Involvement of nectins in the formation of puncta adherentia junctions and the mossy fiber trajectory in the mouse hippocampus

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 31 (2006) 315 – 325

Involvement of nectins in the formation of puncta adherentia junctions and the mossy fiber trajectory in the mouse hippocampus Tomoyuki Honda,a,b Toshiaki Sakisaka,a Tomohiro Yamada,a Noriko Kumazawa,c Takashi Hoshino,a Mihoko Kajita,a Tetsuro Kayahara,b Hiroyoshi Ishizaki,d,e Miki Tanaka-Okamoto,d Akira Mizoguchi,b Toshiya Manabe,c,f Jun Miyoshi,d and Yoshimi Takai a,* a

Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan Department of Anatomy, Faculty of Medicine, Mie University, Tsu 514-8507, Japan c Division of Neuronal Network, Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan d Department of Molecular Biology, Osaka Medical Center for Cancer and Cardiovascular Disease, Osaka 537-8511, Japan e KAN Research Institute Inc., 93 Chudoji-Awatamachi, Shimogyo-ku, Kyoto 600-8815, Japan f CREST, Japan Science and Technology Agency, Japan b

Received 28 April 2005; revised 17 August 2005; accepted 4 October 2005 Available online 21 November 2005

Synapses are specialized intercellular junctions whose specificity and plasticity are mediated by synaptic cell adhesion molecules. In hippocampus, the mossy fibers form synapses on the apical dendrites of the CA3 pyramidal cells where synaptic and puncta adherentia junctions (PAJs) are highly developed. Synaptic junctions are the sites of neurotransmission, while PAJs are regarded as mechanical adhesion sites. Cell – cell adhesion molecules nectin-1 and nectin-3 asymmetrically localize at the pre- and post-synaptic sides of PAJs, respectively. To reveal the definitive role of nectins, we analyzed nectin-1 / and nectin-3 / mice. In both the mutant mice, the number of PAJs at the synapses between the mossy fiber terminals and the dendrites of the CA3 pyramidal cells was reduced. In addition, the abnormal mossy fiber trajectory was observed. These results indicate that nectins are involved in the formation of PAJs, which maintain the proper mossy fiber trajectory. D 2005 Elsevier Inc. All rights reserved.

Introduction Synapses are one of the specialized intercellular junctions whose specificity and plasticity provide neurons with structural and functional basis for formation of neural network. Synapses are asymmetric junctions formed between two different neurons and at least two types of intercellular junctions with different functions have been recognized: synaptic junctions (SJs) and puncta adherentia junctions (PAJs) (Peters et al., 1976). SJs are associated * Corresponding author. Fax: +81 6 6879 3419. E-mail address: [email protected] (Y. Takai). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2005.10.002

with synaptic vesicles docked at the presynaptic active zone where Ca2+ channels localize, and with postsynaptic densities (PSDs) where the specific neurotransmitter receptors localize. PAJs have symmetrical paramembranous dense materials with no association with synaptic vesicles (Peters et al., 1976; Sˇpa*ek and Lieberman, 1974). At the synapses between the mossy fiber terminals and the dendrites of the CA3 pyramidal cells in hippocampus (mossy fiber synapses), both SJs and PAJs are highly developed. The remodeling of these synapses is implicated in synaptic plasticity, a principal mechanism of memory and learning. SJs are regarded as sites for neurotransmission, whereas PAJs are regarded as mechanical adhesion sites between axon terminals and their targets, although their exact physiological functions remain unknown. N-cadherin and catenins have been shown to symmetrically localize at PAJs, but not at SJs, between the mossy fiber terminals and the dendrites of the CA3 pyramidal cells (Mizoguchi et al., 2002; Uchida et al., 1996). N-cadherin is a Ca2+-dependent cell – cell adhesion molecule and catenins are N-cadherin-binding proteins which connect N-cadherin to the actin cytoskeleton (Takeichi, 1991; Tepass et al., 2000). In addition to them, nectin1 and nectin-3 asymmetrically localize at the pre- and post-synaptic sides, respectively, of the PAJs, but not at SJs, while afadin symmetrically localizes at PAJs (Mizoguchi et al., 2002). Nectin-1 and nectin-3 are Ca2+-independent immunoglobulin-like cell – cell adhesion molecules which belong to the nectin family consisting of four members, and afadin is an actin filament-binding protein that connects nectins to the actin cytoskeleton (Takai and Nakanishi, 2003). Nectins form homo- or hetero-trans-dimers in a Ca2+independent manner, causing cell – cell adhesion. In epithelial cells in culture, nectins first form cell – cell adhesion and then recruit Ecadherin to the nectin-based cell – cell adhesion sites to coopera-

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tively form adherens junctions (Takai and Nakanishi, 2003). Disruption of the nectin-based cell – cell adhesion by nectin inhibitors in rat cultured hippocampal neurons results in a decrease in the size of synapses and a concomitant increase in the number of synapses (Mizoguchi et al., 2002). In addition, the mutant nectin-1 gene has reportedly been responsible for human cleft lip/palateectodermal dysplasia, Margarita island ectodermal dysplasia and Zlotogora-Ogu¨r syndrome, characterized by mental retardation, cleft lip/palate, syndactyly, and ectodermal dysplasia (Suzuki et al., 2000). Although available evidence suggests a role of nectins in the formation of synapses in cooperation with N-cadherin, definitive evidence for this role of nectins has not been obtained. We show here the direct evidence, by use of the nectin-1 / and nectin-3 / mice which we have recently made (Inagaki et al., 2005), that these nectins are essential for the formation of PAJs, which serve as a mechanical fixation for maintaining the proper mossy fiber trajectory.

