Wnt signaling: Role in LTP, neural networks and memory

Highlights

• Wnt signaling components are expressed in adult brain. The brain distribution of Frizzled receptor is examined in detail.

• The different effects of Wnt ligands in pre- and postsynaptic region are discussed.

• Wnt ligands are released in spontaneous and activity dependent processes, and probably play a major role in cognition.

• Wnts modulate oscillatory activity in neural networks.

• Wnt signaling stimulate neuroprotection and might be used in the control of neurodegenerative and mental disorders.

Abstract

Wnt components are key regulators of a variety of developmental processes, including embryonic patterning, cell specification, and cell polarity. The Wnt signaling pathway participates in the development of the central nervous system and growing evidence indicates that Wnts also regulates the function of the adult nervous system. In fact, most of the key components including Wnts and Frizzled receptors are expressed in the adult brain. Wnt ligands have been implicated in the regulation of synaptic assembly as well as in neurotransmission and synaptic plasticity. Deregulation of Wnt signaling has been associated with several pathologies, and more recently has been related to neurodegenerative diseases and to mental and mood disorders. In this review, we focus our attention on the Wnt signaling cascade in postnatal life and we review in detail the presence of Wnt signaling components in pre- and postsynaptic regions. Due to the important role of Wnt proteins in wiring neural circuits, we discuss recent findings about the role of Wnt pathways both in basal spontaneous activities as well as in activity-dependent processes that underlie synaptic plasticity. Finally, we review the role of Wnt in vivo and we finish with the most recent data in literature that involves the effect of components of the Wnt signaling pathway in neurological and mental disorders, including a special emphasis on in vivo studies that relate behavioral abnormalities to deficiencies in Wnt signaling, as well as the data that support a neuroprotective role of Wnt proteins in relation to the pathogenesis of Alzheimer's disease.

Introduction

The proto-oncogene int-1 (Wnt-1) was the first member of the Wnt family identified as a common integration site for a mouse mammary tumor virus (Nusse and Varmus, 1982). The int-1 sequence protein proved to be identical to its homologue Drosophila, the Wingless gene, which corresponds to one of the first “segment polarity gene” involved in development as well as in cancer (Nusse and Varmus, 1982, Nusse and Varmus, 2012, Rijsewijk et al., 1987). Later, homologous gene targeting techniques confirmed that knocking down int-1/Wingless family gene causes several developmental impairments in a number of brain regions, corroborating that the Wnt gene is fundamental in determining a normal neural phenotype (McMahon and Moon, 1989b). Now the Wnt family includes 19 members present in mammals and it has been implicated in other major functions, from embryogenesis to different types of cancer, in stem cell development and also in some metabolic disorders such as diabetes mellitus, reflecting its relevance in fundamental biological processes that might be highly observed across species (Clevers and Nusse, 2012).

Although the knowledge about Wnt signaling components and how they are compromised in different phenotypes is vast, there are important aspects of the molecular machinery and how the specificity is established that remains unclear. Historically, Wnt proteins have been classified as either canonical (i.e. Wnt1 and Wnt3a), or non-canonical (i.e. Wnt4, Wnt5 and Wnt11) (Gordon and Nusse, 2006). However, the traditional differentiation between canonical and non-canonical ligands seems to be not as simple as was initially defined, and more aspects should be considered. One of the most important is the recognition that the amount of Wnt ligands and the receptors involved allows enormous possibilities of interactions which are difficult to predict. Moreover, the result of these interactions can differentially activate intracellular cascades, which in turn makes the cellular responses complexes and diverse (van Amerongen and Nusse, 2009). Furthermore, some literature indicates that activation of a canonical or non-canonical pathway in a cell to a given Wnt may depend on the context of how that Wnt couples the receptor and co-receptor, and not by the property of the ligand itself (Grumolato et al., 2010, Mikels and Nusse, 2006b). Finally, it has been shown that Wnt ligands can compete to bind to specific receptors, causing reciprocal pathway inhibition of both signaling pathways (Grumolato et al., 2010). Consequently, the effect of a Wnt ligand depends on a combination of several factors, including the combinatorial probability of binding different types of receptors and co-receptors present in the target cell, which in turn may trigger the activation of a specific intracellular signaling cascade. In the next section we review the details of those interactions.

