Deanna L. Benson

Increasing numbers of synaptic puncta during late-phase LTP: N-cadherin is synthesized, recruited to synaptic sites, and required for potentiation.

It is an open question whether new synapses form during hippocampal LTP. Here, we show that late-phase LTP (L-LTP) is associated with a significant increase in numbers of synaptic puncta identified by synaptophysin and N-cadherin, an adhesion protein involved in synapse formation during development. During potentiation, protein levels of N-cadherin are significantly elevated and N-cadherin dimerization is enhanced. The increases in synaptic number and N-cadherin levels are dependent on cAMP-dependent protein kinase (PKA) and protein synthesis, both of which are also required for L-LTP. Blocking N-cadherin adhesion prevents the induction of L-LTP, but not the early-phase of LTP (E-LTP). Our data suggest that N-cadherin is synthesized during the induction of L-LTP and recruited to newly forming synapses. N-cadherin may play a critical role in L-LTP by holding nascent pre-and postsynaptic membranes in apposition, enabling incipient synapses to acquire function and contribute to potentiation.

Molecular Modification of N-Cadherin in Response to Synaptic Activity

The relationship between adhesive interactions across the synaptic cleft and synaptic function has remained elusive. At certain CNS synapses, pre- to postsynaptic adhesion is mediated at least in part by neural (N-) cadherin. Here, we demonstrate that upon depolarization of hippocampal neurons in culture by K+ treatment, or application of NMDA or alpha-latrotoxin, synaptic N-cadherin dimerizes and becomes markedly protease resistant. These properties are indices of strong, stable, enhanced cadherin-mediated intercellular adhesion. N-cadherin retained protease resistance for at least 2 hr after recovery, while other surface molecules, including other cadherins, were completely degraded. The acquisition of protease resistance and dimerization of N-cadherin is not dependent on new protein synthesis, nor is it accompanied by internalization of N-cadherin. By immunocytochemistry, we found that high K+ selectively induces surface dispersion of N-cadherin, which, after recovery, returns to synaptic puncta. N-cadherin dispersion under K+ treatment parallels the rapid expansion of the presynaptic membrane consequent to the massive vesicle fusion that occurs with this type of depolarization. In contrast, with NMDA application, N-cadherin does not disperse but does acquire enhanced protease resistance and dimerizes. Our data strongly suggest that synaptic adhesion is dynamically and locally controlled, and modulated by synaptic activity.

Making memories stick: cell-adhesion molecules in synaptic plasticity

Synapses are adhesive junctions highly specialized for interneuronal signalling in the central nervous system. The strength of the synaptic signal can be modified (synaptic plasticity), a key feature of the cellular changes thought to underlie learning and memory. Cell-adhesion molecules are important constituents of synapses, with well-recognized roles in building and maintaining synaptic structure during brain development. However, growing evidence indicates that cell-adhesion molecules also play important and diverse roles in regulating synaptic plasticity and learning and memory. This review focuses on recent advances in understanding the molecular mechanisms through which adhesion molecules might regulate synaptic plasticity.

Molecules, maps and synapse specificity

A striking feature of the mature central nervous system is the precision of the synaptic circuitry. In contemplating the mature circuitry, it is impossible to imagine how more than 20 billion neurons in the human brain become precisely connected through trillions of synapses. Remarkably, much of the final wiring can be established in the absence of neural activity or experience; so the algorithms that allow precise connectivity must be encoded largely by the genetic programme. This programme, honed over nearly one billion years of evolution, generates networks with the flexibility to respond to a wide range of physiological challenges. There are several contemporary models of how synapse specificity is achieved, many of them proposed before the identification of guidance or recognition molecules. Here we review a selection of models as frameworks for defining the nature and complexity of synaptogenesis, and evaluate their validity in view of progress made in identifying the molecular underpinnings of axon guidance, targeting and synapse formation.

γ-Protocadherins Are Targeted to Subsets of Synapses and Intracellular Organelles in Neurons

The clustered protocadherins (Pcdhs) comprise >50 putative synaptic recognition molecules that are related to classical cadherins and highly expressed in the nervous system. Pcdhs are organized into three gene clusters (α, β, and γ). Within the α and γ clusters, three exons encode the cytoplasmic domain for each Pcdh, making these domains identical within a cluster. Using an antibody to the Pcdh-γ constant cytoplasmic domain, we find that all interneurons in cultured hippocampal neurons express high levels of Pcdh-γs in a nonsynaptic distribution. In contrast, only 48% of pyramidal-like cells expressed appreciable levels of these molecules. In these cells, Pcdh-γs were associated with a subset of excitatory synapses in which they may mediate presynaptic to postsynaptic recognition in concert with classical cadherins. Immunogold localization in hippocampal tissue showed Pcdh-γs at some synapses, in nonsynaptic plasma membranes, and in axonal and dendritic tubulovesicular structures, indicating that they may be exchanged among synapses and intracellular compartments. Our results show that although Pcdh-γs can be synaptic molecules, synapses form lacking Pcdh-γs. Thus, Pcdh-γs and their relatives may be late additions to the classical cadherin-based synaptic adhesive scaffold; their presence in intracellular compartments suggests a role in modifying synaptic physiology or stability.

