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J. Biol. Chem., Vol. 277, Issue 30, 27227-27231, July 26, 2002

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Mice Deficient in Nervous System-specific Carbohydrate Epitope HNK-1 Exhibit Impaired Synaptic Plasticity and Spatial Learning^*

Shoji Yamamoto, Shogo Oka, Mitsuhiro Inoue, Misa Shimuta^§, Toshiya Manabe^§, Hideki Takahashi^¶, Masaomi Miyamoto^¶, Masahide Asano, Junko Sakagami^**, Katsuko Sudo^**, Yoichiro Iwakura^**, Katsuhiko Ono, and Toshisuke Kawasaki^§§

From the  Department of Biological Chemistry and CREST (Core Research for Educational Science and Technology) Project, Japan Science and Technology Corporation, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan, ^§ Division of Cell Biology and Neurophysiology, Department of Neuroscience, Faculty of Medicine, Kobe University, Kobe 650-0017, Japan and Division of Neuronal Network, Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan, ^¶ Pharmaceutical Research Laboratories I, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., Osaka 532-8686, Japan,  Department of Transgenic Animal Science, Graduate School of Medical Science, Kanazawa University, Kanazawa 920-8640, Japan, ^** Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan, and  Department of Anatomy, Shimane Medical University, Izumo 693-8501, Japan

Received for publication, May 14, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The HNK-1 carbohydrate epitope, a sulfated glucuronic acid at the non-reducing terminus of glycans, is expressed characteristically on a series of cell adhesion molecules and is synthesized through a key enzyme, glucuronyltransferase (GlcAT-P). We generated mice with a targeted deletion of the GlcAT-P gene. The GlcAT-P / mice exhibited normal development of gross anatomical features, but the adult mutant mice exhibited reduced long term potentiation at the Schaffer collateral-CA1 synapses and a defect in spatial memory formation. This is the first evidence that the loss of a single non-reducing terminal carbohydrate residue attenuates brain higher functions.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glycosylation is a major post-translational protein modification, especially for cell surface proteins, which play important roles in a variety of cellular functions including recognition and adhesion. In the last decade, a number of glycosyltransferase genes and related genes have been cloned. Targeted deletion of these genes revealed the roles of cell surface glycans in the modulation of cellular interactions, particularly in the immune system (1, 2). We have been interested in the roles of a neural-specific carbohydrate, the HNK-1 carbohydrate, which is expressed on glycoproteins as well as on glycolipids and is postulated to be associated with cell adhesion, migration, and neurite outgrowth (3-5). The epitope is a sulfated trisaccharide, HSO₃-3GlcA1-3Gal1-4GlcNAc (6, 7), and the inner structure, Gal1-4GlcNAc, is commonly found on various glycoproteins and glycolipids. To elucidate the roles of the HNK-1 carbohydrate more clearly, we cloned two different glucuronyltransferases (GlcAT-P and GlcAT-S)¹ (8-11), which are key enzymes in the biosynthesis of the HNK-1 carbohydrate epitope (12, 13). In this study, we generated mice with a targeted deletion of the GlcAT-P gene and demonstrated clearly that the HNK-1 carbohydrate is in fact required for higher functions of the brain.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Targeted Disruption of the GlcAT-P Gene-- Cloning of the genomic clones of the 129/Sv mouse GlcAT-P gene was described previously (10). Construction of the targeting vector is schematically represented in Fig. 1A. The neomycin resistance gene cassette in vector pPGKneobpA (14) and diphtheria toxin A (DT-A) gene cassette in vector pMC1DT-A (15) were used as positive and a negative selection markers, respectively. The targeting vector was transfected into E14-1 embryonic stem (ES) cells (16) by electroporation. Two ES clones among 525 tested revealed the desired homologous recombination (13C-2 and 22D-5). To generate chimeric mice, both clones were aggregated with C57BL/6 × BDF1 8-cell-stage embryos, and the embryos were transferred into the uteri of pseudopregnant mice. Both clones gave rise to germline chimeras. Mice heterozygous for the mutation were obtained by cross-breeding of the chimeras with C57BL/6 mice. The heterozygotes were further backcrossed with C57BL/6 mice for more than eight generations, and the resulting heterozygous mutants were interbred to obtain wild-type and homozygous littermates. The genotypes of the mice were determined by PCR and Southern blot analysis of genomic DNA prepared from tail tissue using a 5'-external probe (Fig. 1B).

