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Originally published In Press as doi:10.1074/jbc.M705943200 on December 20, 2007

J. Biol. Chem., Vol. 283, Issue 9, 5918-5927, February 29, 2008
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Wnt-7a Modulates the Synaptic Vesicle Cycle and Synaptic Transmission in Hippocampal Neurons*Formula

Waldo Cerpa{ddagger}, Juan A. Godoy{ddagger}, Iván Alfaro{ddagger}, Ginny G. Farías{ddagger}, María J. Metcalfe{ddagger}, Rodrigo Fuentealba{ddagger}, Christian Bonansco§, and Nibaldo C. Inestrosa{ddagger}1

From the {ddagger}Centro de Regulación Celular y Patología "Joaquín V. Luco," Instituto Milenio MIFAB, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile, Alameda 340, 8331010 Santiago, Chile and §Departamento de Fisiología, Facultad de Ciencias, Universidad de Valparaíso, Av. Gran Bretaña 1111, 2360102 Valparaíso, Chile

Received for publication, July 20, 2007 , and in revised form, December 18, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Wnt signaling is essential for neuronal development and the maintenance of the developing nervous system. Recent studies indicated that Wnt signaling modulates long term potentiation in adult hippocampal slices. We report here that different Wnt ligands are present in hippocampal neurons of rat embryo and adult rat, including Wnt-4, -5a, -7a, and -11. Wnt-7a acts as a canonical Wnt ligand in rat hippocampal neurons, stimulates clustering of presynaptic proteins, and induces recycling and exocytosis of synaptic vesicles as studied by FM dyes. Wnt-3a has a moderate effect on recycling of synaptic vesicles, and no effect of Wnt-1 and Wnt-5a was detected. Electrophysiological analysis on adult rat hippocampal slices indicates that Wnt-7a, but not Wnt-5a, increases neurotransmitter release in CA3-CA1 synapses by decreasing paired pulse facilitation and increasing the miniature excitatory post-synaptic currents frequency. These results indicate that the presynaptic function of rat hippocampal neurons is modulated by the canonical Wnt signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Wnt signaling regulates crucial processes in all multicellular organisms, including cell proliferation, differentiation, migration, and morphogenesis. Since its discovery about 25 years ago, Wnt signaling has been extensively studied for its diverse roles in embryogenesis and cancer (1) and, more recently, in neural development and synaptic plasticity (2-5). Several studies suggest that Wnt factors play a role in the formation of neuronal connections, and other reports indicate a specific effect on synapse assembly; for example, in Drosophila embryos overexpression of the Wnt gene DWnt-3, encoding a protein localized in axonal processes, disrupted the formation of commissural tracts (6). Wnt-3 also regulates terminal arborization of neurotrophin-3-responsive spinal sensory neurons before the formation of sensory motoneuron synapses (7). In developing cerebellum cortex it has been found that conditioned medium from granule cells increases the diameter of mossy fiber axons and growth cone complexity, a result mimicked by Wnt-7a (8, 9). Wingless, the prototypical Drosophila Wnt, and its receptor are localized at the larval neuromuscular junction (10). Wingless is secreted by motoneurons and accumulates at both the pre- and postsynaptic terminals. The loss of Wingless leads to reduction in target-dependent synapse formation (10).

The expression of Wnt ligands and proteins of the Wnt signaling machinery in the mature nervous system (11, 12) suggests that Wnt signaling plays a role in neuroprotection and synaptic plasticity in addition to its role in neurite patterning in the developing nervous system (3, 5, 13). Indeed, Wnt ligands can act locally to regulate changes in neuronal cell shape and pre- and postsynaptic terminals, which are thought to underlie changes in synaptic function and learning. Thus, Wnt ligands would appear to be particularly well suited as mediators of synaptic plasticity (5, 14, 15).

In the present study we report that Wnt-7a, a canonical ligand that stimulates vesicle clustering, induces recycling and exocytosis of synaptic vesicles in hippocampal neurons in culture and enhances synaptic transmission in adult hippocampal slices by a presynaptic mechanism. These results are consistent with the idea that the canonical Wnt pathway controls presynaptic function in rat hippocampal neurons.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Detection of Wnt mRNAs in Neurons and Hippocampus—Degenerate oligonucleotides were designed for the identification of rat Wnt genes expressed at the mRNA level: TGA ACC TNC ACA ACA AYG AGG CGG G (I), GYC ACY GGG TGT CAG GCW CCT G (II), RTR GCC GCG GCC ACA GCA CA (III), and GCA GCA CCA GTG GAA CTT GCA (IV). Total RNA from 10 DIV2 hippocampal neurons were extracted, pooled, and treated with DNase I. cDNA was then synthesized using oligo(dT) and Moloney murine leukemia virus reverse transcriptase, and nested PCR amplicons were subcloned in pCRII (Invitrogen). Identity of individual Wnt amplicons was revealed by its unique HaeIII and RsaI restriction endonuclease pattern after PCR reamplification and further confirmed by sequencing. This procedure cannot rule out minor expression of low transcribed Wnt genes. For Wnt-4, Wnt-5a, Wnt-7a, or Wnt-11 determinations, RNA from either adult Sprague-Dawley rat hippocampal tissue or treated hippocampal neurons were subjected to reverse transcription-PCR analysis using primer I and antisense primers ATC TGT ATG TGG CTT GAA CTG, GAA GCG GCT GTT GAC CTG TAC, GCT TCT TGA TCT TCT TCA GAA AGG, and CAA GTG CTT GCG GGT GCC CAT, respectively. β-Actin primers for normalization of cDNA loading were TCT ACA ATG AGC TGC CAG AG and TAC ATG GCT GGG GTG ATG AA.

Constructs—The different hemagglutinin-Wnts constructs were a kind gift of several individuals and made this work possible. Wnt-7a was a gift of Dr. Patricia Salinas, University College London, London, UK. Wnt-3a was a gift of Dr. Roel Nusse, Stanford University, Palo Alto, CA. Wnt-5a was a gift of Dr. Randall T. Moon, University of Washington, Seattle, WA, and soluble Frizzled receptor protein (sFRP-1) was a gift of Dr. Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD.

Conditioned Medium Containing Wnt Ligands—To generate secreting Wnt ligands, human embryonic kidney 293 cells (HEK-293) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen) and 100 µg/ml streptomycin and 100 units/ml penicillin. Then HEK-293 cells were transiently transfected by calcium phosphate precipitation (16) with constant and equal amounts of empty vector pcDNA or pcDNA-containing sequences encoding Wnt-1, Wnt-3a, Wnt-5a, and Wnt-7a constructs. Transiently transfected HEK-293 cells also were used to produce sFRP-1 coupled to the sequence encoding a hemagglutinin tag. Transiently transfected HEK-293 cells were maintained in Neurobasal medium supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin medium for 60 h. Wnt-conditioned or control media or media containing sFRP-1 were prepared as described (9, 17-19). Wnt secretion was verified by Western blot using an anti-hemagglutinin antibody (Upstate Biotechnology, Lake Placid, NY).

