Temporally distinct demands for classic cadherins in synapse formation and maturation

https://doi.org/10.1016/j.mcn.2004.08.008Get rights and content

Classic cadherins are synaptic adhesion proteins that have been implicated in synapse formation and targeting. Brief inactivation of classic cadherin function in young neurons appears to abrogate synapse formation when examined acutely. It remains unknown if such abrogation is unique to young neurons, whether it occurs by stalling neuronal maturation or by directly interfering with the process of synapse assembly, or whether synapse targeting is altered. Here we asked if sustained pan-cadherin blockade would prevent or alter the progression of axonal and dendritic outgrowth, synaptogenesis, or the stereotypic distribution of excitatory and inhibitory synapses on cultured hippocampal neurons. While pre- and postsynaptic cadherins are required for synapse assembly in young neurons, we find that in neurons older than 10 days, classic cadherins are entirely dispensable for joining and aligning presynaptic vesicle clusters with molecular markers of the postsynaptic density. Furthermore, we find that the proportion and relative distributions of excitatory and inhibitory terminals on single neurons are not altered. However, synapses that form on neurons in which cadherin function is blocked are smaller; they exhibit decreased synaptic vesicle recycling and a decreased frequency of spontaneous EPSCs. Moreover, they fail to acquire resistance to F-actin depolymerization, a hallmark of mature, stable contacts. These data provide new evidence that cadherins are required to promote synapse stabilization and structural and functional maturation, but dispensable for the correct subcellular distribution of excitatory and inhibitory synapses.

Introduction

Mature synapses are thought to arise from several temporally and molecularly distinct stages of assembly that culminate in stable junctions. An important component of the process is the segregation of excitatory and inhibitory synapses to different regions of the somatodendritic domain (Andersen et al., 1966, Blackstad and Flood, 1963). Classic cadherins are synaptic adhesion proteins that have been implicated in initial stages of synapse formation based on brief periods of overexpression of dominant-negative mutants in young neurons (Togashi et al., 2002). Differential localization of cadherins implicates them in synapse targeting as well (Arndt and Redies, 1996, Bekirov et al., 2002, Fannon and Colman, 1996, Gil et al., 2002, Suzuki et al., 1997). It is not known whether the actions of classic cadherins are required initially for synapse assembly in neurons of any age, whether their actions on assembly are exerted presynaptically, postsynaptically, or transsynaptically or whether cadherins coordinate the subcellular localization of excitatory and inhibitory synapses.

Classic cadherins comprise a subfamily of the cadherin family of adhesion proteins whose members are suspected to be functionally similar (Tepass et al., 2000). They all engage in strong, calcium-dependent, homophilic binding interactions, and in epithelial cells, they are the principal means for actin recruitment to adherens junctions by virtue of intracellular domain interactions with β- and α-catenin (Takeichi, 1991). Evidence consistent with the idea that cadherins are important for establishing synaptic junctions includes the observations that cadherins and catenins are among the first proteins found concentrated at developing synapses (Benson and Tanaka, 1998, Huntley and Benson, 1999) and that presynaptic terminals fail to form on young neurons that express briefly a dominant-negative N-cadherin mutant lacking the extracellular domain (NcadΔE) (Togashi et al., 2002). Furthermore, in young neurons, F-actin-linked adhesion is critical for stabilizing young synapses (Zhang and Benson, 2001). These data have been broadly interpreted in form of the idea that classic cadherin adhesion across the synaptic cleft is required generally for synapse assembly (Fannon and Colman, 1996, Uchida et al., 1996). Nevertheless, a different view is emerging based on results of experiments carried out on more mature neurons. Transient introduction of NcadΔE into neurons after synapses have formed does not lead to synapse disassembly, but rather produces spikey-shaped dendritic spines (Togashi et al., 2002), similar to the spine morphology observed in mature neurons following actin depolymerization (Allison et al., 1998, Zhang and Benson, 2001) and in neurons cultured from mouse mutants lacking α-N-catenin (Abe et al., 2004). Together, these results suggest that the canonical classic cadherin interaction between the cadherin intracellular domain, β-catenin and α-catenin, and F-actin is critical for the generation of normal spine morphology, but given that synapses can form in the α-N-catenin mutant, is dispensable for initial stages of synapse assembly (Park et al., 2002, Togashi et al., 2002). Selective deletion of β-catenin following synaptogenesis decreases presynaptic vesicle reserve pool size in vivo and alters the shape of presynaptic vesicle clusters in culture (Bamji et al., 2003). Collectively, results from the manipulation of α- and β-catenins have been used to indirectly support the idea that cadherins are not required for synapse development and that cadherin action at synapses is principally modulatory (Abe et al., 2004, Bamji et al., 2003). However, it is important to note that, in both α-N-catenin and β-catenin mouse mutants, N-cadherin remains appropriately localized to synapses (Bamji et al., 2003, Togashi et al., 2002), most likely by virtue of its interaction with a number of additional intracellular binding partners including δ-catenin, p120-catenin, presenilin, and PTPμ; each of which has been localized to synapses, and some of which have links to the F-actin cytoskeleton (Brady-Kalnay et al., 1998, Georgakopoulos et al., 1999, Hirano et al., 2003, Martinez et al., 2003, Yap et al., 1997). Thus, it remains an open question whether cadherins are essential components for synapse assembly, being required in all neurons no matter what their age or whether cadherin contributions to synapses are temporally regulated, being used for discrete purposes at different stages of either neural or synapse differentiation.