The prototypical Wnt receptor is the seven transmembrane-receptor Frizzled (Fz). In its structure is a cystein-rich domain (CRD) which forms the Wnt-binding site (Bhanot et al., 1996), ten of which have been described in mice and humans. Despite the fact that the Fz gene shows many redundancies, Fz proteins show a high degree of affinity to every Wnt protein, resulting in a very specific interaction (Rulifson et al., 2000). In addition to Fz, other proteins have been described as alternative co-receptors for Wnt signaling (Fig. 1), such as the single transmembrane low-density lipoprotein receptor-related protein 5/6 (LRP5/6) (MacDonald et al., 2009, Mao et al., 2001, Tamai et al., 2000, Wehrli et al., 2000) and the single-pass transmembrane receptors tyrosine kinases (RTKs) Ror1, Ror2 and Ryk (Cadigan and Liu, 2006, Gordon and Nusse, 2006, Green et al., 2008, Lu et al., 2004, Oishi et al., 2003), increasing the complexity of Wnt signaling activation. But Wnt signaling has also secreted inhibitors or antagonists such as Dickkopf (Dkk) that binds LRP blocking the interaction of Wnt/Fz (Mao et al., 2001), and inhibitors such as the soluble Fz-related protein (sFRP) that bind directly to Wnt because of their similarity to Fz (Finch et al., 1997, Rattner et al., 1997). This natural antagonism could reflect an additional control of Wnt concentration in the extracellular media. This regulation can be crucial during a signal transduction mechanism.

Downstream of Wnt-receptor binding, a sequence of intracellular responses is elicited. The first Wnt signaling pathway identified, the so called “canonical pathway”, has been focus of intense research. The name “canonical” was given because induction of some embryonic structures in Xenopus (McMahon and Moon, 1989a) and the transformation of mouse mammary cells (Wong et al., 1994) were strongly correlated with the increment in β-catenin/TCF levels (Shimizu et al., 1997). The canonical pathway controls growth and cell fate specification (Cadigan and Nusse, 1997). Because dysregulation of β-catenin/TCF levels is present in a variety of cancers, this Wnt pathway has been the most studied to date. As we know now, Wnt1, Wnt3a, Wnt7a and Wnt8 bind the receptor Frizzled and the LRP5/6 co-receptors, activating the Wnt/β-catenin (MacDonald et al., 2009). Both Fz and LRP5/6 recruit the protein dishevelled (Dvl) usually by phosphorylation, which oligomerizes in the plasma membrane forming a platform for the allocation of the scaffold protein Axin and the glycogen synthase kinase-3β (GSK-3β) (Cliffe et al., 2003, Gao and Chen, 2010, Schwarz-Romond et al., 2007). This fact promotes the phosphorylation of LRP6 by casein kinase 1 (CD1) which is associated with each cluster of proteins to form the signalosome (Bilic et al., 2007). The phosphorylation of LRP5/6 causes the inhibition of the “destruction complex” that not only includes GSK-3β but also the tumor suppressor adenomatous polyposis coli (APC). The consequence of this inhibition is the cytoplasmic stabilization of β-catenin which enters the nucleus and regulates the transcription of Wnt target genes (Logan and Nusse, 2004) through the binding of the transcription factors T cell factor and lymphoid enhancer factor (TCF/LEF) (Clevers and Nusse, 2012). Several Wnt target genes are activated in this process, including c-Myc, cyclin D1, Axin2 and Calcium/calmodulin-dependent protein kinase type IV (CamKIV) (Arrazola et al., 2009, Hodar et al., 2010, Nusse and Varmus, 2012, Toledo et al., 2008). Thus, in this review we refer to the Wnt/β-catenin/TCF pathway when we mention the “canonical” pathway.

In the absence of Wnt stimulation, the cytoplasmic levels of β-catenin are low since it is ubiquitinated and constantly degraded in the proteasome (Aberle et al., 1997). β-catenin is also phosphorylated by casein kinase 1α (CK1α) and GSK-3β in a multiprotein complex composed also for Axin and APC (Nusse and Varmus, 2012). β-catenin phosphorylated by CK1α and GSK-3β also targets the armadillo protein for ubiquitination and subsequent degradation (Liu et al., 2002).