Primary Rat Embryo Hippocampal Neuron Cultures and Treatments—Rat primary hippocampal neurons were prepared as previously described (17, 19-22). Hippocampal neurons were obtained from Sprague-Dawley rat embryos E18. On 3 DIV, hippocampal neurons were treated with 1 µM 1-β-D-ar-abinofuranosylcytosine for 24 h to reduce the number of proliferating non-neuronal cells (21, 22). The treatment were performed in 14-21 DIV. Neurons were first depleted of B-27 with Neurobasal media for 2 h and treated with Wnt and/or sFRP-1 and pcDNA control-conditioned media for different times.

Immunofluorescence for Synaptic Proteins—Immunostaining was carried out using polyclonal anti-postsynaptic density protein-95 (PSD-95), synaptophysin, SV-2, synaptotagmin antibodies, and secondary antibody labeled with 488Alexa, 543Alexa, or 633Alexa (Affinity Bio Reagents Inc., Golden, CO). To study neuronal morphology, phalloidin labeled with TRITC from Molecular Probes (Leiden, The Netherlands) was used. The number of clusters/length neurite was quantified by the software Sigma Scan Pro., and the number of neurites was evaluated using Image-Pro Plus Software Media Cybernetic (Silver Spring, MD). For quantification of cluster size, the area of the clusters was measured using Image J software (National Institutes of Health).

Western Blot for Wnt Components and Synaptic Protein in Hippocampal Neurons—Total extract of hippocampal neurons were used by immunodetection of synaptophysin (1:1000), β-catenin (1:1000), Dvl-3 (1:500), Engrailed-1 (1:500), cyclin-D1 (1:1000), c-Jun (1:1000), β-tubulin (1:1000) (Santa Cruz Biotechnology), phospho-JNK and JNK (1:2000) (Cell Signaling).

Spontaneous Recycling of Synaptic Vesicles Using FM1-43fx and the SV-2 Marker—21-DIV hippocampal neurons were exposed to a 1 µM concentration of steryl dye FM1-43 (Molecular Probes, Eugene, OR) in Neurobasal media. The control conditioned media and the conditioned media with different Wnt ligands were added into the culture at 37 °C at different times. Following the time course, the cells were washed, fixed, and immunostained against SV2 protein. Representative photographs were taken, and the FM1-43fx positive puncta which co-localized with SV-2 were quantified with the Image J program.

Imaging of FM-1-43 Destaining in Presynaptic Terminals of Cultured Hippocampal Neurons—21-DIV hippocampal neurons were incubated for 3 h with Wnt-7a or pcDNA at 37 °C. Neurons on coverslips were then washed with Tyrode modified solution, mounted in a microscope perfusion chamber, and incubated for 30 s with 10 µM FM1-43 (Molecular Probes) followed by 1 min of loading with mild depolarization with 30 mM KCl. Nonspecific and non-synaptic FM-1-43 staining was diminished by washing with 10 min of continue perfusion of Tyrode solution at 1-2 ml/min controlled with a peristaltic pump (Cole Palmer, Vernon Hills, IL). The chamber was adapted at the stage of a Zeiss Axiovert 200M microscope coupled to Pascal LSM5 confocal laser scanning system. Neurons were imaged with a 63 x 1.4 NA oil objective at 512 x 512 full-frame resolution using a 488-nm argon laser to excite the FM1-43 probe, and the fluorescence signals were collected over 505 nm. Then, after a period of 50 s of basal fluorescence acquisition, neurons were depolarized with 60 mM KCl and imaged by 300 s at 1-s intervals. Images from presynaptic loaded puncta were selected for measuring fluorescence intensities using region of interest areas of 1.5 x 1.5 µm. Images of Wnt-7a-treated neurons and control neurons were obtained using identical settings for laser power, confocal thickness, and detector sensitivity. All measures were carried at room temperature (25 °C).

Slice Preparation and Electrophysiology—Hippocampal slices were prepared according to standard procedures from 22- to 30-day-old Sprague-Dawley rats. Transverse slices (250-300 µm) from the dorsal hippocampus were cut under cold artificial cerebrospinal fluid (ACSF) using a Vibroslice microtome (World Precision Instruments) and incubated in ACSF for more than 1 h at room temperature. In all experiments picrotoxin (10 µM) was added to ACSF perfusion media to suppress inhibitory {gamma}-aminobutyric acid, type A transmission. Then slices were transferred to an experimental chamber (2 ml), superfused (3 ml/min, at 22-26 °C) with gassed ACSF, and visualized by transillumination with a binocular stereo microscope (MSZ-10, Nikon). The experiments were carried out at room temperature (21 °C-22 °C), measured at the recording chamber. Two recording methods were used; that is, path clamp (22, 23) and extracellular field potentials recording (24). Single cell recording were made in the whole-cell configuration with fire-polished pipettes (3-5 megaohms) filled with intracellular solution (see below) and connected to a tight seal (>1 gigaohm), and whole-cell a recordings were obtained from the cell body of neurons in the CA1 pyramidal layer. Patch electrodes were made from borosilicate glass and had a resistance of 2-5 megaohms when filled with 97.5 mM potassium gluconate, 32.5 mM KCl, 10.0 mM HEPES, 1.0 mM MgCl2, 5.0 EGTA, and 4.0 mM sodium salt (Na-ATP), pH 7.2 (289 mosM). Neurons were voltage-clamped with an EPC-7 amplifier (Heka Instruments), and the experiments started after a 5-10-min stabilization period after access to the intracellular compartment with patch electrodes. The access resistance (10-25 megaohms) was monitored, and cells were rejected if it changed more than 20% during the experiment. Extracellular field potentials recording (25) were made with a glass pipette (2-4 megaohms, filled with the perfusion medium), connected to an AC amplifier (P-5 Series, Grass), with gain 10,000x, low pass filter 3.0 kHz, and high pass filter 0.30 Hz, which was placed in the middle of stratum radiatum of CA1. The electric pulses (50 µs, 0.3 Hz, 20-100 µA) were applied on Schaeffer collaterals eliciting compound action potentials from the presynaptic axons (fiber volley) followed by field excitatory postsynaptic potentials (fEPSPs). To evoke excitatory postsynaptic currents (EPSCs) and fEPSPs, Schaeffer collaterals fibers were activated by bipolar cathodic stimulation, generated by a stimulator (Master 8, AMPI) connected to an isolation unit (Isoflex, AMPI). The bipolar concentric electrodes (platinum/iridium, 125 µm outer diameter, FHC Inc.) were placed in the stratum radiatum within 100-200 µm from the recording site. Miniature EPSCs (mEPSCs) were recorded in presence of tetrodotoxin (0.5 µM), and the Ca2+/Mg2+ ratio was elevated to increase the probability of neurotransmitter release (4.0 mM Ca2+ and 0.5 mM Mg2+) (26).

The presynaptic origin of the regulation of EPSCs amplitude by Wnt-7a was evaluated with conventional stimulation estimating changes in the paired pulse facilitation, which are considered to be of presynaptic origin (27, 28). The paired pulse facilitation index was calculated by ((R2-R1)/R1), where R1 and R2 were the peak amplitudes of the first and second EPSCs, respectively. Additionally, because the changes in the frequency of mEPSCs are considered to be of presynaptic origin (29, 30), we tested the mean frequency of mEPSCs as an indicator of changes in the probability of the presynaptic transmitter release.