Results Normal protein levels of the typical components of SJs and PAJs in the hippocampus of both the mutant mice We have recently made nectin-1 / and nectin-3 / mice (Inagaki et al., 2005). The nectin-1 / mice are viable and fertile and show microphthalmia. The nectin-3 / mice are viable and male-specifically infertile and show microphthalmia. Overall morphology of brains from both the mutant mice was apparently indistinguishable from that from the wild-type mice (data not shown). We then examined the protein levels of the typical

Fig. 1. Protein levels of various synaptic components in the hippocampus of both the mutant mice. The synaptic proteins extracted from the hippocampus of the wild-type, nectin-1 / , or nectin-3 / mice were analyzed by quantitative immunoblotting with various Abs against the indicated proteins. (A) +/+, wild-type mouse; / , nectin-1 / mouse. (B) +/+, wild-type mouse; / , nectin-3 / mouse. The results shown in all panels are the representative of three independent experiments.

components of SJs and PAJs in the hippocampus of both the mutant mice. In the nectin-1 / hippocampus, nectin-1 was undetectable, but the levels of other marker proteins, including nectin-3, afadin, N-cadherin, h-catenin, synaptophysin, PSD-95, and GluR1, were indistinguishable from those in the wild-type hippocampus (Fig. 1A). PSD-95 and GluR1 are markers for PSDs and synaptophysin is a marker for the synaptic vesicles (Boulter et al., 1990; Cho et al., 1992; Navone et al., 1986). In the nectin-3 / hippocampus, nectin-3 was undetectable, but the levels of the other marker proteins were indistinguishable from those in the wild-type hippocampus (Fig. 1B). These results indicate that nectin-1 deficiency or nectin-3 deficiency does not affect the expression levels of other typical components of SJs or PAJs. Abnormal immunofluorescence signals for nectins, afadin, and N-cadherin at the stratum lucidum in the hippocampus of both the mutant mice In the wild-type hippocampus, the immunofluorescence signals for nectins, afadin, and N-cadherin were found as dots that aligned along the dendrites of the CA3 pyramidal cells at the stratum lucidum as described (Fig. 2) (Mizoguchi et al., 2002). In the nectin-1 / hippocampus, the signal for nectin-1 was indeed undetectable at the stratum lucidum (Figs. 2Aa and b). The signal for nectin-3 was almost undetectable, consistent with the earlier observation that nectin-1 and nectin-3 concentrate there and may form hetero-trans-dimers (Mizoguchi et al., 2002) (Figs. 2Aa and c). The sizes of the signals for afadin and N-cadherin were markedly reduced, but their numbers were not significantly different from those in the wild-type hippocampus (Fig. 2Ab and not shown). Quantitative analysis showed that there was about 50% reduction in the sizes of the signals for afadin and N-cadherin in the nectin-1 / hippocampus (Fig. 2Ad). The signals for afadin and N-cadherin apparently colocalized (data not shown). In the wild-type hippocampus, the immunofluorescence signals for Bassoon, PSD-95, and GluR1 were found as dots that aligned along the dendrites of the CA3 pyramidal cells at the stratum lucidum (data not shown). Their sizes or numbers were apparently indistinguishable between the wild-type and nectin-1 / hippocampus (data not shown). In the nectin-3 / hippocampus, the signal for nectin-3 was indeed undetectable at the stratum lucidum (Figs. 2Ba and b). The signal for nectin-1 was hardly detected, consistent with the earlier observation that nectin-1 and nectin-3 concentrate there and may form hetero-trans-dimers (Mizoguchi et al., 2002) (Figs. 2Ba and c). The sizes of the signals for afadin and Ncadherin were markedly reduced, but their numbers were not significantly different from those in the wild-type hippocampus (Fig. 2Bb and data not shown). Quantitative analysis showed that there was about 50% reduction in the sizes of the signals for afadin and N-cadherin in the nectin-3 / hippocampus (Fig. 2Bd). The signals for afadin and N-cadherin apparently colocalized (data not shown). The sizes or numbers of the signals for Bassoon, PSD-95, and GluR1 were apparently indistinguishable between the wild-type and nectin-3 / hippocampus (data not shown). In the nectin-1+/ or nectin-3+/ hippocampus, the sizes or numbers of the signals for all the molecules described above were not significantly different from those in the wildtype hippocampus (Figs. 2Ac, Ad, Bc, and Bd). The expression pattern of nectin-3 mRNA in the nectin-1 / hippocampus, that of nectin-1 mRNA in the nectin-3 / hippocampus, and those

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Fig. 2. Localization of nectins, afadin, and N-cadherin at the stratum lucidum in the hippocampus of both the mutant mice. (A) The wild-type or nectin-1 / mouse hippocampal sections were double stained with the Abs against the indicated proteins, followed by immunofluorescence microscopy. (a) The staining patterns of nectin-1 and nectin-3. (b) The staining patterns of nectin-1 and N-cadherin. (c) Quantitative analysis of the area of the signal for nectin-3 at the CA3. (d) Quantitative analysis of the ratio of the area of the signals for afadin or N-cadherin at the CA3 to that at the CA1. +/+, Wild-type mouse; +/ , nectin-1+/ mouse; and / , nectin-1 / mouse. (B) The wild-type or nectin-3 / mouse hippocampal sections were double stained with the Abs against the indicated proteins, followed by immunofluorescence microscopy. (a) The staining patterns of nectin-3 and nectin-1. (b) The staining patterns of nectin-3 and N-cadherin. (c) Quantitative analysis of the area of the signal for nectin-3 at the CA3. (d) Quantitative analysis of the ratio of the area of the signals for afadin or N-cadherin at the CA3 to that at the CA1. +/+, Wild-type mouse; +/ , nectin-3+/ mouse; and / , nectin-3 / mouse. *P < 0.05 versus the wild type. Lower left insets are images at a high magnification. SL, stratum lucidum; SP, stratum pyramidale; and SR, stratum radiatum. Scale bars, 30 Am. The results shown in panels Aa, Ab, Ba, and Bb are the representative of three independent experiments. The areas or the ratios shown in panels Ac, Ad, Bc, or Bd are the mean T SEM.

