-4(Fuc1-3)GlcNAcß1-3Galß1-4GlcNAcß1-3Galß1-4Glc1-1'Cer (V3FucnLc6), and Galß1-4(Fuc1-3)GlcNAcß1-3Galß1-4(Fuc1-3)GlcNAcß1-3Galß1-4Glc1-1'Cer (III3V3Fuc2nLc6), as reported previously (Chou et al. 1996). All of the bands were absent soon after birth, as seen in P0.5 and P10.5 embryos (Figure 2B). These results concerning the developmental regulation of the Lex-active glycolipids in the mouse brain are consistent with those reported previously (Dasgupta et al. 1996). We carried out immuno-TLC analysis with anti-SSEA-1 mAb for neutral glycolipids extracted from E18.5 brain of wild-type, Fut9+/–, and Fut9–/– mice (Figure 2B). The intensity of each band from Fut9+/– mice was decreased in comparison with that from wild-type mice. This indicates that the Fut9 gene dosage affects the expression level of the Lex structure. No positive bands could be detected in glycolipids of Fut9–/– mouse brain. These results demonstrate that the Lex structure on glycoproteins and glycolipids in the mouse brain is synthesized by Fut9, not by Fut4.
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Morphological analysis of the CNS of Fut9–/– mice
The tissue architecture of the CNS was investigated using Nissl staining. At the light microscopy level, the general morphology of brains of adult Fut9–/– mice appeared normal and indistinguishable from that of wild-type littermates. Cross sections through the hippocampi of Fut9–/– mice displayed a normal pattern of stratification of ammonic pyramidal cells and dentate granule cells (Figure 3A and B). In addition, we found no abnormalities in the subventricular zones of Fut9–/– mice (data not shown). In the cerebella of Fut9–/– mice, the molecular layer, Purkinje cell layer, and granular layer appeared normal (Figure 3C and D).
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Fut9–/– mice exhibit an increased level of anxiogenic-like behavior
In the open-field test, we evaluated spontaneous locomotor activity and the pattern of each genotype under a novel environment. Male mice 8–15 weeks old were tested at 1–6 PM. The distance in locomotion and the frequency of rearing did not differ between wild-type and Fut9–/– mice (Figure 4A and B). We carried out a light–dark preference test (Figure 4C), which is considered to be a measure of anxiety or fear-related emotion in rodents (Costall et al. 1989
). The mean percentage of time spent on the light side was significantly lower in Fut9–/– mice than in wild-type mice [F(1,270)=8.10, P<0.005, two-way ANOVA]. The mean percentage of time spent in locomotion and the distance of locomotion did not differ between wild-type and Fut9–/– mice (data not shown). Moreover, we investigated the emotionality of mutant mice using an elevated plus maze task, which is a useful test for measuring anxiety or fear (Pellow and File 1986; Lister 1987). Fut9–/– mice spent significantly less time than wild-type mice in the open arms of the maze (Figure 4D; wild-type mice, 43.6±4.1%, n=16; Fut9–/– mice, 32.2±3.4%, n=16; P=0.04). They also entered the open arms significantly fewer times than wild-type mice (Figure 4E; wild-type mice, 30.6±4.2%, n=16; Fut9–/– mice, 18.7±2.2%, n=16; P=0.017). Taken together with data from light–dark preference test and elevated plus maze, these results suggest that Fut9–/– mice display increased anxiety responses in novel environments.
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Evaluation of learning and memory in Fut9–/– mice
We tested wild-type and Fut9–/– mice for spatial learning performance in the Morris water maze. In the hidden platform test, the time to enter the hidden platform (Figure 5A) was significantly longer for Fut9–/– mice than for wild-type mice [F(1
, 872)=8.10, P<0.01, two-way ANOVA]. Both wild-type and Fut9–/– mice showed a high spatial bias for the quadrant where the platform had been fixed (training quadrant) in the pool (Figure 5B). During probe trials, there was no significant difference between groups in the percentage of time spent in the training quadrant. In reversal learning, there was no significant difference between groups in the time to reach the hidden platform (Figure 5C). In the visible platform test, the time required to enter with a visible cue (flag) attached was significantly longer for Fut9–/– mice than for wild-type mice [F(1, 222)=12.8, P<0.0001, two-way ANOVA]. We compared swimming speed during training sessions between the groups of mice. On all sessions, there was no significant difference in swimming speed between wild-type and Fut9–/– mice (Figure 5D). These results obtained from the water maze test demonstrate that Fut9–/– mice had a mild deficit in acquisition on the hidden platform test and the visual test. In -CaMKII deficient mice, abnormal emotional behavior as well as impairments in water maze performance have been reported (Silva et al. 1992; Chen et al. 1994). Moreover, Fyn-deficient mice exhibited impaired spatial learning in water maze tests (Grant et al. 1992) but not in a radial eight-arm maze task, which is a spatial learning task using food as reward but without the potential stressful components (Miyakawa et al. 1996). Fyn-deficient mice also showed increased fearfulness in open field and elevated plus maze tests (Miyakawa et al. 1996). The impaired performance of Fut9–/– mice in water maze test may also be related to abnormalities in their emotional behavior.