Recordings were filtered at 2.0-3.0 kHz, sampled at 4.0 kHz using an A/D converter (ITC-16, Intrutech), and stored with Pulse FIT software (Heka Instruments). Both evoked postsynaptic responses, and mEPSCs were analyzed off-line using an analysis software (Minianalysis, Synaptosoft) which allowed visual detection of events, computing only those events that exceeded and arbitrary threshold. Data are expressed as the means ± S.E. (number of cells) unless otherwise noted. The Student's t test or Kolmogorov-Smirnov analysis at p < 0.05 determined significant differences between control and mutant cells.

Statistical Analysis—Data were expressed as the mean ± S.E. of the values from the number of experiments as indicated in the corresponding figures. Data were evaluated statistically by using Student's t test, with p < 0.05 considered significant. Analysis of variance was used to compare n differences between experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Several Wnts Ligands Are Expressed in Embryonic Hippocampal Neurons and in the Adult Rat Hippocampus—Wnt ligands are secreted glycoproteins encoded by a family of about 19 conserved genes in humans and other mammals (1, 31). Therefore, we first screenED for Wnt genes expressed in hippocampal neurons using a prototype mammalian Wnt gene (Fig. 1A) and we found several Wnt ligands including Wnt-4, Wnt-5a, Wnt-7a, and Wnt-11 (Fig. 1B); moreover, all four Wnt mRNAs detected in primary hippocampal cultures are also present in the hippocampus of adult rats (Fig. 1C). Analysis of mRNA shows that in particular, Wnt-7a and Wnt-5a are modulated by cell density in hippocampal neuronal cultures (Fig. 1, D and E). These results suggest that the Wnt signaling pathway may play a role in the mammalian central nervous system throughout the organism lifespan.

Wnt-7a Increases the Clusters of Synaptic Vesicles in Rat Hippocampal Cultures—Because different Wnt ligands were observed in embryonic and adult hippocampal neurons, we assessed the capacity of two Wnt ligands, Wnt-7a and Wnt-5a, to affect the clustering of synaptic proteins in mature hippocampal neurons. We examined the localization of synaptophysin, a presynaptic vesicle protein (32). Hippocampal neurons exposed to Wnt-7a for 1 h showed a different pattern of synaptophysin localization in comparison with control neurons (Fig. 2A). Wnt-7a-treated neurons present more synaptophysin clusters (Fig. 2Ac) than both Wnt-5a-treated neurons and pcDNA (control) cultures (Fig. 2A, a and b; more details are shown in supplemental Fig. S1A. We quantified the effect of Wnt-7a in the hippocampal cultures, counting the number of synaptophysin clusters in 100 µm of neurite length. The results show that Wnt-7a increases the clustering of synaptophysin (Fig. 2Ad). Moreover, we found that Wnt-7a increases the number of clusters of different presynaptic proteins as synaptotagmin and SV-2 (data not shown). In all cases the maximal effect was obtained after 1 h of incubation with the Wnt-conditioned medium (Fig. 3B; data not shown). The increased number of synaptic vesicle clusters is due to a redistribution of synaptophysin because the amount of synaptophysin protein did not change under the different experimental conditions used as evaluated by Western blots (Fig. 2B).

To determine whether Wnt-7a induces a regulation of postsynaptic proteins, we study the distribution of the PSD-95. Hippocampal neurons exposed to Wnt-7a for 1 h did not show any change in the distribution of PSD-95 (Fig. 2C, a-d). The number of PSD-95 clusters was unaffected by Wnt-7a (Fig. 2Ce). The effect of Wnt-7a in synapse was not due to change in neuronal morphology as observed by phalloidin staining (Fig. 2C, c and d). These results indicate that Wnt-7a ligand regulates the clustering of presynaptic proteins in mature hippocampal neurons cultures.


Figure 1
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  		FIGURE 1.
Different Wnt ligands are present in hippocampus and in hippocampal neuron cultures. A, the scheme shows a diagram of the Wnt gene and the primers used to detect different Wnt ligands. B, reverse transcription-PCR was carried out with hippocampal neurons to detect different Wnt ligands; the results show that Wnt-4, Wnt-5a, Wnt-7a, and Wnt-11 mRNAs were detected as shown in relative arbitrary units (C). Primary rat hippocampal neurons were seeded at 5.0 x 105, 1.0 x 106, and 1.5 x 106 cells/cm2 for low, medium, and high density studies corresponding to Wnt-5a ligand (D) or Wnt-7a ligand (E).

 
Wnt-7a Is a Canonical Wnt Ligand That Increases Presynaptic Protein Clustering Independent of GSK-3β Activity and β-Catenin Stabilization—At least three different signaling pathways are activated by Wnt proteins; the canonical Wnt/β-catenin pathway and two non-canonical Wnt pathways, Wnt/JNK and Wnt/Ca2+. We assessed the capacity of Wnt-7a and Wnt-5a ligands to activate the canonical Wnt pathway. When hippocampal neurons were exposed to Wnt ligands for 1 h, Wnt-7a but not Wnt-5a was able to increase β-catenin protein levels with respect to control as observed by Western blot (Fig. 3A). Similar results were obtained by immunofluorescence (data not shown).

Because Wnt-7a but not Wnt-5a increase the presynaptic protein clustering, hippocampal neurons were exposed to Wnt-7a in a time course experiment to evaluate both the clustering of the presynaptic protein synaptophysin and the stabilization of β-catenin. The clustering of synaptophysin became evident after 30 min with a peak at 60 min of Wnt-7a ligand exposure (Fig. 3B). When the stabilization of β-catenin was evaluated, a similar pattern of increase in β-catenin levels was observed; such change was similar to the increase for synaptophysin clustering. In both cases the induction was observed after 30 min and maintained at 60 min (Fig. 3C). To test the specificity of the Wnt ligand, Wnt-7a was incubated with the sFRP-1. sFRP recaptured the Wnt ligands, thereby preventing their interaction with cellular membrane-bound Frizzled receptor (33). Hippocampal neurons exposed to Wnt-7a in the presence of sFRP-1 showed a decrease in β-catenin levels with respect to Wnt-7a treatment, indicating the specificity of Wnt-7a to activate the Wnt pathway through its interaction with Frizzled receptors.

To determine whether GSK-3β activity and β-catenin stabilization, two components of the canonical Wnt pathway, are required to induce the clustering of presynaptic proteins, we used lithium, the classical GSK-3 inhibitor (34). Lithium induces the canonical Wnt pathway through GSK-3β inactivation that leads to β-catenin stabilization. We first compared the β-catenin stabilization induced by Wnt-7a and lithium. Hippocampal neurons exposed to lithium for 1 h showed an increase in the β-catenin levels as Wnt-7a (Fig. 3D); however, Dvl, a component of the Wnt pathway upstream of the GSK-3β and β-catenin proteins, was activated by Wnt-7a but not by lithium, as evidenced by phosphorylation-dependent mobility shift of Dvl (Fig. 3D). Under these conditions in which both Wnt-7a and lithium induce the stabilization of β-catenin, we study whether the activation of Wnt target genes occurs after 1 h of treatment. As indicated in Fig. 3E, the expression of engrailed, cyclin-D1 and c-Jun were not affected by Wnt-7a or lithium, suggesting that Wnt-7a induces the clustering of presynaptic proteins by a mechanism that did not required changes in the expression of Wnt target genes.