Histochemical Staining-- Mice (6 weeks old) were deeply anesthetized by diethyl ether inhalation and then perfused with phosphate-buffered saline followed by 4% paraformaldehyde in phosphate-buffered saline. The mouse brains were postfixed overnight followed by dipping into a 30% sucrose solution. For histological analysis, the sagittal sections (10 µm thick) were prepared, and the sections were stained with 0.5% thionine. For immunofluorescence staining, sections were incubated with the HNK-1 antibody and then incubated with the anti-mouse IgM antibody conjugated with FITC. These sections were visualized and digitized with a Fluoview laser confocal microscope system (Olympus). For double fluorescence staining, coronal sections of 8-week-old GlcAT-P / brains were prepared and incubated with biotinylated Wisteria floribunda agglutinin (WFA) and the HNK-1 antibody in 5% fetal calf serum. Then reactivity was visualized with rhodamine-avidin and a fluorescein isothiocyanate-conjugated secondary antibody followed by examination by laser confocal microscopy.

Electrophysiology-- To compare GlcAT-P / and +/+ mice, most experiments were performed in a blind fashion, and the results were essentially identical to those of non-blind experiments; so all the data were pooled. 11-20-week-old GlcAT-P / and +/+ mice were decapitated under deep halothane anesthesia. Hippocampal slices (400 µm thick) were prepared with a vibratome slicer and placed in a holding chamber for at least 1 h. A single slice was then transferred to the recording chamber and submerged beneath a continuously perfusing medium that had been saturated with 95% O₂, 5%CO₂. The medium comprised 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO₄, 2.5 mM CaCl₂, 1.0 mM NaH₂PO₄, 26.2 mM NaHCO₃, and 11 mM glucose. All perfusing solutions contained 100 µM picrotoxin to block GABA_A receptor-mediated inhibitory synaptic responses. Field potential recordings were made with a glass electrode (3 M NaCl) placed in the stratum radiatum. An Axopatch-1D amplifier was used, and the signal was filtered at 1 kHz and digitized at 10 kHz. To evoke synaptic responses, a bipolar tungsten stimulating electrode was placed in the stratum radiatum, and Schaffer collateral/commissural fibers were stimulated at 0.1 Hz. The ratio of excitatory postsynaptic potential (EPSP)/fiber volley amplitude (input-output relationship) of basal synaptic responses was measured in the presence of a low concentration of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (1 µM) to partially block AMPA receptors. This enables more accurate measurement of input-output relationships since the amplitude of EPSPs is usually much greater than that of fiber volleys. Furthermore, the use of a low concentration of CNQX reduces the non-linear summation of field EPSPs, especially when strong stimulus strengths are used. All experiments were performed at 25 °C. The data are expressed as means ± S.E. Student's t test was used to determine whether or not there was a significant difference (p < 0.05) in the mean between two sets of data.