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Decreased occurrence of calbindin-immunoreactive cells in the basolateral amygdala of Fut9–/– mice
The amygdala is part of the neural circuitry, which is critical for emotion. To elucidate the neural mechanisms of emotional defects in Fut9–/– mice, immunohistochemical analysis with anti-calbindin antiserum was performed (Figure 6A–D). Calbindin is a member of a large family of intracellular calcium-binding proteins, and calbindin-immunoreactive neurons constitute 40–60% of the
-aminobutylic acid (GABA)-containing population in the amygdalar subdivisions (McDonald and Mascagni 2001; Jinno and Kosaka 2002). Examination of the amygdalar subdivisions revealed statistically significant decreases in the number of calbindin-immunoreactive cells in the basolateral amygdala [lateral, basolateral, and basomedial nuclei in McDonald (1992)] of Fut9–/– mice in comparison with wild-type mice (Figure 6E; basolateral amygdala, wild-type mice, 97.7±10.0, n=7; Fut9–/– mice, 56.4±10.9, n=7; P=0.005). The numbers of calbindin-immunoreactive cells in the central and medial subdivisions of amygdala did not differ significantly between wild-type and Fut9–/– mice. There was no significant difference between wild-type and Fut9–/– mice in the distribution of calbindin-immunoreactive cells in either the cerebral cortex or the hippocampi (data not shown). In regard to the changes in GABAergic neurons in the amygdala, we have examined the distribution and numbers of calretinin-immunopositive cells, in addition to calbindin-positive neurons, both as the subsets of GABAergic neurons (data not shown), because it was rather difficult to examine the numbers of GAD-positive neurons owing to the dense terminals of GABAergic trajectories within the amygdala. In the present study, only calbindin-positive cells in the basolateral complex exhibited the significant difference between wild-type and Fut9–/– mice.
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The basolateral amygdala, where a decrease in calbindin-immunoreactive cells was detected in Fut9–/– mice, receives sensory information from the cerebral cortex and thalamus, processes such emotion-related information, and sends the processed information to the central nucleus (i.e. the amygdalar output nucleus). This nucleus projects to the hypothalamus, midbrain, and brainstem, and emotional reaction is expressed through this system (LeDoux 2000
). Because GABAergic interneurons innervate the projection neurons within the basolateral amygdala (McDonald et al. 2002), the specific reduction in the numbers of calbindin neurons in the basolateral amygdala of Fut9–/– mice is considered to reflect a decrease in the inhibitory input to the excitatory projection neurons within the basolateral amygdala. Taking into account that the excitation of basolateral complex attenuates the excitability of the central nucleus by the mediation of intercalated masses (Collins and Pare 1999), the excitability of the central nucleus of Fut9–/– mice should be altered owing to the reduced inhibitory action on the excitatory output neurons within the basolateral amygdala. Such defects in the intra-amygdalar neural circuit could lead to the abnormal emotion-related behaviors in the Fut9–/– mice. Moreover, the mice with genetically induced deficits of GABA function display increased fear- and anxiety-like behavior (Crestani et al. 1999; Stork et al. 2003). The relation between lack of Lex and the decrease in calbindin-positive cells in the basolateral amygdala could be considered on the developmental aspect and the involvement of Lex-related molecule.
In regard to the amygdalar development, the basolateral complex has been reported to originate from the boundary of the pallium and striatum, referred to as the corticostriatal angle (Medina et al. 2004; Tole et al. 2005). It has also been reported that restricted expression of CD15 (Lex)-immunoreactivity is observed in the corticostriatal angle, which is the origin of the basolateral complex in the mouse embryonic forebrain, and CD15 plays a role in the pattern formation of the forebrain (Mai et al. 1998). Accordingly, the lack of CD15 in the developmental origin could result in the defective formation of the basolateral complex.
The expression of Lex is observed in the entire amygdala of the adult mouse (Figure 1C). On the other hand, the expression of basigin that harbors Lex-antigenic epitope (Fan et al. 1998a) has been reported to exhibit a restricted and intense expression in the basolateral complex among all of the amygdalar subdivisions (Fan et al. 1998b). The loss of Lex in the basolateral complex of Fut9–/– mice could lead to the defective function of Lex-haboring proteins such as basigin, and a subset of GABAergic neurons in the subdivision might be lost. Taking into account the expression of Lex in stem cells and the involvement in the cellular interaction (Capela and Temple 2006), the finding on the amygdalar neurons in the present study suggests the function of Lex in both embryonic and postnatal development.