Because we did not observed any induction on Wnt target genes by Wnt-7a or lithium, we evaluated the requirement of the upstream proteins GSK-3β and β-catenin in the clustering of presynaptic proteins using two strategies, inhibition of GSK-3β activity or activation of GSK-3β. Lithium, the inhibitor of GSK-3β, was not able to induce the synaptophysin clustering as occurs with Wnt-7a (Fig. 3G). Previous evidence indicates that phosphatidylinositol 3-kinase and protein kinase C are inhibitors of GSK-3β activity. Moreover, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and bisindolamide-X, an inhibitor of protein kinase C, are considered GSK-3 activators (35). Hippocampal neurons were exposed to bisindolamide-X or wortmannin for 1 h, and presynaptic and postsynaptic proteins clusters were evaluated. We did not observed any change in the number of synaptic clusters (Fig. 3H). These results suggest that GSK-3β did not participate in the presynaptic protein clustering induced by Wnt-7a at least in a short term study.


Figure 2
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  		FIGURE 2.
Wnt-7a ligand increases the clustering of the synaptic vesicle proteins. A, immunofluorescence labeling for synaptophysin are shown in neurons subjected to treatments with control (a), Wnt-5a (b), or Wnt-7a (c) media for 1 h. Representative photographs show the synaptophysin clustering induced by Wnt-7a, and in d, a quantification of synaptophysin clusters per 100-µm neurite length is shown (n = 4). B, Western blot of synaptophysin (Syp) shows that the total levels of the protein did not change during 1 h of treatment. The graph shows the densitometric analysis carry of different ligands and normalized against β-tubulin (n = 5). C, fluorescence labeling of processes by phalloidin (c and d) and immunofluorescence labeling for PSD-95 (a and b) are shown in neurons exposed to control conditions (a and c) or Wnt-7a ligand (b and d) for 1 h. Representative photographs and a quantification of the clusters number (e) shows no changes in the clustering of the postsynaptic protein PSD-95 by Wnt-7a (n = 3). The bar represents the mean ± S.E. (*, p < 0.01 Student's t test).

 
Finally, we evaluated whether Wnt-7a can also acts through non-canonical Wnt pathways that can explain the increase in the presynaptic protein clustering. No effect of the Wnt-7a in non-canonical Wnt pathways, as evidenced by phosphorylation in JNK (Wnt-JNK pathway) or CAM-K-II (Wnt-Ca2+ pathway), was observed (Fig. 3F; supplemental Fig. S1B). These results taken together indicate that Wnt-7a activates specifically the canonical Wnt pathway; however, the clustering of presynaptic proteins observed in short term experiments occurs upstream of GSK-3β activity and β-catenin stabilization.

Wnt-7a Plays a Role in the Spontaneous Recycling of Synaptic Vesicles—To examine a possible function of Wnt ligands on recycling of synaptic vesicles in mature hippocampal neurons, we used a fixable analogue of FM1-43 (FM1-43fx) (36, 37) to measure the ability of presynaptic sites to endocyte the dye in the presence of Wnt ligands (Fig. 4A). After different times of incubation with the Wnt ligands, we quantified FM1-43fx-positive clusters in presynaptic sites evidenced by co-localization with the presynaptic vesicle protein SV-2. We observed that spontaneous recycling activity only stains a small fraction of total SV2-positive clusters that increase 8 times with 30 min of Wnt-7a incubation (Fig. 4, B and C, a-d); subsequently the number of active recycling sites was maintained for up to 2 h (Fig. 4, B and D). On the other hand, incubation with the non-canonical Wnt-5a ligand did not show a significant effect on the number of presynaptic sites with spontaneous dye uptake (Fig. 4, B and D). To determine whether the effect of the Wnt-7a-conditioned medium was specifically due to the presence of Wnt-7a ligand, a secreted Wnt antagonist, sFRP-1, was used to block the Wnt-7a effect; under these conditions the effect of Wnt-7a on the number of active recycling sites was totally abolished (Fig. 4B). We evaluated whether the FM1-43-positive sites observed by 1 h of treatment with the Wnt ligands was able to destain by depolarization with 90 mM KCl (Fig. 4A). KCl induced the destaining of the FM1-43fx/SV2 presynaptic-positive sites, indicating presynaptic active terminals in mature hippocampal neurons.

To further evaluate other Wnt ligands that can play some role in recycling of synaptic vesicles, we tested others Wnt ligands in their capacity to increase the number of active recycling sites. We first tested Wnt-1, the most studied member of the family (1), and Wnt-3a, which protects hippocampal neurons from Aβ toxicity (17). As Fig. 4D indicates, besides Wnt-7a, only Wnt-3a has a moderate effect on the number of recycling sites. However, no effect of Wnt-1 or Wnt-5a was detected. Interestingly, Wnt-3a ligand-induced β-catenin stabilization in hippocampal neurons also occurs with Wnt-7a (17). These results indicate that Wnt-7a and possibly other canonical Wnt ligands contribute to the formation of new active vesicle recycling sites.

Wnt-7a Modulates the Efficacy of Synaptic Vesicle Exocytosis—To examine whether Wnt-7a modulates the exocytosis of synaptic vesicles from individual presynaptic nerve terminals, we incubated hippocampal neurons with the Wnt-7a ligand for 3 h and then we evaluated the kinetics of release of synaptic vesicles evoked by depolarization with 60 mM KCl in the presynaptic terminals from mature hippocampal neurons (Fig. 5A, c-f). In this experiment, after the treatment with Wnt-7a or control conditioned media, we load the synaptic vesicles with the amphiphatic fluorescent dye FM1-43 (Fig. 5A, b and c) (36, 37) and follow its destaining kinetics induced by depolarization with 60 mM KCl by confocal time lapse microscopy imaging. As indicated in Fig. 5, B and C, incubation of the neurons with Wnt-7a increases the velocity of release of the FM1-43 fluorescence trapped in vesicles with a mean half-life of 60 s versus 100 s for control (pCDNA) in a total of 65 sites of vesicle release. At 250 s, normalized fluorescence values reach the plateau in Wnt-7a-treated and control cells. These results suggest that a Wnt-7a-dependent signaling affects the machinery that mediates the release of synaptic vesicles, probably increasing the probability of evoked transmitter release.

Wnt-7a Enhances Synaptic Transmission in CA3-CA1 Synapses of Hippocampal Slices by a Presynaptic Mechanism—To further establish that the canonical Wnt-7a modulates the release of the presynaptic neurotransmitter, we first recorded the effect of Wnt-7a on normal synaptic transmission in hippocampal slices (CA3-CA1 synapses) taken from P24-P30 rats by extracellular field recording. All experiments were performed in ACSF at 22 °C in the presence of the {gamma}-aminobutyric acid antagonist picrotoxin continuously perfused with Wnt-7a. To address whether Wnt-7a exerts its effect on a presynaptic mechanism, we used the paired-pulse test. As Fig. 6Aa shows, Wnt-7a increased the amplitude of the fEPSP almost three times (Fig. 6Ab). This increase in amplitude occurs concomitant with a decrease of the paired-pulse facilitation, which is considered to be of presynaptic origin (27, 28) (Fig. 6Ac), suggesting that a presynaptic change has been induced by Wnt-7a. Moreover, when we studied the fiber volley amplitude in the presence of Wnt-7a, we found no change when compared with control conditions (Fig. 6Ad), indicating that Wnt-7a did not change the number of activated fibers and suggesting that the effects of Wnt-7a on the decrease facilitation occurs by a change in neurotransmitter release. In the case of Wnt-5a, we observed only a 50% increased in fEPSP amplitude (Fig. 6B, a and b); however, paired pulse facilitation did not change (Fig. 6Bc), indicating a possible effect in synaptic transmission by a mechanism independent of the presynaptic site. These results show again a differential effect between a canonical Wnt ligand (Wnt-7a) and a non-canonical ligand (Wnt-5a).