Behavioral Tests-- Behavioral tests were conducted in a blind fashion during the light phase at approximately the same time each day. Data were calculated as means ± S.E. and analyzed by means of one-way analysis of variance and Student's t test; p values greater than 0.05 were taken as not significant. The Morris water maze test (17) has been used for analysis of spatial navigation and hippocampus-dependent memory formation of rodents. The apparatus consisted of a circular pool (120-cm diameter, 30-cm depth) filled with water at 30 °C. A transparent platform (10-cm diameter) was placed at a fixed location in one quadrant of the pool, 0.5 cm below the surface of the water. Using a video tracking system with computerized data (Target/2, Neuroscience), we analyzed the escape latency (platform search time), path length (the distance traveled to reach the platform), and swimming velocity for each trial. 12-15-week-old mice were trained with blocks of four trials per day for 4 days. The water-filled multiple T-maze was used as another test for evaluating the spatial learning of mice. The apparatus was similar to that described previously (18). The time taken to reach the goal, as the escape latency, was recorded. 17-20-week-old mice were trained through three trials per day for 3 days.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Generation of GlcAT-P-deficient Mice-- Mice with targeted deletion of the GlcAT-P gene were generated using the strategy outlined in Fig. 1A. Most of the catalytic region of GlcAT-P, i.e. exons 4 and 5 (10), was replaced by a PGK-neo gene. The disrupted region is essential for the glucuronyltransferase activity (8). The homologous recombination was confirmed by Southern blot analysis using an external 5' probe (Fig. 1B) and 3' probe (data not shown). Two lines of GlcAT-P / mice were generated from two independent ES clones. They gave essentially the same results in all experiments described below. Genotyping of 207 progeny from F1 heterozygous intercrosses suggested that GlcAT-P / mice exhibit normal embryonic development. GlcAT-P / mice appeared to be normal, and there was no significant difference in body weight or brain size between GlcAT-P / and +/+ mice up to 20 weeks after birth. Northern blot analysis of total RNA from 10-week-old mouse brains revealed that the expression of GlcAT-P mRNA was completely abolished in GlcAT-P / mice (Fig. 1C). In GlcAT-P +/ mice, GlcAT-P mRNA was reduced by ~50%. The same blot probed with the cDNA of GlcAT-S, which is the second glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate (9), indicated that the expression level of GlcAT-S mRNA in GlcAT-P +/ and / mice remained as low as that in GlcAT-P +/+ mice (Fig. 1C).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Generation and analysis of GlcAT-P / mice. A, schematic diagram of the targeting construct. The primers used for the screening of ES cell clones and mice are indicated by arrows and arrowheads, respectively. The probes used for Southern blot analysis of ES cells and mice are also shown. B, Southern blot analysis. Genomic DNAs isolated from mouse tail tissue from GlcAT-P +/+, +/, and / littermates were digested with the combination of ScaI and XhoI and then hybridized with the 32P-labeled 5' probe shown in A. Two independent lines of GlcAT-P / mice were analyzed. C, Northern blot analysis. The total RNAs were extracted from the whole brains of 10-week-old littermates and probed with GlcAT-P or GlcAT-S cDNA according to the procedure described previously (8). Arrowheads indicate the specific signals. D, glucuronyltransferase activity. The Nonidet P-40 extract was prepared from the whole brains of 10-week-old littermates as described previously (13) and was used as the enzyme source. The radioactivity of [14C]GlcA incorporated into asialoorosomucoid (ASOR) and paragloboside was measured. E, Western and lectin blot analyses. The membrane proteins were prepared from whole brains of 10-week-old littermates. Protein bands were stained with the HNK-1 antibody (left panel) or various biotinylated lectins (right panel). mAb, monoclonal antibody; MAM, M. amurensis mitogen; SSA, S. sieboldiana agglutinin; RCA, R. communis agglutinin.

GlcAT-P Is Responsible for Biosynthesis of the HNK-1 Carbohydrate on Both Glycoproteins and Glycolipids in Vivo-- Since the HNK-1 carbohydrate is expressed on both glycoproteins and glycolipids, the effect of GlcAT-P gene disruption on the overall ability to synthesize the HNK-1 carbohydrate in the brain was examined. As shown in Fig. 1D, the glucuronyltransferase activity of GlcAT-P / mice toward the glycolipid acceptor (paragloboside) disappeared almost completely as did the activity toward the glycoprotein acceptor (asialoorosomucoid) in 10-week-old mice. The glucuronyltransferase activity in GlcAT-P +/ mice was reduced to about half of that in the GlcAT-P +/+ mice for both acceptor substrates. Thus, GlcAT-P is the most predominant glucuronyltransferase responsible for the biosynthesis of the HNK-1 carbohydrate, and the contribution of GlcAT-S to the HNK-1 carbohydrate biosynthesis is marginal at this age.

GlcAT-P-deficient Mice Lack the HNK-1 Carbohydrate in the Central Nervous System-- As shown in Fig. 1E, the GlcAT-P / mice almost completely lost the HNK-1 carbohydrate as revealed on Western blot analysis. Several protein bands corresponding to over 100 kDa detected for GlcAT-P +/+ and +/ mice had disappeared in GlcAT-P / mice. The intensity of several protein bands in GlcAT-P +/ mice decreased to about half of that in GlcAT-P +/+ mice. Similarly immunohistochemical staining of 6-week-old mouse brains with the HNK-1 antibody revealed high levels of HNK-1 carbohydrate expression widely in the GlcAT-P +/+ mice brains including the cerebral cortex, hippocampus, and cerebellum (Fig. 2, A-C). In contrast, the immunoreactivity of the HNK-1 carbohydrate in the GlcAT-P / mice was reduced to almost negligible levels in all regions of the nervous system (Fig. 2, D-F). These findings clearly indicated that GlcAT-P is the principal enzyme responsible for the biosynthesis of the HNK-1 carbohydrate in the mature brain. There is at least the possibility that the lack of glucuronic acid at the terminus of glycans has an overall effect on the glycan structures in the brain since lectin blot analysis with the MAM (Maackia amurensis mitogen), SSA (Sambucus sieboldiana agglutinin), and RCA (Ricinus communis agglutinin) lectins, which specifically recognize the terminal structures of glycans, i.e. Sia2-3Gal, Sia2-6Gal, and -Gal, respectively, exhibited almost indistinguishable profiles in wild-type and mutant mice (Fig. 1E). In GlcAT-P / mice, non-reducing terminal galactose is presumably substituted by sialic acid, resulting in a common terminal disaccharide, Sia-Gal.