Another Wnt pathway identified is the β-catenin-independent pathway (also referred as “non-canonical pathway”). Wnt4, Wnt5a and Wnt11 have been identified as members of this non-canonical pathway because they bind to RTKs co-receptors (Green et al., 2008). There are at least two of them: the planar cell polarity (PCP) pathway and the Ca²⁺ pathway. The Wnt/PCP pathway was originally identified in Drosophila where it regulates tissue polarity and cell migration during development (Adler, 2002, Veeman et al., 2003). The PCP pathway signals through the JNK pathway to control cell polarity, which is why it is also known as the Wnt/JNK pathway (Boutros et al., 1998, Yamanaka et al., 2002). During activation, Wnt binds the receptor Fz on the membrane surface, and Dvl is followed by the activation of Rho/Rac small GTPase and c-Jun N-terminal kinase (JNK). Particularly, it is required that Wnt5a physically bridges Ror2 to Fz extracellular domains to activate the downstream pathway (Grumolato et al., 2010). Like Fz, Ror1/2 binds Wnt by its CRDs (Grumolato et al., 2010, Logan and Nusse, 2004). The downstream effect of activating this pathway is the regulation of cytoskeletal organization and gene expression (Fig. 1). Instead, the Wnt/Ca²⁺ pathway is mostly a G-protein dependent signaling pathway (Kohn and Moon, 2005). The activation of the Wnt/Ca²⁺ pathway requires binding of Wnt to Fz on the membrane surface to trigger stimulation of heterotrimeric G-proteins (Slusarski et al., 1997a, Slusarski et al., 1997b), which activate phospholipase-C (PLC). PLC causes the increment on intracellular release Ca²⁺, decreases cyclic guanosine monophosphate (cGMP) and activates the protein kinases Ca²⁺-Calmodulin-dependent Protein kinase II (CaMKII) and protein kinase-C (PKC) (Kohn and Moon, 2005, Montcouquiol et al., 2006, Veeman et al., 2003). This process activates the nuclear factor NFAT and transcription factors like cAMP Response Element-Binding Protein-1 (CREB). Therefore, the classification of individual Wnt proteins in canonical and non-canonical terms is used now to describe the activation of a signaling pathway, either β-catenin/TCF dependent or independent (Mikels and Nusse, 2006a, Mikels and Nusse, 2006b, van Amerongen and Nusse, 2009).

In this review we will only superficially refer to the importance that the Wnt/Frizzled signaling pathway plays in the development of the nervous system. Instead, the purpose of this review is to highlight the fact that much of the literature suggests that Wnt signaling also plays a key role during postnatal life (Chen et al., 2006, Gogolla et al., 2009, Li and Pleasure, 2005, Stranahan et al., 2010, Wayman et al., 2006). In following sections we will review the evidence, showing first that expression of Wnt ligands and proteins of the Wnt signaling machinery occurs in the mature nervous system. Because Shimogori and Cols published a very detailed paper describing the postnatal expression pattern of Wnt, Fz and FRP (Shimogori et al., 2004), we will focus this section mainly on their results.

The expression of Wnt ligands and proteins of the Wnt signaling machinery in the adult brain has been identified in the major subdivisions of the cerebral cortex, from olfactory bulb (OB), hippocampal formation, neocortex and thalamus (Shimogori et al., 2004). In situ hybridization has shown that expression of different Wnts is particularly high in those areas where the neurons are continuously renewed, such as OB and dentate gyrus (DG) in the hippocampus. It has been shown that Wnt5a, Wnt7a and Wnt1 are expressed at least from birth in OB and also in the piriform cortex and other olfactory related areas, and last as late as P20 (Shimogori et al., 2004). Wnt2b is also expressed in piriform, somatosensory and in the entorhinal cortex (EC) of postnatal and young adult mouse brains. Wnt5a has also been found in pre- and parasubiculum and EC until young adulthood (P20). The hippocampi of young adult rodents express several Wnt ligands, including Wnt1, Wnt2, Wnt4, Wnt5a, Wnt7a and b, Wnt8b and Wnt11 (Fig. 2) (Cerpa et al., 2008, Shimogori et al., 2004, Wayman et al., 2006). Particularly, Wnt2 is well expressed in CA1, CA2 and CA3 until the third week after birth, precisely during the period of dendrite development in the hippocampus (Wayman et al., 2006), while the expression of Wnt5a, Wnt7a and Wnt8b in specific layers of DG where postnatal neurogenesis occurs, may be related to their function in this area.

In the neocortex the expression is strikingly particular, with layer- and region-specific expression patterns for Wnt2b, Wnt5a and Wnt7b in the neocortex (Shimogori et al., 2004). Those regions include expression of Wnt2b in close relation to the whisker barrel pattern in layer 4 and 6 of the somatosensory cortex, and also in layer 6 in the visual and auditory cortex in young adulthood. Wnt5a and Wnt7a also appear in layer specific pattern expression in other cortices, including prefrontal, parietal and temporal neocortex (Shimogori et al., 2004).