Figure 3
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  		FIGURE 3.
Wnt-7a ligand stabilizes β-catenin but induces the clustering of presynaptic proteins independent of GSK-3β activity and β-catenin stabilization. A, representative Western blot of hippocampal neurons stimulated with Wnt-5a, Wnt-7a, or pcDNA (control) for 1 h show that only Wnt-7a ligand stabilized β-catenin (cat) levels. The densitometric analysis of β-catenin was normalized against β-tubulin (tub; n = 4). Hippocampal neurons were exposed to Wnt-7a for different times to study the clustering of synaptophysin (B) or β-catenin stabilization (C). Wnt-7a significantly increases synaptophysin clustering (n = 2) and stabilization of β-catenin (n = 4) in a similar time-course dependent form. D-F, Western blot of hippocampal neurons exposed to control (pcDNA), Wnt-7a, or 20 mM LiCl (GSK-3β inhibitor) for 1 h were tested for different component of Wnt pathways. D, Wnt-7a and LiCl induce the β-catenin levels as showed in the densitometric analysis (n = 4); however, Dvl, an upstream component in the Wnt pathway, is only activated by Wnt-7a as indicated by the increase in the phosphorylated state. E, Wnt target genes as Eng-1, cyclin-D1, and c-Jun are not affected by Wnt-7a or LiCl at 1 h of treatment (n = 2). F, JNK, a non-canonical Wnt component, is not induced by Wnt-7a at 1 h of treatment, suggesting that Wnt-7a acts mainly as a canonical Wnt ligand. p-, phospho-. G, immunofluorescence labeling for synaptophysin in neurons subjected to treatments with control, Wnt-7a, or LiCl for 1 h indicates that Wnt-7a but not LiCl is able to induce the clusters number of synaptophysin as shown in representative photographs and in quantification (n = 2). H, fluorescence labeling of processes by phalloidin and immunofluorescence labeling for PSD-95 and for synaptophysin are shown in neurons exposed to control conditions or in the presence of wortmannin (WT; 100 nM) or bisindolamide-X (BSD-X;1 µM) (GSK-3β activators) for 1 h. Representative photographs and the quantification of the Syp clusters and PSD-95 clusters show not changes in the number of clusters by Wnt-7a (n = 3). The bar represents the mean ± S.E. (*, p < 0.01 Student's t test). Eng-1, Engrailed-1; Dvl, Dishevelled; Syp, synaptophysin.

 
We also analyzed the effect of Wnt-7a directly on CA1 pyramidal neurons by intracellular recording. In agreement with previous data, the normalized amplitude of response was significantly greater 2-3 times after Wnt-7a application (Fig. 7C). The facilitation index decreases after Wnt-7a treatment (Fig. 7D). The Wnt-7a effect was specifically reverted with Wnt-7a plus the Wnt antagonist sFRP-1 (33) and blocks the effect of Wnt-7a over synaptic transmission (Fig. 7E).


Figure 4
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  		FIGURE 4.
Wnt-7a increases the number of active spontaneous recycling sites of synaptic vesicles in hippocampal neurons. A, experimental procedure. Mature hippocampal neurons were loaded with FM1-43fx 30 min before adding the Wnt ligand, the ligands were added in a time-dependent manner, and then the cells were washed and fixed. B, to measure the number of active recycling presynaptic sites, we examined the effects of Wnt-5a and Wnt-7a ligands on the spontaneous incorporation of FM1-43fx into hippocampal neurons. Then we stained the neurons by immunofluorescence with an anti-SV-2 antibody. The graph shows a representative time course of the density of FM1-43 active recycling sites that colocalize with SV2 marker in the presence of Wnt ligands. Wnt-7a ligand shows an increase in the number of spontaneous recycling sites, and Wnt-5a ligand does not show the effect on spontaneous vesicle recycling. The effect of Wnt-7a is specific because it was abolished by a sFRP-1. Another set of neurons was loaded as indicated above after 1 h, and the cells were washed for 5 min and destained with 90 mM K+ and then wash and fixed as indicated in A. FM1-43-positive sites loaded in the presence of Wnt ligands were able to destain by depolarization 90 mM KCl for 5 min, indicating active presynaptic sites. C, representative photographs of hippocampal neurons treated with control (a) or Wnt-7a for 60 min (b) are shown; c and d, the regions enclosed in the rectangles in a and b are shown. The fluorescence encoded in pseudo-color shows FM1-43 dye spontaneous uptake (white arrowhead). D, the spontaneous synaptic vesicles recycling was tested with different Wnt ligands as described above. Analysis was performed by quantifying FM1-43 dye uptake sites (FM1-43 site/neuron) and are shown in the table. The results show that Wnt-7a and Wnt-3a induce a spontaneous vesicle recycling in a time-dependent manner; however, Wnt-3a has a modest effect in recycling respect to Wnt-7a ligand, and no effects were observed with Wnt-1a or Wnt-5a. The bar represents the mean ± S.E. (*, p < 0.01 Student's t test). Multiple comparative data were made using analysis of variance HOLM-SIDAK methods; the results are significantly different in relation to initial time (p < 0.05).