View larger version (48K): [in this window] [in a new window]   Fig. 2.   A-F, immunohistochemical analysis of expression of the HNK-1 carbohydrate in mouse brain. Sagittal brain sections from 6-week-old wild-type (+/+, A-C) and GlcAT-P-deficient (/, D-F) mice were immunostained with the HNK-1 antibody. The HNK-1 carbohydrate was markedly diminished in the cerebral cortex (A and D), hippocampus (B and E), and cerebellum (C and F) of GlcAT-P / mice. A small amount of remaining HNK-1 carbohydrate was also detected in the cerebral cortex and hippocampus of GlcAT-P / mice (arrowheads in D and E). G-L, the remaining HNK-1 immunoreactivity in perineuronal nets of GlcAT-P (/) mice. Coronal sections of the cerebral cortex of adult GlcAT-P / mice were stained doubly with the HNK-1 antibody (G and J) and biotinylated WFA (H and K). Equivalent subpopulations of cortical neurons were stained with the HNK-1 antibody and WFA lectin (I and L). Images of magnified regions are also shown (J-L). Scale bars, 100 µm.

Remaining HNK-1 Carbohydrate in the GlcAT-P / Mice-- In GlcAT-P / mice, the HNK-1 carbohydrate disappeared almost completely as described above. However, further studies revealed that a trace of HNK-1 immunoreactivity remained on the surfaces of soma and proximal dendrites of a subset of neurons in some limited regions (Fig. 2, D and E, arrowheads). Some parts of interneurons were likely to be HNK-1-positive in the cerebral cortex, and such remaining immunoreactivity was not so significant up to 2 weeks after birth but seemed to increase gradually thereafter. These signal patterns and morphological features imply that the remaining HNK-1 carbohydrate in the GlcAT-P / mice corresponded to the perineuronal nets, which are known to comprise a lattice-like accumulation of the extracellular matrix on an unidentified subset of neurons (19). This was confirmed by double fluorescence staining of adult GlcAT-P / mice with the HNK-1 antibody and an N-acetylgalactosamine-binding lectin, WFA, a well known marker of perineuronal nets (19). In the cerebral cortex, both signals were detected in a restricted population of cortical neurons that were observed mainly in layers III-V (Fig. 2, G-L), although not all of the WFA-positive neurons were immunoreactive for the HNK-1 antibody (Fig. 2, I and L). These results revealed that the HNK-1 carbohydrate expressed in the perineuronal net structure was synthesized by an enzyme(s) other than GlcAT-P, presumably GlcAT-S.