of afadin and N-cadherin mRNAs in the hippocampus of both the mutant mice were not significantly different from those in the wild-type hippocampus (Fig. 3). These results suggest that nectin-1 deficiency or nectin-3 deficiency causes impaired localization of nectins, afadin, and N-cadherin. Impairment of the formation of PAJs in the hippocampus of both the mutant mice We next examined morphological change electron microscopically in the mossy fiber synapses of both the mutant mice. The mossy fiber terminals, spines, dendritic trunks, and PAJs were identified according to the previous electron microscopic studies (Amaral and Dent, 1981; Blackstad, 1963; Hamlyn, 1962). In the wild-type hippocampus, the mossy fiber terminals appeared as a vesicle-rich

large expansion, SJs had synaptic vesicles and PSDs at the pre- and post-synaptic sides, respectively, and well-developed PAJs had symmetrical paramembranous dense materials with no association with synaptic vesicles (Fig. 4A). In the hippocampus of both the mutant mice, the number of the well-developed PAJs was markedly reduced (Fig. 4). Quantitative analysis showed that there was about 70% reduction in the number of PAJs per bouton and in the percentage of the perimeter occupied by PAJs at the stratum lucidum in the hippocampus of both the mutant mice (Table 1). The morphological difference of the spines, PSDs, or synaptic vesicles was not markedly observed between the wild-type and nectin-1 / hippocampus and between the wild-type and nectin-3 / hippocampus (Fig. 4 and Table 1). These results indicate that nectins are necessary for the formation of PAJs at the stratum lucidum but not likely necessary for the formation of the spines or SJs.

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Fig. 3. Expression patterns of nectins, afadin, and N-cadherin mRNA in the hippocampus of both the mutant mice. The expression patterns of nectins, afadin, and N-cadherin mRNA at the indicated area were analyzed by in situ hybridization. (A) The expression pattern of nectin-3 mRNA. (a) Wild-type mouse. (b) Nectin-1 / mouse. (B) The expression pattern of nectin-1 mRNA. (a) Wild-type mouse. (b) Nectin-3 / mouse. (C) The expression patterns of afadin and Ncadherin mRNA. (a1 – c1 and a2 – c2) Afadin mRNA. (a3 – c3 and a4 – c4) N-cadherin mRNA. (a1 – a4) Wild-type mouse. (b1 – b4) Nectin-1 / mouse. (c1 – c4) Nectin-3 / mouse. DG, dentate gyrus; ML, molecular layer; SG, stratum granulosum; DH, dentate hilus; SL, stratum lucidum; SP, stratum pyramidale; and SO, stratum oriens. Scale bars, 30 Am. The results are the representative of three independent experiments.

Abnormal mossy fiber trajectory in the hippocampus of both the mutant mice To examine a possible involvement of the nectin-based PAJs in the mossy fiber trajectory, we visualized the mossy fibers at the stratum lucidum in the hippocampus by using biotylated dextran amine (BDA) as an anterograde tracer. After the injection of BDA into the dentate gyrus, anterogradely labeled mossy fibers were observed in the CA3 area. At the curvature of the CA3 in the wildtype hippocampus, the labeled mossy fibers were mostly confined to the stratum lucidum (Fig. 5Aa). In contrast, at the curvature of the CA3 in the hippocampus of both the mutant mice, the labeled

mossy fibers were not confined to the stratum lucidum, and some of the labeled mossy fibers were also observed in the stratum oriens (Figs. 5Ab and c). To quantify this phenotype, we visualized all the mossy fibers by calbindin staining. Calbindin is a Ca2+-binding protein which shows a characteristic spatial pattern of expression in hippocampus and is a marker of the mossy fibers (Baimbridge and Miller, 1982). In the wild-type hippocampus, the most mossy fibers were found in a bundle above the stratum pyramidale (suprapyramidal bundle, SPB) (Figs. 5Ba1 and a2). In addition, a small number of the mossy fibers were found underneath the stratum pyramidale (infrapyramidal bundle, IPB). The IPB crossed the stratum pyramidale to join the SPB before extending to the apex of

Fig. 4. Electron microscopic morphology of the synapse in the hippocampus of both the mutant mice. The wild-type, nectin-1 / , or nectin-3 / mouse hippocampal sections were subjected to electron microscopy. (A) The synapse from the wild-type mouse. (B) The synapse from the nectin-1 / mouse. (C) The synapse from the nectin-3 / mouse. Arrows, PAJs; arrowheads, PSDs; D, dendritic trunks; and S, dendritic spines. Scale bars, 500 nm. The results are the representative of four independent experiments.