Recognition between pre- and postsynaptic elements is suspected to be a regulated stage of synapse assembly (Benson et al., 2001, Vaughn, 1989). While individual neurons express several cadherins simultaneously, particular cadherins tend to predominate within interconnected groups of neurons (Arndt and Redies, 1996, Suzuki et al., 1997, Bekirov et al., 2002, Gil et al., 2002), leading to the widely touted notion that adhesion between like-cadherins may be important for synapse recognition (Fannon and Colman, 1996, Serafini, 1999, Shapiro and Colman, 1999). In support of this idea, N-cadherin is important for generating layer-specific connections (Inoue and Sanes, 1997, Poskanzer et al., 2003), N- and E-cadherin parse to different dendritic domains in hippocampus (Fannon and Colman, 1996), and N-cadherin becomes concentrated solely at excitatory synapses in several brain areas (Benson and Tanaka, 1998). Such specificity can be considered at many levels, one being that of subcellular position. For example, in adult hippocampus, nearly all synapses on cell bodies are inhibitory, while spine synapses are all excitatory (Andersen et al., 1966, Blackstad and Flood, 1963), a distribution of inputs that profoundly affects the firing behavior of individual neurons (Freund, 2003, Harris et al., 2002). This is a property that is at least partly intrinsic to hippocampal neurons in that, when they are dissociated and allowed to form synapses in culture, inhibitory synapses predominate on cell somata, while excitatory synapses predominate more distally (Benson and Cohen, 1996).

Here we have examined directly the temporal requirements for classic cadherins in synapse assembly and whether cadherins generate the ordered distribution of excitatory and inhibitory synapses upon individual neurons. Our data show that there are maturationally distinct demands for cadherins at synapses. Cadherin function is required for the earliest stages of synapse assembly in young neurons, but in older neurons, compensatory or alternative mechanisms suffice. Cadherins are also dispensable for the precise targeting of excitatory and inhibitory synaptic terminals to different regions of the somatodendritic domain. Beyond initial assembly, classic cadherin functions are required to generate synaptic complexes having normal levels of synaptic vesicle recycling and spontaneous neurotransmitter release, as well as for synapses to progress to a stage of maturation in which they become resistant to disassembly by actin-depolymerizing reagents.

Section snippets

NcadΔE prohibits classic cadherin-mediated cell–cell interactions, synaptic accumulation of β-catenin, and N-cadherin-mediated outgrowth

It is well established that overexpression of a membrane-targeted, mutant N-cadherin lacking most or all of the extracellular domain effectively blocks N-cadherin-mediated cell–cell adhesion by both competing for β-catenin binding at the membrane, which would disconnect cadherins from the actin cytoskeleton, as well as by downregulating cadherin production (Kintner, 1992, Fujimori and Takeichi, 1993, Nieman et al., 1999). We generated a similar construct from mouse N-cadherin, tagged at its

Discussion

Here we report that functional interactions between pre- and postsynaptic classic cadherins are essential for normal synapse growth, for the acquisition of mature presynaptic function, and for resistance to disassembly by actin-depolymerizing reagents, all hallmarks of mature and stable synapses. While classic cadherins are important contributors to the initiation of synapse assembly, cadherin blockade in young neurons delays rather than prevents synapse formation. Over time, cadherin-based

Hippocampal culture

Hippocampal neurons were cultured from embryonic day 18 Sprague–Dawley rats, using the methods described by Goslin and Banker (1991), and plated at a density of 7200 cells/cm2 on poly-l-lysine and laminin-coated coverslips unless indicated otherwise in the text. Neurons were inverted over a confluent layer of astrocytes and maintained in Neurobasal media (Invitrogen, Carlsbad, CA) supplemented with 1× B-27 (Invitrogen).

Transfections

Hippocampal neurons were transfected at times indicated in text using

Acknowledgments

This research was supported by NIH USPHS grants NS37731 and AA12971, and an Irma T. Hirschl Career Scientist Award. We thank Drs. G. W. Huntley, G. Phillips, and T. Anderson for their comments on the manuscript and Insoo Kim and Pamela Shah for technical assistance. We thank Dr. S. T. Suzuki at the Institute for Developmental Research in Aichi, Japan, for generously providing us with cadherin 8-expressing L-cells, and Drs. Yoshinaga Saeki and E. A. Chiocca at Massachusetts General Hospital in

References (84)

  • G.W. Huntley et al.