Neural progenitor cells in the subventricular zone contain Lex-expressing cells and generate GABAergic interneurons in the hippocampus (Aguirre et al. 2004). The precursor cells of calbindin-immunoreactive cells in the basolateral amygdala of Fut9–/– mice may be abnormal in stem cell behavior, cell differentiation, or cell migration.
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Materials and methods |
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Mice
Fut9–/– mice were generated using standard gene-targeting techniques described previously (Kudo et al. 2004
) and backcrossed for five generations into the C57BL/6 genetic background. Experimental animals were bred and housed in a specific pathogen-free animal facility and provided with sterilized chow and water ad libitum.
Western blotting and immuno-TLC with anti-SSEA-1 mAb
The whole brain was solubilized in 50 mM Tris–HCl buffer (pH 7.4), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride by brief sonication. After determination of the protein concentration, the samples were suspended in the Laemmli buffer. Solubilized protein for each sample was subjected to 10% SDS–PAGE and then transferred to a polyvinylidene fluoride membrane. The primary antibody, anti-SSEA-1 mAb (anti-Lex; Developmental Studies Hybridoma Bank, University of Iowa, Ames, IA; 1:1000), and a secondary antibody, horseradish peroxidase-conjugated donkey anti-mouse IgM (Jackson ImmunoResearch, West Grove, PA), were diluted 5000-fold in phosphate-buffered saline containing 0.1% Tween20 (PBST). The positive signals were detected using the ECL plus system (Amersham Bioscience, Buckinghamshire, UK). Crude glycolipids were extracted twice from the brain tissues of E16.5, E18.5, P0.5, and P10.5 wild-type mice, the E18.5 Fut9+/– mouse, and the E18.5 Fut9–/– mouse, first with chloroform–methanol (2:1) and secondly with chloroform–methanol–water (30:60:8). Samples dried with an N2 evaporator were dissolved in methanol, then subjected to a mild alkaline treatment in 0.1 N KOH/methanol at 40 °C for 2 h, and neutralized with 1 N acetic acid. After the free fatty acids had been removed with n-hexane, the remaining fractions were dried with the N2 evaporator and then subjected to Folch's partition. The lower neutral glycolipid fractions were dried with the N2 evaporator and subjected to immuno-TLC analysis. Neutral glycolipids were separated by TLC (HPTLC Kieselgel 60, 5641; Merck, Damstadt, Germany) with mixtures of chloroform–methanol–water (60:35:8), and immuno-TLC analysis with anti-SSEA-1 mAb was performed as described previously (Kimura et al. 1997).
Histochemistry
Adult mouse brains (6–7 months old) were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) (w/v) overnight and then embedded in paraffin. Sections of 10 µm were cut and stained with cresyl violet.
Paraffin sections (3–4 µm) were deparaffinized in xylene and rehydrated in ethanol and water. After antigen retrieval (10 min of autoclaving in PBS), the sections were rinsed in PBS and treated with 0.3% H2O2 in methanol for 30 min. Then, the sections were washed in PBS and incubated with the anti-SSEA-1 antibody (1:500) overnight at 4 °C. The sections were then washed in PBS and incubated with EnvisionTM+peroxidase-linked anti-mouse immunoglobulin (Dako, Kyoto, Japan) for 30 min at room temperature. They were washed in PBS and color was developed in 3,3'-diaminobenzidine (Wako, Osaka, Japan) with H2O2. The sections were then washed in running water, counterstained in hematoxylin, dehydrated in ethanol and xylene, and mounted.
Immunohistochemical analysis with anti-calbindin Ab was performed basically according to Yuasa et al. (2004). Adult mice, 6–7 months old, were transcardially perfusion-fixed with 4% paraformaldehyde and 0.5% picric acid in PBS. Coronal vibratome sections (70 µm) were immunostained with rabbit anti-calbindin antiserum (1:1000 dilution, Swant, Switzerland), a marker for a subset of GABAergic neurons, as the primary antibody, and Cy2-conjugated anti-rabbit IgG (1:100 dilution, Jackson Immunoresearch) was used as the secondary antibody. The localization of antigens was examined and the number of stained cells was counted under a fluorescence microscope. Immunofluorescence microscopy photographs were taken under the same conditions of brightness and contrast in regard to the separated parts of amygdala at a higher magnification. Subsequently, the images of each part of the amygdala were merged using Adobe Photoshop 7.0 software. By this method, the numbers of immunopositive cells were counted more accurately than if a single picture at lower magnification were used.