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Table 1 Quatitative analysis of the fine structure of the mossy fiber synapses Wild-type Perimeter (Am) Bouton area (Am2) Number of PSDs/bouton Length of PSDs (nm) Cumulative length of PSDs/perimeter Number of PAJs/bouton Length of PAJs (nm) Cumulative length of PAJs/perimeter Number of synaptic vesicles/Am2 Number of docked vesicles/synaptic vesicles Number of spines/boutonb

12.5 4.45 3.48 227 0.0645 3.45 239 0.0630 128 0.0326 2.03

T T T T T T T T T T T

6.0 4.56 1.91 81 0.0385 2.00 137 0.0302 114 0.0242 0.89

Nectin-1

/

Nectin-3

/

11.0 T 3.53 T 3.69 T 220 T 0.0704 T 1.00 T 250 T 0.0228 T 133 T 0.0255 T 2.12 T

2.6 1.28 2.07 61 0.0391 1.83a 120 0.0443a 35 0.0148 1.18

12.4 T 6.6 4.91 T 4.15 3.38 T 1.82 240 T 129 0.0650 T 0.0347 1.13 T 1.15a 148 T 56.8 0.0156 T 0.0179a 112 T 52 0.0290 T 0.0168 1.63 T 0.89

40 mossy fiber terminals from each genotype were examined. The means T SD are shown. a P < 0.00001 versus the wild type. b The spine was defined as an expansion that was smaller than 8 Am round and had at least one PSD.

the curvature of the CA3. In the nectin-1 / or nectin-3 / hippocampus, there were also both the SPB and the IPB, but the IPB was abnormally long, extending to the apex of the curvature of the CA3 and appearing to fail to cross the stratum pyramidale (Figs. 5Bb1, b2, c1, and c2). The abnormally long IPB is likely to form ectopic synapses with improper neurons somewhere. Quantitative analysis showed that the IPB in the nectin-1 / or nectin-3 / hippocampus was longer than that in the wild-type mice (Figs. 5C and D). These results suggest that the nectin-based PAJs are involved in maintaining the proper mossy fiber trajectory. We then examined the mossy fiber trajectory during development. The SPB appears firstly after the birth, and the IPB appears secondly after P7 during normal development of mossy fibers (Amaral and Dent, 1981). Consistently, on P5, only the SPB, but not the IPB, was observed at the CA3 in the wild-type, nectin-1 / , and nectin-3 / hippocampus (data not shown). Significant difference was not observed in the SPB between the wild-type and nectin-1 / hippocampus and between the wild-type and nectin-3 / hippocampus. On P12, both the SPB and the IPB were observed in the wild-type, nectin-1 / , and nectin-3 / hippocampus (Fig. 6). The abnormally long IPB was observed in the hippocampus of both the mutant mice as shown in the hippocampus of both the adult mutant mice in Fig. 5. These results indicate that the impairment of the mossy fiber trajectory in the hippocampus of both the mutant mice appears during development. Apparently indistinguishable electrophysiological properties at the mossy fiber synapses in the hippocampus of both the mutant mice from those in the wild-type hippocampus To examine possible involvement of the nectin-based PAJs in the properties of synaptic transmission, we finally focused on excitatory synaptic transmission at the mossy fiber synapses. We first examined the input – output relationship at the mossy fiber synapses by recording field excitatory post-synaptic potentials (EPSPs) evoked by electrical stimulation of the mossy fibers with variable stimulus strengths. The input – output relationship of excitatory synaptic transmission was apparently indistinguishable between the wild-type and nectin-1 / hippocampus (Fig. 7A). Furthermore, paired-pulse facilitation (PPF) evoked by stimulating the mossy fibers twice at short intervals, which is a presynaptic parameter associated with the probability of transmitter release, was also apparently indistinguishable (Fig. 7B). We next

examined synaptic plasticity in the nectin-1 / hippocampus. At the mossy fiber synapses, high-frequency stimulation of afferent fibers induces N-methyl-d-aspartate (NMDA) receptor-independent long-term potentiation (LTP) (Nicoll and Malenka, 1995). In the nectin-1 / hippocampus, LTP was normally induced by tetanic stimulation in the presence of 2-amino-5-phosphonovaleric acid (D-APV), which was apparently indistinguishable from that in the wild-type hippocampus (Fig. 7C). Thus, although the number of PAJs was reduced and the mossy fiber trajectory was abnormal in the nectin-1 / hippocampus, basal synaptic transmission and long-term synaptic plasticity at the mossy fiber synapses were apparently indistinguishable from those in the wild-type hippocampus under our experimental conditions. We have obtained similar results in the nectin-3 / hippocampus (data not shown).

Discussion Immunofluorescence microscopy has revealed that the signals for nectin-1 and nectin-3 are undetectable at the stratum lucidum in the hippocampus of both the mutant mice, although normal protein levels and mRNA expression patterns of nectin-1 and nectin-3 are detected in the nectin-3 / and the nectin-1 / hippocampus, respectively. These results, together with the earlier observations that nectin-1 and nectin-3 asymmetrically localize at the pre- and post-synaptic sides, respectively, of the PAJs (Mizoguchi et al., 2002), indicate that they form hetero-trans-dimers at the PAJs of the mossy fiber synapses in hippocampus. In the absence of nectin1, remaining nectin-3 is not able to form hetero-trans-dimers with nectin-1, resulting in dispersion of nectin-3 clusters and reduction of the immunofluorescence signal for nectin-3 at the stratum lucidum. Similarly, in the absence of nectin-3, remaining nectin-1 is not able to form hetero-trans-dimers with nectin-3, resulting in dispersion of nectin-1 clusters and reduction of the immunofluorescence signal for nectin-1 at the stratum lucidum. We have revealed that the sizes of the immunofluorescence signals for afadin and N-cadherin are markedly reduced in the hippocampus of both the mutant mice. The signals for afadin and N-cadherin apparently co-localize, and their numbers are not significantly different from those of the wild-type hippocampus. In addition, the hippocampus of both the mutant mice show the normal expression patterns of afadin and N-cadherin mRNAs. We have previously