 
In an alternative way to explore whether the effects of Wnt-7a modulates the release of neurotransmitter, we evaluated the frequency of the mEPSC in the CA3-CA1 synapses. Results carried out in the presence of tetrodotoxin indicated that the frequency of mEPSC increased almost 60% in the presence of Wnt-7a (Fig. 8, A-C) without affecting the amplitude of mEPSP (Fig. 8D). This increase in the frequency was also observed after comparing the cumulative probability plots of the mEPSC frequency 30 min before and after the addition of Wnt-7a in the perfusion media (Fig. 8E). Previous studies by Nicoll and co-workers (29, 30) indicated that an increase in the frequency of mEPSCs is considered to be of presynaptic origin. These results indicate that Wnt-7a (a) is a physiologically active Wnt ligand in postnatal hippocampal slices and (b) increases synaptic transmission by a presynaptic mechanism, probably involving an increase in neurotransmitter release.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Several important observations were obtained in the present work. 1) We have found that several Wnt ligands are expressed in the adult nervous system, particularly in the rat hippocampus. Recent studies, indicated that Wnt-3a is a key factor in hippocampal stem cell neurogenesis in adult rat (38) and modulates long-term potentiation in mouse adult hippocampal slices (5); therefore, the persistent expression of Wnts in the adult nervous system and the modulation of neurogenesis and synaptic plasticity strongly suggest that the Wnt signaling pathway plays a role in the mammalian central nervous system during its entire lifespan, maintenance, and neuroprotection. Moreover, our results are consistent with the idea that Wnt signaling may be affected in the brain of Alzheimer patients. In fact, we have previously reported that activation of Wnt signaling prevent Aβ-induced neurotoxicity both in primary hippocampal neuron cultures (17-19) as well as in vivo, where Wnt activation blocks the behavioral impairments induced by Aβ fibrils (39, 40). 2) We demonstrated that the canonical Wnt-7a ligand modulates the recycling and release of synaptic vesicles in functionally mature excitatory synapses in vitro. Concerning the recycling of synaptic vesicles, Wnt-7a was the more active Wnt factor, and its ability to increase the number of active recycling sites was completely abolished by the Wnt antagonist sFRP-1. Wnt-3a has a moderate effect, and no effect for Wnt-1 was observed. No effect was detected for the non-canonical Wnt ligand Wnt-5a. We also found that hippocampal neurons treated with Wnt-7a increase the initial velocity of synaptic vesicle exocytosis without affecting the amplitude of total FM1-43 evoked destaining. These results suggest that the presence of the canonical Wnt-7a ligand in mature hippocampal neurons modulates transmitter release in presynaptic terminals without affecting the size of the total vesicle pool. Synaptic vesicles in the nerve terminal are found in three subpopulations of vesicles: the readily releasable pool, the reserved pool, and the resting pool (41). Substantial evidence indicates that the subpopulation of vesicles that recycles rapidly modulate the probability of neurotransmitter release. Future studies will tell which is the Wnt signaling component that participates in the recycling of SV and whether Wnt signaling acts specifically modulating the size or activity of readily releasable pool. 3) We have found that Wnt-7a regulates neurotransmitter release. In fact, analysis of mEPSCs recorded in the presence of tetrodotoxin was used to estimate the frequency of spontaneous vesicle fusion in adult hippocampal slices of 30-day-old postnatal rats. Whole-cell recordings indicate that in the presence of Wnt-7a the mean mEPSC frequency was consistently higher than in control experiments. In our experiments we showed an important decrease paired pulse facilitation together with an increase in mEPSC frequency, suggesting that Wnt-7a modulates paired pulse facilitation by an increase in the release probability. These results indicate that Wnt-7a stimulates neurotransmitter release from presynaptic terminals. Our results provide direct evidence that Wnt-7a modulates the function of mature presynaptic terminals. Consistent with our studies, electrophysiological recordings in cerebellar slices from Wnt-7a/Dishevelled (Dvl) double mutant mice reveal a defect in neurotransmitter release at mossy fiber-granule cell synapses (42). 4) Herein, we show that short-term exposure to Wnt-7a induces the clustering of presynaptic proteins and the stabilization of β-catenin; however, at this time Wnt target genes are not induced, suggesting that the clustering of presynaptic proteins does not required β-catenin-dependent transcription. The Wnt pathway activated by Wnt-7a appears to be mainly Wnt-canonical, because Dvl was activated, and β-catenin was stabilized. Dvl is also a component of the non-canonical Wnt pathways; however, Wnt-7a does not induce Wnt/JNK or Wnt/Ca2+, two non-canonical Wnt pathways.


Figure 5
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  		FIGURE 5.
Wnt-7a stimulates synaptic vesicle exocytosis in old hippocampal-cultured neurons. 21-DIV hippocampal neurons were treated with Wnt-7a ligand or pcDNA control-conditioned media for 3 h at 37 °C. Then the coverslips were mounted in a imaging chamber and loaded with the FM-1-43 probe (10 µM) by 30 s followed by 30 s of moderated depolarization with 30 mM KCl. Synaptic vesicle release induced by Wnt-7a and control-treated neurons was imaged by time lapse imaging of FM-1-43 fluorescence at intervals of 1 s in a period of 5 min induced by depolarization with 60 mM KCl and constant perfusion at 1-2 ml/min. A, composed image of optic and fluorescence images of a 21-DIV hippocampal neuron synaptically loaded with FM-1-43 probe (FM-1-43 in red) (a and b); c, d, and f, a boxed of the image shown in b is followed in time. A sequence of pseudocolored images of FM-1-43 fluorescence of representative synaptic vesicle exocytosis experiments is shown in hippocampal neurons exposed to Wnt-7a. B, pseudocolored images of FM-1-43 fluorescence of representative synaptic vesicle exocytosis in Wnt-7a (b, d, f, and h) and control (a, c, e, and g)-treated neurons at different times 0 s (a and b), 50 s (c and d), 100 s (e and f), and 200 s (g and h). C, graphics of the normalized FM-1-43 fluorescence decrease of Wnt-7a and control-treated neurons shows that Wnt-7a decreases the FM-1-43 stain more quickly than the control-treated neurons. Data represent the mean and S.D. for 20 quantified boutons.

 


Figure 6
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  		FIGURE 6.
Wnt-7a presynaptically enhances the synaptic transmission in CA3-CA1 synapses of postnatal rat hippocampus. A, field recording of CA1 pyramidal cells treated with Wnt-7a. a, average traces of fEPSP evoked by paired pulse protocol before (control) and after 30 min of continued perfusion with Wnt-7a. b, normalized amplitude of fEPSPs evoked by the first stimulus in control and after Wnt-7a treatment. c, index of facilitation measured before and after Wnt-7a application. d, effect of Wnt-7a on fiber volley; average traces of fiber volley of control and Wnt-7a treatment. The average values of normalized amplitude of fiber volley in both conditions are shown in the right graph. B, field recording of CA1 pyramidal cells treated with Wnt-5a. a, average traces of fEPSP evoked by paired pulse protocol before (control) and after 30 min of continued perfusion with Wnt-5a. b, normalized amplitude of fEPSPs evoked by the first stimulus in control and after Wnt-5a treatment. c, index of facilitation measured before and after Wnt-5a application.

 


Figure 7
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  		FIGURE 7.
Intracellular recording of a CA1 pyramidal cell after Wnt-7a treatment. Shown are 10 superimposed EPSCs (gray) and the corresponding average traces (black), evoked by Schaffer collaterals stimulation, before (control) (A) and after 30 min of Wnt-7a treatment (B). C, mean of normalized amplitude of evoked EPSCs in control and Wnt-7a-treated slices. D, mean of facilitation index before and after Wnt-7a application. E, sFRP-1 blocks the effect of Wnt-7a on synaptic transmission.

 


Figure 8
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  		FIGURE 8.
Miniature EPSCs recorded in presence of tetrodotoxin (1 µM) before and after continuous application of Wnt-7a. Shown are nine representative traces recording in control conditions at a holding potential of -60 mV (A) and 30 min after the superfusion of Wnt-7a (B). C, increase of mean values of relative frequency of mEPSC (C) in both conditions without affecting the amplitude of mEPSC (D). E, cumulative probability plot of the mEPSC frequency, recorded from a same cell during 90 s of sampling in control and after Wnt-7a treatment.

 
Both GSK-3β and β-catenin have been described to participate in synaptic processes. β-Catenin has two principal functions, transcription via interaction with the transcription factor TCF/LEF and interaction with cadherins at cell adhesions. The participation of the β-catenin-cadherins complex has been strongly implicated in synaptic processes, where regulates excitatory post-synaptic strength at hippocampal neurons (43). Moreover, the NMDA activity in the post-synaptic site regulates N-cadherin endocytosis mediated by β-catenin providing plasticity (44). Presynaptic function of β-catenin has been described; β-catenin participates in the presynaptic assembly through recruitment of PDZ proteins to cadherins at synapses (45) and through induction of the canonical-Wnt pathway, where long-term exposure to Wnt-7a and lithium are able to induce the clustering of presynaptic proteins in young mouse primary cerebellar cultures (8, 9).