    Structural remodeling of the synapse in response to physiological activity

    Cell

    (2002)
  • Y. Iwai et al.

    DN-cadherin is required for spatial arrangement of nerve terminals and ultrastructural organization of synapses

    Mol. Cell. Neurosci.

    (2002)
  • N. Kaufmann et al.

    Drosophila liprin-alpha and the receptor phosphatase Dlar control synapse morphogenesis

    Neuron

    (2002)
  • C. Kintner

    Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain

    Cell

    (1992)
  • R. Mohrmann et al.

    Developmental maturation of synaptic vesicle cycling as a distinctive feature of central glutamatergic synapses

    Neuroscience

    (2003)
  • M. Morales et al.

    Actin-dependent regulation of neurotransmitter release at central synapses

    Neuron

    (2000)
  • S. Murase et al.

    Depolarization drives beta-catenin into neuronal spines promoting changes in synaptic structure and function

    Neuron

    (2002)
  • S. Naisbitt et al.

    Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin

    Neuron

    (1999)
  • R.J. O'Brien et al.

    Synaptic clustering of AMPA receptors by the extracellular immediate– early gene product Narp

    Neuron

    (1999)
  • R. Riehl et al.

    Cadherin function is required for axon outgrowth in retinal ganglion cells in vivo

    Neuron

    (1996)
  • P. Scheiffele et al.

    Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons

    Cell

    (2000)
  • T. Serafini

    Finding a partner in a crowd: neuronal diversity and synaptogenesis

    Cell

    (1999)
  • L. Shapiro et al.

    The diversity of cadherins and implications for a synaptic adhesive code in the CNS

    Neuron

    (1999)
  • S.C. Suzuki et al.

    Neuronal circuits are subdivided by differential expression of type-II classic cadherins in postnatal mouse brains

    Mol. Cell. Neurosci.

    (1997)
  • L. Tang et al.

    A role for the cadherin family of cell adhesion molecules in hippocampal long-term potentiation

    Neuron

    (1998)
  • H. Togashi et al.

    Cadherin regulates dendritic spine morphogenesis

    Neuron

    (2002)
  • M. Wyszynski et al.

    Interaction between GRIP and liprin-alpha/SYD2 is required for AMPA receptor targeting

    Neuron

    (2002)
  • A.S. Yap et al.

    Lateral clustering of the adhesive ectodomain: a fundamental determinant of cadherin function

    Curr. Biol.

    (1997)
  • K. Abe et al.

    Stability of dendritic spines and synaptic contacts is controlled by alphaN-catenin

    Nat. Neurosci.

    (2004)
  • D.W. Allison et al.

    Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors

    J. Neurosci.

    (1998)
  • P. Andersen et al.

    Location and identification of excitatory synapses on hippocampal pyramidal cells

    Exp. Brain Res.

    (1966)
  • K. Arndt et al.

    Restricted expression of R-cadherin by brain nuclei and neural circuits of the developing chicken brain

    J. Comp. Neurol.

    (1996)
  • D.L. Benson et al.

    Activity-independent segregation of excitatory and inhibitory synaptic terminals in cultured hippocampal neurons

    J. Neurosci.

    (1996)
  • D.L. Benson et al.

    N-cadherin redistribution during synaptogenesis in hippocampal neurons

    J. Neurosci.

    (1998)
  • D.L. Benson et al.

    Molecules, maps and synapse specificity

    Nat. Rev., Neurosci.

    (2001)
  • T. Biederer et al.

    SynCAM, a synaptic adhesion molecule that drives synapse assembly

    Science

    (2002)
  • L.I. Binder et al.

    Differential localization of MAP2 and tau in mammalian neurons in situ

    Ann. N. Y. Acad. Sci.

    (1986)
  • T.W. Blackstad et al.

    Ultrastructure of hippocampal axo-somatic synapses

    Nature

    (1963)
  • M.E. Blue et al.

    The formation and maturation of synapses in the visual cortex of the rat: I. Qualitative analysis

    J. Neurocytol.

    (1983)
  • T.M. Boeckers et al.

    Proline-rich synapse-associated protein-1/cortactin binding protein 1 (ProSAP1/CortBP1) is a PDZ-domain protein highly enriched in the postsynaptic density

    J. Neurosci.

    (1999)
  • S.M. Brady-Kalnay et al.

    Dynamic interaction of PTPmu with multiple cadherins in vivo

    J. Cell Biol.

    (1998)
  • S. Brenowitz et al.

    Maturation of synaptic transmission at end-bulb synapses of the cochlear nucleus

    J. Neurosci.

    (2001)
  • Cited by (0)

    1

    These authors contributed equally.

    View full text