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Fig. 5. The mossy fiber trajectory at the stratum lucidum in the hippocampus of both the mutant mice. (A) The mossy fibers were labeled by the injection of BDA into the dentate gyrus of the wild-type, nectin-1 / , or nectin-3 / mouse. (a) The BDA-labeled mossy fibers in the wild-type mouse. (b) The BDA-labeled mossy fibers in the nectin1 / mouse. (c) The BDA-labeled mossy fibers in the nectin-3 / mouse. (B) The wild-type, nectin-1 / , or nectin-3 / mouse hippocampal sections were stained with the anti-calbindin Ab, followed by immunofluorescence microscopy. (a1) The mossy fiber trajectory in the wild-type mouse. (a2) High-magnification of the boxed region shown in a1. (b1) The mossy fiber trajectory in the nectin-1 / mouse. (b2) High magnification of the boxed region shown in panel (b1). (c1) The mossy fiber trajectory in the nectin-3 / mouse. (c2) High magnification of the boxed region shown in panel (c1). Arrows, the abnormally long IPB; SPB, suprapyramidal bundle; and IPB, infrapyramidal bundle. Scale bars, 500 Am. (C) Quantitative analysis of the normalized length of the IPB in the nectin-1 / mice. +/+, Wild-type mouse; / , nectin-1 / mouse. (D) Quantitative analysis of the normalized length of the IPB in the nectin-3 / mice. +/+, Wild-type mouse; / , nectin-3 / mouse. *P < 0.01 versus the wild type. The results shown in panels A and B are the representative of three independent experiments. The normalized lengths of the IPB shown in panels C and D are the mean T SEM.

shown that nectins and cadherins are associated through afadin and a-catenin in epithelial cells and fibroblasts in culture (Takai and Nakanishi, 2003). Therefore, this co-localization of the signals for afadin and N-cadherin is likely to be the association of afadin through aN-catenin with N-cadherin which has formed trans-

dimers in small clusters by itself in the absence of the transinteraction of nectin-1 and nectin-3. We have shown here immunohistochemically that marked difference is not observed in the staining patterns of synaptic markers, including Bassoon, PSD-95, and GluR1, at the stratum lucidum between the wild-type and nectin-1 / hippocampus and between the wild-type and nectin-3 / hippocampus. These results indicate that nectins are not at least apparently essential for the formation of SJs. We have previously shown that disruption of the nectin-based cell – cell adhesion by the nectin inhibitors results in a decrease in the size of synapses in rat cultured hippocampal neurons (Mizoguchi et al., 2002). The exact reason for this discrepancy for the roles of nectins in the formation of SJs in vivo and synapses in the cultured neurons is unknown but may be just due to the different properties between in vivo and dissociated cultured neurons. Another explanation for this discrepancy is that nectins may be involved in the formation of SJs during their development but not after the maturation of SJs. It has been shown that blocking the N-cadherin activity by its dominant-negative mutant results in impaired synaptogenesis in cultured hippocampal neurons (Togashi et al., 2002). However, we have shown here that, although the accumulation of N-cadherin to the cell – cell contact sites at the stratum lucidum is reduced in the hippocampus of both the mutant mice, SJs are still formed at the stratum lucidum. The reduced level of N-cadherin at the cell – cell contact sites may be sufficient for the formation of SJs in vivo. It may be noted that the number and the size of PAJs are reduced in the hippocampus of both the mutant mice. During the mossy fiber development, distinct PAJs are not formed on P4 but formed on P5 (Amaral and Dent, 1981; Mizoguchi et al., 2002). On the other hand, nectins and N-cadherin localize at the cell – cell contact sites in the stratum lucidum by P4 (Mizoguchi et al., 2002). In the absence of nectins, the accumulations of N-cadherin and other important components for the formation of PAJs to the cell – cell contact sites at the stratum lucidum may be reduced, and then the formation of PAJs may be impaired. This interpretation is consistent with the earlier observations that nectins recruit cadherins to the nectin-based cell – cell adhesion sites and enhance the velocity of the formation of the cadherin-based adherens junctions in epithelial cells and fibroblasts in culture (Takai and Nakanishi, 2003). By analogy, it is likely that nectins enhance the velocity of the formation of PAJs by recruiting N-cadherin to the nectin-based cell – cell adhesion sites, and that N-cadherin alone is not sufficient for and nectins are additionally necessary for the maturation of PAJs. We have shown here that the mossy fiber trajectory is impaired in the hippocampus of both the mutant mice. The mutant nectin-1 gene has reportedly been responsible for mental retardation (Suzuki et al., 2000). In the nectin-1 / hippocampus, the mossy fibers in the IPB do not join the SPB, and reduction in the number of mossy fibers in the SPB results in alteration of the neural networks at the CA3 area. It has been reported that alterations of neural networks at the CA3 area show impairments of memory and information processing (McEwen, 1999; Nakazawa et al., 2002). Therefore, this alteration of the neural networks at the CA3 area may explain at least partly why nectin-1 is one of the responsible genes for mental retardation, although there still remains possible involvement of nectin-1 expressed in other areas of brain than the CA3 area in the mental retardation induced by the mutant nectin-1 gene. The mossy fibers underneath the stratum pyramidale in hippocampus (IPB) appear after P7 during the normal development