In the present work we showed that lithium, an inhibitor of GSK-3β that induces β-catenin stability, or GSK-3β activators do not affect the clustering of presynaptic proteins in a short-term exposure. It is possible that prolonged exposure to Wnt ligand involves the expression of Wnt target genes important for presynaptic assembly. However, it has been shown that TCF/LEF transcription factors, which regulate Wnt target genes transcription, are down-regulated in mature neurons with respect to young neurons (11). The short-term changes in the clustering of presynaptic proteins induced by Wnt-7a indicates upstream regulation of GSK-3β and β-catenin, suggesting that Wnt-7 ligand activates a fast non-conventional Wnt-signaling that diverges to function in presynaptic assembly and function.

Concerning the possible mechanism by which Wnt-7a modulates the dynamics of synaptic vesicle content, one possibility may involves Wnt activation and subsequent interaction of Dvl with a molecular component of neurotransmitter release machinery. Recent studies indicate that Dvl apparently regulates endo- and exocytic processes throughout the binding to the calcium sensor synaptotagmin (46). Previous studies showed that Wnt-7a and Wnt-7b regulate presynaptic morphology through Dvl; moreover, synaptic Dvl increases the number of Bassoon clusters, suggesting that Dvl regulates the assembly of the presynaptic apparatus (42). On the other hand, it has been observed that the tumor suppressor APC protein, a component of the canonical Wnt pathway, interacts with synaptotagmin (47); however, no functional consequence of this interaction has been demonstrated. Recently, we have shown that Wnt-7a induces the interaction of APC and the {alpha}7-nicotinic acetylcholine receptor ({alpha}7-nAChR) and localize {alpha}7-nAChR to presynaptic sites in an APC-dependent manner (20). Interestingly, {alpha}7-nAChR contributes significantly to hippocampal function where influence neurotransmitter release interacts with a molecular component of the neurotransmitter release machinery such as synaptotagmin. According to the function of {alpha}7-nAChR in neurotransmitter release, we found that Wnt-7a induces the interaction of {alpha}7-nAChR with VAMP-1/2, a component of synaptic vesicles that form the SNARE (soluble N-ethylmaleimide factor attachment protein) fusion complex enabling vesicle exocytosis. This suggest a role of Wnt-7a/APC in neurotransmitter release through the localization of {alpha}7-nAChR in presynaptic sites (20). The present study indicate that the canonical Wnt ligand, Wnt-7a, plays a key role in the presynaptic events associated to neurotransmitter release observed in hippocampal synapses.


   FOOTNOTES
 
* This work was supported by Fondo de Estudios Avanzados en Areas Prioritarias and Millennium Institute (El Instituto Milenio de Biología Fundamental y Aplicada) (to N. C. I.), Fondo Nacional de Ciencia y Tecnologia (Fondecyt) Grant 1061074 and DIPUV-REG 08/2005 grants (to C. B.), and predoctoral fellowships from Fondecyt (to W. C., I. A., and G. G. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence should be addressed: CRCP-Biomedical Center, Pontificia Universidad Católica de Chile, Alameda 340, 8331010 Santiago, Chile. Tel.: 56-2-686-2724/2720; Fax: 56-2-686-2959; E-mail: ninestrosa{at}bio.puc.cl.