The presence of Wnt signaling components in the thalamus is also very interesting. The thalamus brings most of the sensory information to the cortex. Gene expression of Wnt machinery appears in the postnatal thalamus with a very conspicuous pattern of specific Wnt along its different sections (Shimogori et al., 2004). Due to the fact that the sensory information is continuously modified during an animal's life, it is possible that Wnt signaling plays an important role in thalamocortical connectivity.

There is also evidence of postnatal expression of Fz receptors. Expression of Fz3 in the olfactory system includes expression from P0 to P7 and in the related areas including piriform cortex until adulthood. In the hippocampus, Fz3 expresses ubiquitously in the pyramidal cell layer and granular cells of DG until adulthood, and during this time is always expressed in EC (Shimogori et al., 2004). Instead, the pattern of Fz3 in the cortex is clear at P20 and ascribes to a layer pattern at different cortices, from prefrontal, cingulated, parietal and temporal, matching with Wnt2b in whisker barrel in the somatosensory cortex (Shimogori et al., 2004).

Fz7 is expressed in olfactory nerves through P20 and then decreases the expression in the piriform cortex. In the hippocampus, Fz7 expresses only in a pyramidal layer at P0, in CA1 and CA3 at P7 and expresses only at CA3 and the entorhinal cortex at P20. There appears to be no postnatal expression of Fz7 in the neocortex (Shimogori et al., 2004).

The remaining Fz receptors, including Fz1, Fz2, Fz8, Fz9 and Fz10, are mainly ubiquitously expressed in the neocortex and hippocampus until young adulthood. Fz1, Fz2 and Fz9 expression resembles Wnt5a expression in OB and DG. Fz4, Fz5 and Fz6 do not show postnatal expression except in the thalamus, showing – like Wnt – a striking cellular- and subregion-defined expression that allows us to speculate about its function (Shimogori et al., 2004).

The presence of Wnt components in many brain areas, including those that are actively participating in neurogenesis, or those involved in sensory processing and superior cognitive processes, suggests that Wnt pathway is involved in more than only structural functions. In the next sections we review the evidence that relates Wnt to active structural and functional processing in mature synapses, and we discuss the implications that this can have in modulating active dependent processes, such as synaptic plasticity.

Wnt ligands have been linked to the assembly of structural components in presynaptic compartments. In the cerebellum, Wnt7a is expressed in granular cells (GC) at the same time as the mossy fiber (MF) axon, which is the presynaptic contact (Lucas and Salinas, 1997). Several changes remodel the connectivity between both areas to increase the contact surface. Wnt7a induces axonal spreading and incremental growth of cone size and branching, leading to the accumulation of synaptic proteins (Hall et al., 2000, Lucas and Salinas, 1997). Wnt7a probably contributes to the formation of active zones because it increases the clustering of synapsin I, a protein located in the presynaptic membrane involved in synapse formation and function (Hall et al., 2000). Because this effect of Wnt7a is mimicked by lithium application, it seems to involve GSK-3β (Lucas and Salinas, 1997). This effect has been blocked by the Wnt antagonist sFRP and a mutant mice deficient in Wnt7a shows a delayed synaptic maturation (Hall et al., 2000). Then in the cerebellum, Wnt7a can act as a retrograde signal from GC to induce presynaptic differentiation in MF, working as a synaptogenic factor (Ahmad-Annuar et al., 2006, Hall et al., 2000). Like Wnt7a, Wnt7b and Wnt3a increase the number of pre-synaptic puncta suggesting a role for these ligands in presynaptic assembly (Ahmad-Annuar et al., 2006, Cerpa et al., 2008, Davis et al., 2008). Wnt7a also increases the clustering of presynaptic proteins such as synaptophysin, synaptotagmin and SV-2, but does not affect postsynaptic clustering of proteins like PSD-95 (Cerpa et al., 2008). Despite Wnt7a clustering induction correlates with β-catenin stabilization, this does not involve Wnt gene target expression – an effect that is also mimicked by Wnt3a. Unexpectedly, GSK-3β is also not required for presynaptic clustering induced by Wnt7a, suggesting that an upstream mechanism is involved (Cerpa et al., 2008). It has been suggested that Wnt7a requires Dvl1 to mediate the normal recycling rate of synaptic vesicles, and the deficiency of both proteins (double null mutant) significantly reduces miniature excitatory postsynaptic current (mEPSC) frequency, an indication of a defect in neurotransmitter release (Ahmad-Annuar et al., 2006). Additionally, the use of FM dyes has shown that Wnt7a stimulates recycling and accelerates exocytosis of synaptic vesicles (Ahmad-Annuar et al., 2006, Cerpa et al., 2008). Moreover, Wnt7a increases the frequency of mEPSCs, suggesting that Wnt7a increases the dynamic of neurotransmitter release (Ahmad-Annuar et al., 2006, Cerpa et al., 2008). Furthermore, Wnt7a/Dvl1 double null mutant mice exhibit reduced mEPSC frequency at the mossy fiber-granule cell synapses, revealing a defect in neurotransmitter release as a consequence of this mutation (Ahmad-Annuar et al., 2006). Electrophysiological recordings on hippocampal rat slices also show that, in the CA3-CA1 synapse Wnt7a, but not Wnt5a, increases the frequency of field excitatory postsynaptic potentials (fEPSP) and decreases the rate of paired pulse facilitation (PPF) (Cerpa et al., 2008), a protocol used to distinguish the involvement of the presynaptic from the postsynaptic terminal (Foster and McNaughton, 1991). In addition, a similar modulation has been shown with nanomolar concentrations of Wnt3a, which modulates the recycling and exocytosis of synaptic vesicles in hippocampal synapses, increasing the frequency of mEPSC through a mechanism that involves Ca²⁺ entrance from extracellular media (Avila et al., 2010, Cerpa et al., 2008). Most of the ligands that are able to modulate presynaptic differentiation have shown to activate the Wnt/β-catenin signaling pathway. Although Wnt3a has been designated as a classical “canonical” Wnt, the latest evidence suggests a cross-talk between Wnt3a and Wnt/Ca²⁺ (Avila et al., 2010), strongly supporting the idea that some of the components associated with the non-canonical pathway may be involved in the functionality of the presynaptic nerve terminal.