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Fig. 6. The mossy fiber trajectory in the hippocampus of both the mutant mice during development. (A) The wild-type, nectin-1 / , or nectin-3 / P12 mouse hippocampal sections were stained with the anti-calbindin Ab, followed by immunofluorescence microscopy. (a) The mossy fiber trajectory in the wild-type P12 mouse. (b) The mossy fiber trajectory in the nectin-1 / P12 mouse. (c) The mossy fiber trajectory in the nectin-3 / P12 mouse. Scale bars, 250 Am. (B) Quantitative analysis of the normalized length of the IPB in the nectin-1 / hippocampus on P12. +/+, Wild-type mouse; / , nectin-1 / mouse. (C) Quantitative analysis of the normalized length of the IPB in the nectin-3 / hippocampus on P12. +/+, Wild-type mouse; / , nectin-3 / mouse. *P < 0.05 versus the wild type. The results shown in panel A are the representative of three independent experiments. The normalized lengths of the IPB shown in panels B and C are the mean T SEM.

of the mossy fibers (Amaral and Dent, 1981). Consistent with this observation, the IPB is not detected on P5. Distinct PAJs appear after P5 and get matured by P21 (Amaral and Dent, 1981). Abnormal mossy fiber trajectory in the hippocampus of both the mutant mice is found on P12, when distinct PAJs are getting matured. Taken together, the formation of PAJs precedes the formation of the IPB and is likely to be required for the proper mossy fiber trajectory. In the absence of nectin-1 or nectin-3, the formation of PAJs is impaired, causing the formation of the

abnormal IPB. Based on available evidence, we hypothesize the role of the nectin-based PAJs in the mossy fiber trajectory (Fig. 8). In the wild-type hippocampus, the mossy fibers may be tightly connected to the apical dendrites of the pyramidal cells by the nectin-based PAJs. This is why the most mossy fibers are found in a bundle above the stratum pyramidale (SPB). In the nectin-1 / or nectin-3 / hippocampus, the mossy fibers may be weakly connected to the apical dendrites of the pyramidal cells because there are no nectin-based PAJs. As a result, the mossy fibers may

Fig. 7. Basic properties of synaptic transmission and synaptic plasticity at the mossy fiber synapses in the nectin-1 / hippocampus. (A) Input – output relationship of the wild-type (n = 7) and nectin-1 / (n = 4) hippocampus. There were no statistical differences in any data points with various stimulus strengths ( P > 0.5). (B) PPF induced by paired-pulse stimulation at various inter-stimulus intervals in the wild-type (n = 7) and nectin-1 / (n = 4) hippocampus. There were no statistical differences in any data points ( P > 0.2). (C) LTP induced by tetanic stimulation (100 Hz, 1 s) in the presence of D-APV (50 AM). Tetanic stimulation was delivered at time 0. In the inset, representative EPSPs are shown, which were recorded at the times indicated by the numbers in the graph. There was no statistical difference ( P = 0.13) in the magnitude of LTP measured 50 – 60 min after the tetanic stimulation between the wild-type (131.3 T 6.8% of baseline, n = 7) and nectin-1 / (150.1 T 8.4% of baseline, n = 6) hippocampus. After the LTP experiment, DCG-IV (1 AM), a group II metabotropic glutamate receptor agonist, was routinely applied to confirm that the recorded EPSP was originated mostly from the mossy fiber synapses. If the amplitude of the residual EPSP was more than 10% of that of the original EPSP, the data were discarded. Finally, 6-cyano-7-nitro-quinozaline-2,3-dione (CNQX; 10 AM), a non-NMDA receptor antagonist, was applied to isolate the presynaptic fiber volley component.

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deficiency of nectin functions might be necessary, but not sufficient, for the development of mental retardation. Additional factor, such as a specific genetic background, might be required for the development of mental retardation. In either case, further studies might be necessary to draw a definitive conclusion about the role of nectins in synaptic transmission.

Experimental methods Animals Fig. 8. A possible role of the nectin-based PAJs in the mossy fiber trajectory. N, nucleus. In the wild-type hippocampus, the mossy fibers may be tightly connected to the apical dendrites of the CA3 pyramidal cells by the nectin-based PAJs. In the nectin-1 / or nectin-3 / hippocampus, the mossy fiber may be weakly connected to the apical dendrites of the CA3 pyramidal cells because there are no nectin-based PAJs. As a result, the mossy fibers may be misguided. The nectin-based PAJs may recruit Ncadherin to fix the mossy fibers to the apical dendrites of the CA3 pyramidal cells, which may maintain the proper mossy fiber trajectory.

be misguided. The nectin-based PAJs may play a role, cooperatively with N-cadherin, in mechanical fixation of the mossy fibers to the apical dendrites of the CA3 pyramidal cells. These results are consistent with the earlier observations that the nectin-based cell – cell contacts between the commissural axons and the floor plate cells play a role in regulation of the commissural axon trajectory as a mechanical fixation of the axons (Okabe et al., 2004). Deficiency of other molecules, such as CHL1, NCAM, Semaphorin 3F, and Plexin-A3, shows the similar abnormal mossy fiber trajectory (Cheng et al., 2001; Cremer et al., 1997; Montag-Sallaz et al., 2002; Sahay et al., 2003). However, involvement of these molecules in the formation of the abnormal mossy fiber trajectory induced by nectin-1 and nectin-3 deficiency is unlikely because these molecules and nectins are distributed differently, CHL1 and NCAM have been shown to play a role in axon fasciculation at axon – axon contacts, and Semaphorin 3F and Plexin-A3 have been shown to play a role in axon guidance at growth cones. We have finally investigated here the properties of synaptic transmission as other possible functional consequences of the reduced PAJs in the hippocampus of both the mutant mice. However, under our experimental conditions, we could not detect any significant difference in the properties of synaptic transmission between the wild-type and nectin-1 / hippocampus and between the wild-type and nectin-3 / hippocampus at this time. These results are apparently consistent with the immunohistochemical data that the staining patterns of synaptic markers in the hippocampus of both the mutant mice are not significantly different from those in the wild-type hippocampus and the electron microscopic data that the spines and the PSDs in the hippocampus of both the mutant mice are not so different from those in the wildtype hippocampus. In human, nectin-1 is one of the responsible genes for mental retardation (Suzuki et al., 2000). However, we could not detect any significant difference in the properties of synaptic transmission between the wild-type and nectin-1 / hippocampus. The exact reason for these apparently inconsistent data is not known, but one explanation is that mentality is a highly organized brain function especially for human, not for mice, and therefore, it is hard to detect mental retardation in mice by our present electrophysiological methods. Another explanation is that