Reduced LTP at the Schaffer Collateral-CA1 Synapses-- The HNK-1 epitope is commonly expressed on a series of cell adhesion molecules (CAMs) such as the neural cell adhesion molecule (NCAM), L1, telencephalin, and tenascin-R (20). These CAMs are important constituents of the synaptic structure and play important and diverse roles in the regulation of synaptic plasticity. Aberrations of LTP in the hippocampal CA1 region occurred in mice deficient in NCAM and telencephalin (21, 22). Mice lacking extracellular matrix molecule tenascin-R were also shown to exhibit aberrant LTP in the CA1 region (23). With this background, we analyzed LTP in the CA1 region of GlcAT-P +/+ and / mice to examine the effect of HNK-1 carbohydrate deficiency on synaptic plasticity (Fig. 3). EPSPs were evoked by stimulating afferent fibers in the stratum radiatum of the CA1 region using the extracellular field potential recording technique. High frequency stimulation of afferent fibers (100 Hz for 1 s) gave rise to LTP of excitatory synaptic transmission in the GlcAT-P +/+ mice (161.7 ± 7.3% of baseline, n = 14), while the magnitude of LTP in the GlcAT-P / mice was significantly lower (131.9 ± 8.4% of baseline, n = 14; p < 0.02, t test) than that in the GlcAT-P +/+ mice (Fig. 3, A and B). In contrast, the initial potentiation after tetanus and the depolarization during tetanic stimulation were similar in magnitude to those in GlcAT-P +/+ mice (data not shown). The input-output relationship of AMPA receptor-mediated synaptic responses in the CA1 region was not significantly different between GlcAT-P +/+ and / mice (Fig. 3C), suggesting that basal synaptic transmission in GlcAT-P / mice is normal. We then examined three forms of presynaptic short term plasticity in the presence of D-()-2-amino-5-phosphonovaleric acid (D-APV), which blocks any N-methyl-D-aspartate receptor-dependent postsynaptic modification (Fig. 3, D-F). The magnitude of post-tetanic potentiation induced by tetanic stimulation (100 Hz for 1s) (Fig. 3D), the paired-pulse facilitation induced by a pair of afferent fiber stimuli at 50-, 100-, or 200-ms intervals (Fig. 3E), and the synaptic responses to repetitive afferent fiber stimulation (5 Hz for 3 min) (Fig. 3F) were indistinguishable between GlcAT-P +/+ and / mice. These results indicated that the presynaptic function is intact in GlcAT-P / mice and suggested that presynaptic changes are not involved in the impairment of LTP in GlcAT-P / mice.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   A-C, LTP, but not basal synaptic transmission, is impaired in GlcAT-P / mice. A, the averaged time course of LTP in GlcAT-P +/+ (open circles, n = 14) and / (closed circles, n = 14) mice. Initial EPSP slopes were normalized in each experiment to the mean slope value during the control period (30-0 min). A train of high frequency stimuli (100 Hz for 1s) was applied at time 0. Sample traces of field EPSPs (average of 10 consecutive responses) recorded at the times indicated by the numbers are shown in the inset. B, the summary of LTP calculated as the percent increase in the mean EPSP slope from 50 to 60 min after high frequency stimulation compared with the mean EPSP slope during the control period (30-0 min). LTP in GlcAT-P / mice (closed bar) was significantly lower than that in GlcAT-P +/+ mice (open bar) (p < 0.02, t test). C, the input-output relationship of GlcAT-P / (closed circles) and +/+ (open circles) mice. 25 µM D-APV was present to block N-methyl-D-aspartate receptor-mediated synaptic responses. A low concentration of CNQX (1 µM) was also present. The data were first sorted by the range of fiber volley amplitudes, and then EPSP amplitudes were averaged within each range. Sample traces with various stimulus strengths are shown in the inset. D-F, the impairment of LTP in GlcAT-P / mice is not mediated by the changes in presynaptic release mechanisms. Analysis of the presynaptic short term plasticity in GlcAT-P +/+ (open circles) and / (closed circles) mice was performed in the presence of 50 µM D-APV. D, post-tetanic potentiation recorded in GlcAT-P +/+ (n = 9) and / (n = 12) mice. A train of high frequency stimuli (100 Hz for 1s) was delivered at time 0. E, paired-pulse facilitation in GlcAT-P +/+ (n = 13) and / (n = 13) mice. The ordinate indicates the ratio of the second field EPSP slope to the first field EPSP slope. With any interstimulus interval (50, 100, 200 ms), no significant difference was observed between the two genotypes. F, responses to prolonged low frequency stimulation (5 Hz for 3min) in GlcAT-P +/+ (n = 12) and / (n = 13) mice. amp., amplitude.