Behavioral tests
Littermates of wild-type and Fut9–/– mice were used, and their genotypes were determined by polymerase chain reaction of tail DNA samples. All the mice were male and 3–5 months old. The mice were kept on a 12-h light–dark cycle at a constant temperature (23±1 °C). The behavioral tests were always conducted between 1300 and 1800 hours. One week before the beginning of tests, the mice were housed in individual cages and were handled once a day for 5 days.
General activity
Locomotion and rearing behavior were measured by the method described previously (Ema et al. 1999). An open-field box (50x50x40 cm) was placed in a sound-attenuation room. Two pairs of 24x24 array infrared photosensors were set against the outer wall and equally spaced in the lower and upper rows at intervals of 2.5 and 6.5 cm above the floor. The frequency of photobeam interruption caused by animal movement was recorded by a computer. Each mouse was kept in the box for 30 min.
Light–dark preference test
The apparatus consisted of two compartments and was placed in a darkened sound-attenuating room. One was a bright (250 lx) chamber (25x50x40 cm) illuminated by a white bulb (60 W) and the other was a dark (1 lx) chamber of the same dimension. The two compartments were separated by a wall and connected by a small opening (8x16 cm). Each mouse was placed in the center of the light chamber and its behavior was recorded for 30 min. The frequency of photobeam interruption caused by animal movement in each compartment was recorded by a computer. The following behavioral measures were scored: the time spent in the light and dark compartments, the number of transitions between the two compartments, and the latency of the initial movement from the light to the dark room.
Elevated plus maze
The elevated plus maze consisted of two open (25x5 cm) and two enclosed arms of the same size with 15-cm-high transparent walls. The arms and central square were constructed of white plastic plates and elevated to a height of 50 cm above the floor. The mouse was placed on the central platform of the maze, with its head facing the open arm. The frequency of entry to open and closed arms and the time spent in open arms were recorded during the 10-min test.
Morris water maze task
Mice were trained on the hidden platform version of the Morris water maze task, as described previously (Ema et al. 1999). Mice were first trained to find the hidden platform and escape onto the platform fixed in the center of one of the four quadrants of the pool for six trials per day with an intertrial interval of 30 min over four consecutive days. The start positions were selected semirandomly from seven of eight equally spaced wall locations, excluding the point nearest the platform. The animals were allowed to swim until they mounted the platform and spent 30 s on it before returning to their cages. If the mice failed to find the platform within the 120-s limit, they were placed on to the platform for 30 s. A probe test was given 30 min after the last trial on day 4. For this test, the platform was removed from the pool, and the mouse was allowed to swim freely for 60 s. The time spent in each of the quadrants was measured by the automatic tracking system. On day 5, the hidden platform was switched to the opposite quadrant for reversal training. The mice received six trials on day 5 and two trials on day 6, followed by a reverse probe test 30 min later. On day 7, for the visible platform tests, the position of the platform was signaled by the presence of a white flag (10x10 cm) above the platform. The platform position varied among four possible positions, and the mice were tested on a total of six trials at an intertrial interval of 30 min with a different starting point.
Statistical analysis
Data were analyzed by two-way ANOVA, and comparison of paired groups was carried out by the Fisher LSD test. All values in the text and figure legends are expressed as means±SEM.
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Acknowledgments |
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We thank Mrs Keishin Hayashida and Tsutomu Inoue of Animal House, Institute of Life Science, Soka University for assistance in mouse breeding. This work was performed as a part of the R&D Project of Industrial Science and Technology Frontier Program (R&D for Establishment and Utilization of a Technical Infrastructure for Japanese Industry) supported by the New Energy and Industrial Technology Development Organization (NEDO).
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Footnotes |
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None declared.
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Abbreviations |
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CNS, central nervous system; Fuc, fucose; Fut, fucosyltransferase; GABA,
-aminobutyric acid; Gal, galactose; GlcNAc, N-acetylglucosamine; III3FucnLc4, Galß1-4(Fuc1-3)GlcNAcß1-3Galß1-4Glc1-1'Cer; III3V3Fuc2nLc6, Galß1-4(Fuc1-3)GlcNAcß1-3Galß1-4(Fuc1-3)GlcNAcß1-3Galß1-4Glc1-1'Cer; mAb, monoclonal antibody; PBS, phosphate-buffered saline; RPTPß, receptor-type protein–tyrosine phosphatase; SSEA-1, stage-specific embryonic antigen-1; TLC, thin layer chromatography; V3FucnLc6, Galß1-4(Fuc1-3)GlcNAcß1-3Galß1-4GlcNAcß1-3Galß1-4Glc1-1'Cer. |
References |
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