C57BL/6 mice were purchased from CLEA Japan Inc. (Tokyo, Japan). The nectin-1 / and nectin-3 / mice were generated as described (Inagaki et al., 2005), and were maintained on a C57BL/ 6 background. Experiments were performed on post-natal day (P) 5, P12, and 6- to 12-week-old mice. The animal experiments and procedures used in this study were in accordance with the guidelines of each institution and approved by Osaka University Medical School and University of Tokyo Animal Care and Experimentation Committee. Antibodies A rat anti-nectin-1 monoclonal Ab (mAb), a rat anti-nectin-3 mAb, a rabbit anti-nectin-1 polyclonal Ab (pAb), a rabbit antinectin-3 pAb, and a rabbit anti-afadin pAb were prepared as described (Mandai et al., 1997; Mizoguchi et al., 2002; SatohHorikawa et al., 2000; Takahashi et al., 1999). A mouse anti-Ncadherin mAb (Transduction Laboratories), a mouse anti-h-catenin mAb (Zymed), a mouse anti-synaptophysin mAb (Chemicon), a mouse anti-PSD-95 mAb (Alexis, Switzerland), a rabbit antiGluR1 pAb (Chemicon), a mouse anti-actin mAb (Chemicon), a mouse anti-Bassoon mAb (Stressgene), a mouse anti-calbindin mAb (Swant, Switzerland), and secondary Abs (Chemicon) were purchased from commercial sources. Assay for protein levels of synaptic components Mouse hippocampal tissues homogenized in a lysis buffer of 20 mM Tris/Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 AM aphenylmethanesulfonyl fluoride hydrochloride, 10 Ag/ml leupeptin, and 10 Ag/ml aprotinin. 10 Ag of proteins was separated by SDS-PAGE and transferred to Immobilon membrane (Millipore) sheet. The sheet was incubated with the Abs in TBS containing 5% skim milk for 1 h, followed by the incubation with peroxidaseconjugated secondary Abs for 1 h. The blots were developed with ECL (Amersham Pharmacia). Immunofluorescence microscopy Immunofluorescence microscopy was performed as described with minor modifications (Inagaki et al., 2003; Mizoguchi et al., 2002). In brief, mice were deeply anesthetized by ether and perfused with ice-cold 0.1 M phosphate-buffered saline (PBS), pH 7.4. Brains were dissected out, embedded in OCT compound (Sakura Finetechnical Co., Japan), frozen in liquid nitrogen, and then sectioned using a cryostat. The sections (10-Am thick) were mounted on glass slides, treated with 99% ethanol at 20-C for 30 min and with 100% acetone at 4-C for 1 min, and then washed three times with PBS containing 0.05% saponin. After being

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blocked in PBS containing 20% Block Ace (Dainippon Seiyaku Co., Japan) and 0.05% saponin for 30 min, the samples were incubated at 4-C 2 overnight with the primary Abs described above in PBS containing 5% Block Ace and 0.05% saponin. After being washed three times for 2 min with PBS containing 0.05% saponin, the samples were incubated at 4-C overnight with the secondary Abs described above. After being washed three times, the samples were mounted in 50% glycerol and viewed with Radiance 2000 confocal laser scanning microscope. The areas of the signals for nectin-1, nectin-3, afadin, and N-cadherin per 324 Am2 at the stratum lucidum of the CA3 or the stratum radiatum of the CA1 were quantitated by NIH image. The statistical significance of differences between the genotypes was analyzed by the two-tailed t test. At the CA1 area of the nectin-1+/ , nectin-1 / , nectin-3+/ , or nectin-3 / hippocampus, the signals for all the molecules described above were not significantly different from those of the wild-type hippocampus (data not shown). In situ hybridization In situ hybridization was performed as described (Okabe et al., 2004; Takemoto et al., 2002) with some modifications. In brief, RNA probes were labeled with digoxigenin-UTP by RNA in vitro transcription according to the manufacturer’s protocol (Roche Diagnostics). Mice were perfused with 4% paraformaldehyde (PFA) in PBS. Brains were fixed in 4% PFA in PBS at 4-C for 3 h, followed by the incubation with 15% and 30% sucrose solutions. The cryosections were prepared as described above. The sections were air dried, washed with water for 3 min, and treated with 0.25% dehydrated acetic acid in 0.1 M triethanolamine (pH 8.0) for 10 min. The sections were washed twice with PBS for 3 min and incubated with a prehybridization solution [50% deionized formamide, 5 SSPE (pH 7.5), 5% SDS, and 1 mg/ml yeast tRNA]. Each RNA probe was mixed with the prehybridization solution and added to the sections. After the sections were incubated at 60-C overnight, the sections were washed with 5 SSC and then with 2 SSC containing 50% formamide at 60-C for 30 min for three times. After being blocked with 1.5% Blocking Reagent (Roche Diagnostics) in TNT buffer [100mM Tris/Cl (pH 7.5), 150mM NaCl, and 0.01% Tween-20], the sections were incubated with the anti-digoxigenin Ab conjugated to alkaline phosphatase at 4-C overnight. The sections were washed five times with TNT buffer at room temperature for 30 min. Hybridized probes were visualized by BM purple (Roche Diagnostics). We used the probes for nectin-1, nectin-3, afadin, and N-cadherin as described previously (Okabe et al., 2004; Radies and Takeichi, 1993). pbMN3 was kindly supplied by Dr. M. Takeichi (RIKEN Center for Developmental Biology, Kobe, Japan).