Impaired Performance in Spatial Learning Tasks in GlcAT-P-deficient Mice-- In view of the reduced LTP, two types of spatial learning tests were carried out with 11-15-week-old GlcAT-P +/+ and / mice (Fig. 4). In the Morris water maze test, the time taken to reach the hidden platform (escape latency) was significantly longer for the GlcAT-P / mice than for the GlcAT-P +/+ mice during 4 days of training (Fig. 4A), although the swimming speed in the task was not significantly different between GlcAT-P +/+ and / mice (data not shown). In the water-filled multiple T-maze task, the GlcAT-P / mice showed increased escape latencies to the goal arm compared with the GlcAT-P +/+ mice, most significantly on day 2 during 3 days of training (Fig. 4B). These results suggest that the differences in performance of GlcAT-P / mice in the spatial learning tasks were related to impaired spatial learning, not to decreased motor activity.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Impaired spatial learning in GlcAT-P / mice. GlcAT-P +/+ (n = 17, open circles) and / (n = 18, closed circles) mice were tested with spatial learning tasks. A, mice were trained in a Morris water maze for 4 consecutive days. Learning performance is expressed as the mean escape latency of four trials per day. GlcAT-P / mice showed a significantly longer escape latency than GlcAT-P +/+ mice (p < 0.05, one-way analysis of variance (ANOVA)). B, learning performance in a water-filled multiple T-maze. Mice were given one session of three trials per day for 3 consecutive days. The mean escape latency on training day 2 is significantly different between the two genotypes (p < 0.05, t test).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The abnormality in higher brain functions of the GlcAT-P / mice, such as reduced LTP at the Schaffer collateral-CA1 synapses and defect in spatial memory formation, was similar to those in NCAM-deficient mice (21, 24), suggesting that the HNK-1 carbohydrate may function through modulation of the functions of CAMs. Besides NCAM, CAMs bearing the HNK-1 carbohydrate, such as telencephalin and tenascin-R, have also been shown to play important roles in the induction and expression of LTP (22, 23). The detailed molecular mechanisms by which the HNK-1 carbohydrate modulates the functions of CAMs are not clear at the moment. However, it should be noted that the HNK-1 carbohydrate on NCAM and L1 was shown to negatively regulate their homophilic binding activity.² The HNK-1 carbohydrate may weaken the homophilic interaction of CAMs expressed on the synapses and facilitate synaptic plasticity. It should be noted that only a subpopulation of CAMs express the HNK-1 carbohydrate and that the expression is independently regulated from the biosynthesis of CAMs, indicating that the HNK-1 carbohydrate is the characteristic functional component in vivo as a fine tuner that regulates synaptic plasticity or other brain functions. Alternatively the HNK-1 carbohydrate itself may be involved in LTP via interaction with binding proteins (receptors) on the cell surface or in the cell matrix. Several HNK-1 carbohydrate-binding proteins have been identified, such as laminin, selectins, SBP-1, and brevican (25-28). However, the association of these receptors or binding proteins with LTP has not been proved.

Recently Saghatelyan et al. (29) reported that a monoclonal antibody that recognizes the HNK-1 carbohydrate decreased perisomatic inhibitory postsynaptic currents and enhanced LTP in acute slices of the mouse hippocampus. This inhibition of perisomatic inhibitory postsynaptic currents by the antibody was also observed in NCAM-deficient mice but not in tenascin-R-deficient mice. The authors suggested that tenascin-R is a carrier molecule for the HNK-1 carbohydrate involved in the regulation of perisomatic inhibition of CA1 pyramidal neurons by GABAergic interneurons (29). In the present study, however, we detected reduced LTP in the presence of 100 µM picrotoxin that blocks the GABA_A receptor-mediated inhibitory synaptic response, demonstrating that a mechanism other than the reduction of GABAergic inhibition of CA1 pyramidal neurons is involved in the attenuation of LTP in GlcAT-P / mice.

In the present study, we produced mice deficient in GlcAT-P, a glucuronyltransferase, which is involved in biosynthesis of the HNK-1 carbohydrate. The GlcAT-P / mice exhibited gross defects in functions of the nervous system such as LTP in the CA1 region and hippocampus-dependent spatial learning. This is the first study to demonstrate the involvement of a carbohydrate, notably of only a single non-reducing terminal carbohydrate residue, in higher ordered brain functions including learning and memory.

	FOOTNOTES

^* This work was supported in part by Grants-in-aid for Scientific Research B-12470497 (to T. K.), A-12308043 (to T. M.), and C-13680688 (to S. O.) from the Japan Society for the Promotion of Sciences and Grants-in-aid for Scientific Research on Priority Areas A-12053251 (to T. M.) and A-12033204 (to S. O.), and Special Coordination Funds for Promoting Science and Technology (to T. M.) from the Ministry of Education, Culture, Sports and Technology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§§ To whom correspondence should be addressed: Dept. of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan. Tel.: 81-75-753-4572; Fax: 81-75-753-4605; E-mail: kawasaki@pharmsun.pharm.kyoto-u.ac.jp.

Published, JBC Papers in Press, May 24, 2002, DOI 10.1074/jbc.C200296200

² K. Ohtsubo, S. Oka, A. Nakamura, Y. Mitsumoto, S. Yamamoto, K. Ono, J. Lütjohann, M. Schachner, U. Rutishauser, and T. Kawasaki, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: GlcAT, glucuronyltransferase; GlcA, glucuronic acid; CAM, cell adhesion molecule; NCAM, neural cell adhesion molecule; EPSP, excitatory postsynaptic potential; LTP, long term potentiation; WFA, W. floribunda agglutinin; DT-A, diphtheria toxin A; ES, embryonic stem; GABA, -aminobutyric acid; GABA_A, GABA, type A; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; D-APV, D-()-2-amino-5-phosphonovaleric acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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