Wnt7a has been also involved in trafficking of receptors, increasing the number and size of co-clusters of presynaptic α₇-nicotinic acetylcholine receptors (α₇-nAChR) and APC in hippocampal neurons, as well as in the modulation of the α₇-nAChR trafficking to the nerve terminal (Farias et al., 2007), indicating that Wnt pathway components are actively involved in the functional availability of receptors in the synaptic terminal.

Wnt signaling can actively modulate the assembly of the postsynaptic region. Our laboratory has shown strong evidence supporting the role of the β-catenin independent pathway in remodeling of mature synapses. Wnt signaling activation modulates excitatory and inhibitory synaptic transmission (Cerpa et al., 2010, Cerpa et al., 2011, Cuitino et al., 2010). Wnt5a increases the evoked glutamatergic synaptic transmission without changes in the presynaptic compartment, as shown by PPF (Cerpa et al., 2010). Instead, Wnt5a acutely and specifically up-regulates synaptic currents through NMDA receptors, not AMPA receptors, involving PKC and JNK, and facilitating the induction of excitatory LTP (Cerpa et al., 2011). Moreover, Wnt5a is actively modulating inhibitory synapses in the hippocampus, increasing the turnover of GABAA receptors (GABAAR) on the surface, increasing GABA currents by postsynaptic mechanisms that involve Wnt/Ca²⁺/CaMKII (Cuitino et al., 2010). More evidence indicates that Wnt5a can induce de novo dendrite spine formation and increase the volume and density of pre-existent spines, enhancing the efficacy of hippocampal glutamatergic synapses (Varela-Nallar et al., 2010), suggesting that Wnt5a has a role in synaptic structure and function. Wnt5a can transiently induce formation of dendritic protrusions, which results in a net increase in mature dendrite spines (Varela-Nallar et al., 2010). Interestingly, the treatment with the soluble CRD region of Fz2, which act as a Wnt scavenger, decreases spine density in cultured neurons, supporting the idea that Wnt ligands participates in dendrite spine morphogenesis (Varela-Nallar et al., 2010). Wnt5a also increases calcium recruitment to synaptic puncta of culture cells, suggesting the activation of the Wnt/Ca²⁺ signaling pathway in cultured hippocampal neurons (Varela-Nallar et al., 2010) through a mechanism that involves fast phosphorylation of CaMKII induced by Wnt5a, as we demonstrated previously (Farias et al., 2009). As it does with GABA_AR, Wnt5a modulates postsynaptic assembly increasing the clustering of the postsynaptic density protein-95 (PSD-95) via Wnt/JNK signaling pathway (Farias et al., 2009), inducing a fast increase in the number of PSD-95 clusters without affecting total levels of PSD-95 protein or presynaptic protein clustering in hippocampal cultured neurons (Farias et al., 2009). However, Wnt5a does not only induce protein clustering. It has also been shown that Wnt3a is able to induce recruitment of AChRs in motoneurons at the moment of neuromuscular innervations (Henriquez et al., 2008). This effect requires Dvl1 and Agrin, a protoglycan released by motoneurons, but does not involve the Wnt/β-catenin pathway. Instead, aggregation is induced through activation of Rac1 (Henriquez et al., 2008). However, Wnt3a inhibits Agrin-induced AChR clusters through the activation of the Wnt/β-catenin pathway, suggesting that Wnt signaling dynamically regulates the interaction between postsynaptic components during the establishment of neuromuscular junctions (Wang et al., 2008).