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electron microscope (H-7100, HITACHI, Japan). Photographs were randomly taken from 5 sections per each mouse. The mossy fiber bouton was defined as a vesicle-rich expansion that was larger than 8 Am around and contacted with one dendrite in the section. Quantification was performed on 10 boutons per mouse. The statistical significance of differences between the genotypes was analyzed by the two-tailed t test. BDA injection Mice were anesthetized and fixed on stereotaxic apparatus. 5% biotylated dextran amine (BDA, 10000 MW, Molecular Probes, Inc.) in physiological saline was injected into the dentate gyrus through a glass micropipette using a pneumatic picopump (PV800, World Precision Instruments). A period of 5 days later, the mice were anesthetized again and fixed by perfusion with 2% PFA in 0.1 M PB. After the perfusion, the brains were isolated and placed in 25% Sucrose in 0.1 M PB at 4-C for 2 days. Serial coronal sections (50 Am thick) were cut with a freezing microtome. The serial sections were rinsed with PBS and incubated with streptavidine in PBS containing 0.5% Triton X-100 overnight. Then, the sections were incubated for 10 min with diaminobenzidine in 50 mM Tris/ Cl (pH 7.6) containing 0.2% nickel ammonium, added H2O2 at a final concentration of 0.00033%, and incubated until the color was developed. The sections were counterstained with neutral red. Calbindin staining P5, P12, and adult mice were perfused with 4% PFA in PBS. Brains were fixed in 4% PFA in PBS at 4-C overnight, followed by the incubation with 10% sucrose solution for 12 h, 20% sucrose solution for 12 h, and 30% sucrose solution for 12 h. After being washed with PBS containing 0.1% Triton X-100, the cryosections were prepared as described above. The sections (20-Am thick) were mounted on glass slides. After being washed with PBS containing 0.3% Triton X-100 (PBST), the sections were blocked in PBST containing 1% BSA for 30 min. The sections were incubated with the anti-calbindin Ab, followed by the incubation with a secondary Ab. Quantification of the IPB length was performed using the ratio of the IPB length to the length of the CA3 (‘‘normalized IPB length’’) as described (Bagri et al., 2003). The IPB length was measured from the tip of the inferior blade of the granule cell layer (‘‘a’’). The length of the CA3 was measured from the tip of the inferior blade to the apex of the curvature of the CA3 pyramidal cell layer (‘‘b’’). Normalized IPB length was taken as a/b. The statistical significance of differences in normalized IPB length between the genotypes was analyzed by the two-tailed t test. Electrophysiology

Electron microscopy Electron microscopy was performed as described (Inagaki et al., 2003; Mizoguchi et al., 2002; Yamada et al., 2003). In brief, mice (n = 4) were perfused with 2% PFA and 2% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.3. Brains were cut to 1-mm-thick coronal sections with a razor. The sections were fixed in 2% PFA and 2% glutaraldehyde in 0.1 M PB for 1 h and then in 2% osmium tetroxide in PB for 1 h. The sections were stained in black in 1% uranyl acetate, dehydrated in ethanol and propylene oxide, and embedded in epoxy resin. Ultrathin sections from the stratum lucidum of the CA3c area were cut, stained, and examined with an

Male mutant mice and their male littermates (6 – 12 weeks old) were used. Hippocampal slices (400 Am thick) were cut with a Vibratome tissue slicer and placed in a humidified interface-type holding chamber for at least 1 h. A single slice was then transferred to the recording chamber and submerged in a continuously perfusing medium that had been saturated with 95% O2/5% CO2. The medium contained 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 experiments were conducted at 25 – 26-C. Electrical stimuli were delivered through a tungsten bipolar electrode inserted into the stratum granulosum of the dentate gyrus,

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and the resulting field EPSPs were recorded from the stratum lucidum in the CA3 area with glass microelectrodes filled with 3 M NaCl (1 – 2 MV). PPF of excitatory synaptic transmission was induced by stimulating afferent fibers twice at short intervals of 30, 50, 100, 300, and 500 ms. LTP of excitatory synaptic transmission was induced in the presence of 50 AM D-APV, an NMDA receptor antagonist, by applying tetanic stimulation (100 Hz, 1 s) to the mossy fibers. All the experiments were performed in a blind fashion. The statistical significance of differences between the genotypes was analyzed by the two-tailed t test. Other procedures Protein concentrations were determined with bovine serum albumin as a reference protein (Bradford, 1976). SDS-PAGE was done as described (Laemmli, 1970).

Acknowledgments We are grateful to Dr. N. Yamamoto (Osaka University, Osaka, Japan) for the useful discussion. We also thank Dr. M. Takemoto (Osaka University, Osaka, Japan) for the advice regarding the in situ hybridization experiments. This work was supported by grants-in-aid for Scientific Research (Y. Takai) from the Ministry of Education, Culture, Sports, Science, and Technology, and RISTEX (T. Manabe) from Japan Science and Technology Agency, Japan (2003, 2004). T. Sakisaka is a recipient of a Human Frontier Science Program Career Development Award (2003).

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