2 The abbreviations used are: DIV, days in vitro; HEK-293, human embryonic kidney 293 cells; sFRP-1, soluble Frizzled receptor protein; ACSF, artificial cerebrospinal fluid; fEPSP, field excitatory postsynaptic potentials; EPSC, field excitatory postsynaptic current; mEPSC, miniature EPSC; Dvl, Dishevelled; {alpha}7-nAChR, {alpha}7-nicotinic acetylcholine receptor; TRITC, tetramethylrhodamine isothiocyanate; PSD-95, postsynaptic density protein-95; GSK, glycogen synthase kinase; JNK, c-Jun NH2-terminal kinase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Patricia Salinas from the University College of London, UK and Dr. Randall Moon from the University of Washington, Seattle, WA, for generously providing Wnt-7a and Wnt-5a, to Dr. Roel Nusse, Stanford University, Palo Alto, CA, for the Wnt-3a, and Dr. Jeremy Nathans from the Johns Hopkins University, Baltimore, MD for the sFRP-1 construct.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Logan, C. Y., and Nusse, R. (2004) Annu. Rev. Cell Dev. Biol. 20, 781-810[CrossRef][Medline] [Order article via Infotrieve]
  2. Patapoutian, A., and Reichardt, L. F. (2000) Curr. Opin. Neurobiol. 10, 392-399[CrossRef][Medline] [Order article via Infotrieve]
  3. Fradkin, L. G., Garriga, G., Salinas, P. C., Thomas, J. B., Yu, X., and Zou, Y. (2005) J. Neurosci. 25, 10376-10378[Free Full Text]
  4. Ciani, L., and Salinas, P. C. (2005) Nat. Rev. Neurosci. 6, 351-362[Medline] [Order article via Infotrieve]
  5. Chen, J., Park, C. S., and Tang, S. J. (2006) J. Biol. Chem. 281, 11910-11916[Abstract/Free Full Text]
  6. Fradkin, L. G., Noordermeer, J. N., and Nusse, R. (1995) Dev. Biol. 168, 202-213[CrossRef][Medline] [Order article via Infotrieve]
  7. Krylova, O., Herreros, J., Cleverley, K. E., Ehler, E., Henriquez, J. P., Hughes, S. M., and Salinas, P. C. (2002) Neuron 35, 1043-1056[CrossRef][Medline] [Order article via Infotrieve]
  8. Lucas, F. R., and Salinas, P. C. (1997) Dev. Biol. 192, 31-44[CrossRef][Medline] [Order article via Infotrieve]
  9. Hall, A. C., Lucas, F. R., and Salinas, P. C. (2000) Cell 100, 525-535[CrossRef][Medline] [Order article via Infotrieve]
  10. Packard, M., Koo, E. S., Gorczyca, M., Sharpe, J., Cumberledge, S., and Budnik, V. (2002) Cell 111, 319-330[CrossRef][Medline] [Order article via Infotrieve]
  11. Shimogori, T., VanSant, J., Paik, E., and Grove, E. A. (2004) J. Comp. Neurol. 473, 496-510[CrossRef][Medline] [Order article via Infotrieve]
  12. Tissir, F., and Goffinet, A. M. (2006) Eur. J. Neurosci. 23, 597-607[CrossRef][Medline] [Order article via Infotrieve]
  13. Fuentealba, R. A., Farias, G., Scheu, J., Bronfman, M., Marzolo, M. P., and Inestrosa, N. C. (2004) Brain Res. Brain Res. Rev. 47, 275-289[CrossRef][Medline] [Order article via Infotrieve]
  14. Scheiffele, P. (2003) Annu Rev. Neurosci. 26, 485-508[Medline] [Order article via Infotrieve]
  15. Dabdoub, A., Donohue, M. J., Brennan, A., Wolf, V., Montcouquiol, M., Sassoon, D. A., Hseih, J. C., Rubin, J. S., Salinas, P. C., and Kelley, M. W. (2003) Development 130, 2375-2384[Abstract/Free Full Text]
  16. Conroy, W. G., and Berg, D. K. (1998) Mol. Pharmacol. 53, 392-401[Abstract/Free Full Text]
  17. Alvarez, A. R., Godoy, J. A., Mullendorff, K., Olivares, G. H., Bronfman, M., and Inestrosa, N. C. (2004) Exp. Cell Res. 297, 186-196[CrossRef][Medline] [Order article via Infotrieve]
  18. Quintanilla, R. A., Munoz, F. J., Metcalfe, M. J., Hitschfeld, M., Olivares, G., Godoy, J. A., and Inestrosa, N. C. (2005) J. Biol. Chem. 280, 11615-11625[Abstract/Free Full Text]
  19. Garrido, J. L., Godoy, J. A., Alvarez, A., Bronfman, M., and Inestrosa, N. C. (2002) FASEB J. 16, 1982-1984[Abstract/Free Full Text]
  20. Farias, G. G., Valles, A. S., Colombres, M., Godoy, J. A., Toledo, E. M., Lukas, R. J., Barrantes, F. J., and Inestrosa, N. C. (2007) J. Neurosci. 27, 5313-5325[Abstract/Free Full Text]
  21. Banker, G. A., and Cowan, W. M. (1977) Brain Res. 126, 397-442[CrossRef][Medline] [Order article via Infotrieve]
  22. Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981) Pfluegers Arch. Eur. J. Physiol. 391, 85-100[CrossRef][Medline] [Order article via Infotrieve]
  23. Bonansco, C., and Buno, W. (2003) Hippocampus 13, 150-163[CrossRef][Medline] [Order article via Infotrieve]
  24. Hu, H., Shao, L. R., Chavoshy, S., Gu, N., Trieb, M., Behrens, R., Laake, P., Pongs, O., Knaus, H. G., Ottersen, O. P., and Storm, J. F. (2001) J. Neurosci. 21, 9585-9597[Abstract/Free Full Text]
  25. Andersen, P., Silfvenius, H., Sundberg, S. H., Sveen, O., and Wigstrom, H. (1978) Brain Res. 144, 11-18[CrossRef][Medline] [Order article via Infotrieve]
  26. Hsia, A. Y., Malenka, R. C., and Nicoll, R. A. (1998) J. Neurophysiol. 79, 2013-2024[Abstract/Free Full Text]
  27. Clark, K. A., Randall, A. D., and Collingridge, G. L. (1994) Exp. Brain Res. 101, 272-278[Medline] [Order article via Infotrieve]
  28. Kuhnt, U., and Voronin, L. L. (1994) Neuroscience 62, 391-397[CrossRef][Medline] [Order article via Infotrieve]
  29. Manabe, T., Renner, P., and Nicoll, R. A. (1992) Nature 355, 50-55[CrossRef][Medline] [Order article via Infotrieve]
  30. Wyllie, D. J., Manabe, T., and Nicoll, R. A. (1994) Neuron 12, 127-138[CrossRef][Medline] [Order article via Infotrieve]
  31. Moon, R. T., Bowerman, B., Boutros, M., and Perrimon, N. (2002) Science 296, 1644-1646[Abstract/Free Full Text]
  32. Fletcher, T. L., Cameron, P., De Camilli, P., and Banker, G. (1991) J. Neurosci. 11, 1617-1626[Abstract]
  33. Rattner, A., Hsieh, J. C., Smallwood, P. M., Gilbert, D. J., Copeland, N. G., Jenkins, N. A., and Nathans, J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2859-2863[Abstract/Free Full Text]
  34. Jope, R. S. (2003) Trends Pharmacol. Sci. 24, 441-443[CrossRef][Medline] [Order article via Infotrieve]
  35. Liu, S. J., Zhang, A. H., Li, H. L., Wang, Q., Deng, H. M., Netzer, W. J., Xu, H., and Wang, J. Z. (2003) J. Neurochem. 87, 1333-1344[Medline] [Order article via Infotrieve]
  36. Cochilla, A. J., Angleson, J. K., and Betz, W. J. (1999) Annu. Rev. Neurosci. 22, 1-10[CrossRef][Medline] [Order article via Infotrieve]
  37. Kay, A. R., Alfonso, A., Alford, S., Cline, H. T., Holgado, A. M., Sakmann, B., Snitsarev, V. A., Stricker, T. P., Takahashi, M., and Wu, L. G. (1999) Neuron 24, 809-817[CrossRef][Medline] [Order article via Infotrieve]
  38. Lie, D. C., Colamarino, S. A., Song, H. J., Desire, L., Mira, H., Consiglio, A., Lein, E. S., Jessberger, S., Lansford, H., Dearie, A. R., and Gage, F. H. (2005) Nature 437, 1370-1375[CrossRef][Medline] [Order article via Infotrieve]
  39. De Ferrari, G. V., Chacon, M. A., Barria, M. I., Garrido, J. L., Godoy, J. A., Olivares, G., Reyes, A. E., Alvarez, A., Bronfman, M., and Inestrosa, N. C. (2003) Mol. Psychiatry 8, 195-208[CrossRef][Medline] [Order article via Infotrieve]
  40. Inestrosa, N., De Ferrari, G. V., Garrido, J. L., Alvarez, A., Olivares, G. H., Barria, M. I., Bronfman, M., and Chacon, M. A. (2002) Neurochem. Int. 41, 341-344[CrossRef][Medline] [Order article via Infotrieve]
  41. Sudhof, T. C. (2004) Annu. Rev. Neurosci. 27, 509-547[CrossRef][Medline] [Order article via Infotrieve]
  42. Ahmad-Annuar, A., Ciani, L., Simeonidis, I., Herreros, J., Fredj, N. B., Rosso, S. B., Hall, A., Brickley, S., and Salinas, P. C. (2006) J. Cell Biol. 174, 127-139[Abstract/Free Full Text]
  43. Okuda, T., Yu, L. M., Cingolani, L. A., Kemler, R., and Goda, Y. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 13479-13484[Abstract/Free Full Text]
  44. Tai, C. Y., Mysore, S. P., Chiu, C., and Schuman, E. M. (2007) Neuron 54, 771-785[CrossRef][Medline] [Order article via Infotrieve]
  45. Bamji, S. X., Shimazu, K., Kimes, N., Huelsken, J., Birchmeier, W., Lu, B., and Reichardt, L. F. (2003) Neuron 40, 719-731[CrossRef][Medline] [Order article via Infotrieve]
  46. Kishida, S., Hamao, K., Inoue, M., Hasegawa, M., Matsuura, Y., Mikoshiba, K., Fukuda, M., and Kikuchi, A. (2007) Genes Cells 12, 49-61[Abstract/Free Full Text]
  47. Matsumine, A., Ogai, A., Senda, T., Okumura, N., Satoh, K., Baeg, G. H., Kawahara, T., Kobayashi, S., Okada, M., Toyoshima, K., and Akiyama, T. (1996) Science 272, 1020-1023[Abstract]

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G. G. Farias, I. E. Alfaro, W. Cerpa, C. P. Grabowski, J. A. Godoy, C. Bonansco, and N. C. Inestrosa
Wnt-5a/JNK Signaling Promotes the Clustering of PSD-95 in Hippocampal Neurons
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