Besides its role in presynaptic functions, Wnt7a has also been shown to regulate the postsynaptic compartment stimulating the morphogenesis and function of excitatory dendritic spines, without affecting the inhibitory connections in the hippocampus (Ciani et al., 2011). To do this, Wnt7a requires Dvl and activation of CamKII at dendritic spines (Ciani et al., 2011). Interestingly, Dvl expressed only in postsynaptic spines and not in innervating presynaptic axons is enough to induce spine growth, suggesting that it is the activation of postsynaptic Wnt signaling which induces spine maturation (Ciani et al., 2011). Moreover, Dvl promotes the assembly of pre- and postsynaptic structures at pre-existing spines because this does not change the number of spines (Ciani et al., 2011). This evidence supports the idea that an extracellular signal such as Wnt7a can generate a divergent intracellular product, using a common molecule such as Dvl to support processes like synaptic differentiation (Gao and Chen, 2010), and a new role for Wnt7a inducing the formation and function of excitatory synapses through CaMKII.

In addition, Wnt7b and Dvl can activate Rac, which in turn stimulates downstream JNK to increase dendritic length and branching in immature hippocampal neurons (Rosso et al., 2005). This effect is independent of Wnt/GSK-3β and is mimicked by over-expression of Dvl. The use of a dominant-negative Rac or dominant-negative JNK also blocks Dvl-dependency of dendritic growth (Rosso et al., 2005). This confirms the activation of the β-catenin independent pathway by Wnt7b, demonstrating that the non-canonical Wnt pathway, the Wnt/JNK, participates in dendrite development (Rosso et al., 2005).

Previous studies showed that β-catenin enhances dendritic arborization and this effect does not require Wnt/β-catenin/TCF dependent transcription (Yu and Malenka, 2003). This effect could be explained depending on the requirements during synaptic formation and maturation. Wnt/β-catenin dependent pathways might be relevant during developmental processes and synapse formation processes, while β-catenin independent signaling might be more relevant during activity-dependent processes, such as synaptic remodeling during plasticity.

Besides the role of Wnt in synaptic differentiation and function, is the role of Fz receptors. It has been shown that Fz1 clusters are co-localized with presynaptic proteins like synapsin-1 (Varela-Nallar et al., 2012) in functional synapses of cultured hippocampal neurons and also with the postsynaptic PSD-95 scaffold (Varela-Nallar et al., 2009). Over-expression of Fz1 increases the number of Bassoon clusters, a protein component of the active zone. The treatment with Wnt3a, a Fz1 ligand, also induces presynaptic clustering, increasing presynaptic recycling sites and the kinetics of vesicle release. These effects are prevented by incubation with CRD of Fz1 that acts as a scavenger (Varela-Nallar et al., 2009). Thus, Wnt acting through Fz1 modulates synaptic differentiation and function (Varela-Nallar et al., 2009). A more detailed study has shown that different Fz expresses differently along hippocampal development, from E18 to P60 (Varela-Nallar et al., 2012) and some but not all have synaptic distribution in culture. In fact, some of them, like Fz7 and Fz 9, localize in soma and all the processes, while Fz9 and Fz5 also concentrate in the growth cone (Varela-Nallar et al., 2012). The particular temporal and location pattern of Fz could amplify the range of possible interactions during development and later during maturation, but also brings specificity to the interaction of each different Wnt. More studies are required to elucidate how and when these pathways become relevant for the system. In the next section, we address some of these questions by studying the physiological processes involved in the interaction of multiple cells during activity and non-activity dependent processes.