Functional binding interaction identified between the axonal CAM L1 and members of the ERM family

Ayeast two-hybrid library was screened using the cytoplasmic domain of the axonal cell adhesion molecule L1 to identify binding partners that may be involved in the regulation of L1 function. The intracellular domain of L1 bound to ezrin, a member of the ezrin, radixin, and moesin (ERM) family of membrane–cytoskeleton linking proteins, at a site overlapping that for AP2, a clathrin adaptor. Binding of bacterial fusion proteins confirmed this interaction. To determine whether ERM proteins interact with L1 in vivo, extracellular antibodies to L1 were used to force cluster the protein on cultured hippocampal neurons and PC12 cells, which were then immunolabeled for ERM proteins. Confocal analysis revealed a precise pattern of codistribution between ERMs and L1 clusters in axons and PC12 neurites, whereas ERMs in dendrites and spectrin labeling remained evenly distributed. Transfection of hippocampal neurons grown on an L1 substrate with a dominant negative ERM construct resulted in extensive and abnormal elaboration of membrane protrusions and an increase in axon branching, highlighting the importance of the ERM–actin interaction in axon development. Together, our data indicate that L1 binds directly to members of the ERM family and suggest this association may coordinate aspects of axonal morphogenesis.

Temporally Distinct Demands for Classic Cadherins in Synapse Formation and Maturation

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.

Persistence of Coordinated Long-Term Potentiation and Dendritic Spine Enlargement at Mature Hippocampal CA1 Synapses Requires N-Cadherin

Persistent changes in spine shape are coupled to long-lasting synaptic plasticity in hippocampus. The molecules that coordinate such persistent structural and functional plasticity are unknown. Here, we generated mice in which the cell adhesion molecule N-cadherin was conditionally ablated from postnatal, excitatory synapses in hippocampus. We applied to adult mice of either sex a combination of whole-cell recording, two-photon microscopy, and spine morphometric analysis to show that postnatal ablation of N-cadherin has profound effects on the stability of coordinated spine enlargement and long-term potentiation (LTP) at mature CA1 synapses, with no effects on baseline spine density or morphology, baseline properties of synaptic neurotransmission, or long-term depression. Thus, N-cadherin couples persistent spine structural modifications with long-lasting synaptic functional modifications associated selectively with LTP, revealing unexpectedly distinct roles at mature synapses in comparison with earlier, broader functions in synapse and spine development.

Neural (N)-Cadherin at Developing Thalamocortical Synapses Provides an Adhesion Mechanism for the Formation of Somatopically Organized Connections

Thalamic projections from the ventrobasal (VB) nucleus to rodent somatosensory cortex develop highly ordered terminations that form discrete, clustered patches localized to layer IV cellular aggregates that are termed “barrels.” The molecular signaling and adhesion events that occur at the synapse as barrel‐clustered thalamic connections form are unknown. Here, we show that neural (N)‐cadherin, a membrane glycoprotein mediating strong homophilic adhesion, is concentrated at the developing thalamocortical synaptic junctional complex and demarcates these synaptic junctions as they form their characteristic barrel clusters during the first postnatal week. Furthermore, experimentally altering the distribution of thalamocortical axon terminals by peripheral manipulation leads to an identically altered N‐cadherin distribution pattern, which is significant in establishing that N‐cadherin does not define region‐specific patterns of synapse distribution proactively but, rather, conforms to patterning imposed by thalamic axons through instructional cues conveyed through several synaptic relays. At postnatal day 9, levels of N‐cadherin expression rapidly decrease, leading to loss of N‐cadherin labeling of the barrels and, at adulthood, elimination from VB thalamocortical synapses. However, αN‐ and β‐catenin, which are critical binding partners of the classic cadherins, persist at the adult synapse, suggesting the presence of another classic cadherin as the thalamocortical synapse matures. This is the first evidence linking a synapse adhesion molecule with the establishment of patterned thalamocortical synapse distribution, suggesting strongly that N‐cadherin performs a critical role in this process by adhering presynaptic and postsynaptic membranes as ingrowing thalamic axon terminals and postsynaptic thalamorecipient sites link and stabilize into mature synaptic junctional complexes distributed with precise topographic order. It is speculated that the developmental redistribution of N‐cadherin may reflect dynamic regulation of synaptic membrane adhesion, which, in turn, might modulate plasticity of thalamocortical synaptic function. J. Comp. Neurol. 407:453–471, 1999.

Identification and localization of multiple classic cadherins in developing rat limbic system

Classic cadherins are multifunctional adhesion proteins that play roles in tissue histogenesis, neural differentiation, neurite outgrowth and synapse formation. Several lines of evidence suggest that classic cadherins may establish regional or laminar recognition cues by virtue of their differential expression and tight, and principally homophilic, cell adhesion. As a first step toward investigating the role this family plays in generating limbic system connectivity, we used RT-PCR to amplify type I and type II classic cadherins present in rat hippocampus during the principal period of synaptogenesis. We identified nine different cadherins, one of which, cadherin-9, is novel in hippocampus. Using in situ hybridization, we compared the cellular and regional distribution of five of the cadherins (N, 6, 8, 9 and 10) during the first two postnatal weeks in hippocampus, subiculum, entorhinal cortex, cingulate cortex, anterior thalamus, hypothalamus and amygdala. We find that each cadherin is differentially distributed in distinct, but highly overlapping fields that largely correspond to known anatomical boundaries and are often coordinately expressed in interconnected regions. For example, cadherin-6 expression defines CA1 and its principal target, the subiculum; cadherin-10 is differentially expressed in CA1 and CA3 in a manner correlating with the organization of interconnecting Schaffer collateral axons; and cadherin-9 shows a striking concentration in CA3. Some cadherin mRNAs are highly restricted to particular anatomical fields over the entire time course, while others are more broadly expressed and become concentrated within particular domains coincident with the timing of afferent ingrowth. Our data indicate that classic cadherins are sufficiently diverse and differentially distributed to support a role in cell surface recognition and adhesion during the formation of limbic system connectivity.